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Oxygen
Sensing
Emily E. Weinert Editor
Methods in
Molecular Biology 2648
Methods and Protocols

METHODS INMOLECULARBIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For f
olumes:
http://www.springer.com/series/7651

Forover35years, biological scientists havecome torelyontheresearch protocols and
methodologies inthecritically acclaimed Methods inMolecular Biology series.Theseries was
theﬁrsttointroduce thestep-by-step protocols approach thathasbecome thestandard inall
biomedical protocol publishing. Eachprotocol isprovided inreadily-reproducible step-by
stepfashion, opening withanintroductor yoverview,alistofthematerials andreagents
needed tocomplete theexperiment, andfollowed byadetailed procedure thatissuppor ted
withahelpful notes section offering tipsandtricks ofthetradeaswellastroubleshooting
advice. These hallmark features wereintroduced byseries editor Dr.John Walkerand
constitute thekeyingredient ineachandeveryvolume oftheMethods inMolecular Biology
series.Tested andtrusted, comprehensive andreliable, allprotocols from theseries are
indexed inPubMed.

OxygenSensing
Methods and Protocols
Edited by
Emily E. Weinert
Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA, USA

Editor
Emily E. Weinert
Department of Biochemistry and
Molecular Biology
Penn State University
University Park, PA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods inMolecular Biology
ISBN 978-1-0716-3079-2 ISBN 978-1-0716-3080-8 (eBook)
https://doi.org/10.1007/978-1-0716-3080-8
©Theditor(s)ifpplicabe)ndheuthors),nderxclusiveicenseopringercience+Buinessedia,LC,ar
ofpringerature023
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ofheaterialsoncerned,peciﬁcallyheightsfranslation,eprinting,eusefllustratins,ecitation,
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ission orinformation storage and
retrieval, electronic adaptation ,computer software, orbysimilar ordissimilar methodol ogynowknown orhereafter
developed. Theuseofgeneral descriptive names, registered names, trademarks, servicemarks, etc.inthispublication doesnotimply,
evenintheabsence ofaspeciﬁc statement, thatsuchnames areexempt fromtherelevant protective lawsandregulatio ns
andtherefore freeforgeneral use.
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,andtheeditors aresafetoassume thattheadvice andinformation inthisbookarebelieved to
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Preface
This volume aims to provide the necessary protocols for researchers investigating known or
putative O
2sensing proteins and pathways. Oxygen sensing pathways are found within
organisms from all domains of life due to the essential role of O
2in aerobic metabolism and
the potential for oxidative damage. However, investigating O
2responsive systems in the
laboratory can be challenging, as most standard laboratory techniques are performed in air,
and therefore require specialized methods to ensure the absence or controlled concentration
of O
2. By detailing methods for O
2sensing protein and pathway characterization within this
volume, we aim to make these techniques available to a wide audience, ranging from
microbiologists and cell biologists to protein biochemists. Furthermore, the chapters
describe techniques ranging from anaerobic redox midpoint measurement to approaches
to control the expression of globin genes, which should provide detailed protocols for
researchers interested in expanding their investigations of O
2sensing systems.
University P
ark, PA , USA Emily E. Weinert
v

Contents
Preface . .................................................................... v
Contributors................................................................. ix
1Integrating UV-Vis Spectroscopy and Oxygen Optode
for Accurate Determination of Oxygen Afﬁnity of Proteins........... ........ 1
Anoop Rama Damodaran and Ambika Bhagi-Damodaran
2Measurement of O
2Binding by Sensory Hemeproteins....... ....... ........ 11
Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
3Resonance Raman Characterization of O
2-Binding Heme Proteins............ 27
Samuel N. Snyder, Tapiwa Chiura, and Piotr J. Mak
4Anaerobic Infrared Spectroelectrochemical Methods
for Studying Oxygen-Sensitive [FeFe] Hydrogenases......... ....... ........ 43
Patrick Corrigan and Alexey Silakov
5Monitoring the Kinase Activity of Heme-Based Oxygen
Sensors and Its Dependence on O
2and Other Ligands Using
Phos-Tag Electrophoresis........... ....... ....... .
...... ....... .. ....... 63
Jakub Va´vra, Artur Sergunin, Toru Shimizu, and Marke´ta Martı´nkova´
6In Vitro Measurement of Gas-Dependent and Redox-Sensitive
Diguanylate Cyclase Activity......... ....... ....... ........ ....... ........ 75
Nushrat J. Hoque, Madison P. Helm, and Emily E. Weinert
7Spectrophotometric Method for the Quantiﬁcation and Kinetic
Evaluation of In Vitro c-di-GMP Hydrolysis in the Presence
and Absence of Oxygen............. ....... ....... ........ ....... ........ 87
Dayna C. Patterson and Emily E. Weinert
8Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor
Proteins............ ........ ....... ........ 99
Jakub Va´vra, Artur Sergunin, Martin Stra´nˇava,
Alan Ka´dek, Toru Shimizu, Petr Man, and Marke´ta Martı´nkova´
9Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily........ 123
Molly J. McBride, Sarah R. Pope, Mrutyunjay A. Nair, Debangsu Sil,
Xavier E. Salas-Sola´, Carsten Krebs, J. Martin Bollinger Jr,
and Amie K. Boal
10Analyzing Iron and Oxygen-Regulated Protein Complex Formation
Using Proteomic Mass Spectrometry........ ....... ........ ....... ........ 155
Vijaya Pandey, Adarsh K. Mayank, and James A. Wohlschlegel
11CRISPR Activator Approaches to Study Endogenous Androglobin
Gene Regulation........... ........ ....... ......
......... ....... ........ 167
Te
i Koay, Johannes Scho¨del, and David Hoogewijs
vii

viii Contents
12Assays to Study Hypoxia-Inducible Factor Prolyl Hydroxylase
Domain 2 (PHD2), a Key Human Oxygen Sensing Protein.......... ........ 187
Yan Ying Chan, Naasson M. Mbenza, Mun Chiang Chan,
and Ivanhoe K. H. Leung
13Kinetic Measurements to Investigate the Oxygen-Sensing
Properties of Plant Cysteine Oxidases....... ....... ........ ....... ........ 207
Anna Dirr, Dona M. Gunawardana, and Emily Flashman
14Methods for Culturing Anaerobic Microorganisms........... ....... ........ 231
Michel G
antiago-Martı´nez and James Gregory Ferry
Index . ......... ........ ....... ........ ....... ....... ........ ....... ........ 239

Contributors
AMBIKABHAGI-DAMODARAN •Department of Chemistry, University of Minnesota-Twin
Cities, Minneapolis, MN, USA
A
MIEK. BOAL•Department of Chemistry, The Pennsylvania State University, University
Park, PA, USA; Department of Biochemistry and Molecular Biology, The Pennsylvania
State University, University Park, PA, USA
M
UNCHIANGCHAN•Department of Molecular Medicine, Universiti Malaya, Kuala
Lumpur, Malaysia; GlaxoSmithKline, Stevenage, Hertfordshire, UK
Y
ANYINGCHAN•Department of Molecular Medicine, Universiti Malaya, Kuala Lumpur,
Malaysia
T
APIWACHIURA•Department of Chemistry, Saint Louis University, Saint Louis, MO, USA
P
ATRICKCORRIGAN •Department of Chemistry, Penn State University, University Park, PA,
USA
A
NOOPRAMADAMODARAN •Department of Chemistry, University of Minnesota-Twin Cities,
Minneapolis, MN, USA
A
NNADIRR•Department of Chemistry, University of Oxford, Oxford, UK
J
AMESGREGORYFERRY•Department of Biochemistry and Molecular Biology, Pennsylvania
State University, University Park, PA, USA
E
MILYFLASHMAN •Department of Chemistry, University of Oxford, Oxford, UK;
Department of Biology, University of Oxford, Oxford, UK
M
ARIEA. GILLES-GONZALEZ •Department of Biochemistry, University of Texas Southwestern
Medical Center, Dallas, TX, USA
D
ONAM. GUNAWARDANA •Department of Chemistry, University of Oxford, Oxford, UK
M
ADISONP. HELM•Department of Chemistry, Penn State University, University Park, PA,
USA
D
AVIDHOOGEWIJS •Section of Medicine, Department of Endocrinology, Metabolism and
Cardiovascular System, University of Fribourg, Fribourg, Switzerland
N
USHRATJ. HOQUE •Department of Chemistry, Penn State University, University Park, PA,
USA
A
LANKA
´
DEK •Institute of Microbiology of the Czech Academy of Sciences, v.v.i., BIOCEV,
Vestec, Czech Republic
T
ENGWEIKOAY•Section of Medicine, Department of Endocrinology, Metabolism and
Cardiovascular System, University of Fribourg, Fribourg, Switzerland
C
ARSTENKREBS•Department of Chemistry, The Pennsylvania State University, University
Park, PA, USA; Department of Biochemistry and Molecular Biology, The Pennsylvania
State University, University Park, PA, USA
I
VANHOEK. H. LEUNG •School of Chemistry and the Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia
P
IOTRJ. MAK•Department of Chemistry, Saint Louis University, Saint Louis, MO, USA
PETRMAN
•Department of Biochemistry, Faculty of Science, Charles University, Prague,
Czech Republic; Institute of Microbiology of the Czech Academy of Sciences, v.v.i., BIOCEV,
Vestec, Czech Republic
ix

x Contributors
J. MARTINBOLLINGERJR•Department of Chemistry, The Pennsylvania State University,
University Park, PA, USA; Department of Biochemistry and Molecular Biology, The
Pennsylvania State University, University Park, PA, USA
M
ARKE
´
TA MARTI
´
NKOVA
´ •Department of Biochemistry, Faculty of Science, Charles University,
Prague 2, Czech Republic
A
DARSHK. MAYANK•Department of Biological Chemistry, David Geffen School of Medicine,
University of California–Los Angeles, Los Angeles, CA, USA
N
AASSONM. MBENZA•Department of Laboratory Medicine and Pathobiology, Temerty
Faculty of Medicine, University of Toronto, Toronto, ON, Canada
M
OLLYJ. MCBRIDE•Department of Chemistry, The Pennsylvania State University,
University Park, PA, USA
M
RUTYUNJAYA. NAIR•Department of Chemistry, The Pennsylvania State University,
University Park, PA, USA
V
IJAYAPANDEY•Department of Biological Chemistry, David Geffen School of Medicine,
University of California–Los Angeles, Los Angeles, CA, USA
D
AYNAC. PATTERSON •Department of Chemistry, Penn State University, University Park,
PA, USA
S
ARAHR. POPE•Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, PA, USA
X
AVIERE. SALAS-SOLA
´ •Department of Chemistry, The Pennsylvania State University,
University Park, PA, USA
M
ICHELGEOVANNISANTIAGO-MARTI
´
NEZ •Department of Biochemistry and Molecular
Biology, Pennsylvania State University, University Park, PA, USA; Department of
Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA
J
OHANNESSCHO¨DEL •Department of Nephrology and Hypertension, Universit€atsklinikum
Erlangen and Friedrich-Alexander-Universit€at Erlangen-Nu¨rnberg, Erlangen, Germany
A
RTURSERGUNIN •Department of Biochemistry, Faculty of Science, Charles University,
Prague 2, Czech Republic
T
ORUSHIMIZU •Department of Biochemistry, Faculty of Science, Charles University, Prague
2, Czech Republic
D
EBANGSUSIL•Department of Chemistry, The Pennsylvania State University, University
Park, PA, USA
A
LEXEYSILAKOV•Department of Chemistry, Penn State University, University Park, PA,
USA
S
AMUELN. SNYDER •Department of Chemistry, Saint Louis University, Saint Louis, MO,
USA
E
DUARDOH. S. SOUSA•Department of Organic and Inorganic Chemistry, Federal
University of Ceara, Center for Sciences, Fortaleza, Ceara, Brazil
M
ARTINSTRA
´
N
ˇ
AVA •Department of Biochemistry, Faculty of Science, Charles University,
Prague, Czech Republic
J
AKUBVA
´
VRA •Department of Biochemistry, Faculty of Science, Charles University, Prague 2,
Czech Republic
E
MILYE. WEINERT•Department of Chemistry, Penn State University, University Park, PA,
USA; Department of Biochemistry & Molecular Biology, Penn State University, University
Park, PA, USA
JAMESA.W OHLSCHL EGEL •Depar tment ofBiological Chemistr y,David GeffenSchool of
Medicine, University ofCalifor nia–LosAngeles, LosAngeles, CA,USA

Chapter1
Integrating UV-Vis Spectroscopy and Oxygen Optode
for Accurate Determination of Oxygen Afﬁnity of Proteins
Anoop Rama Damodaran and Ambika Bhagi-Damodaran
Abstract
Protein-based oxygen sensors exhibit a wide range of afﬁnity values ranging from low nanomolar to high
micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate
their oxygen afﬁnity (
d) to a value that is appropriate for their biological function is an important question
in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis
spectrometer with an oxygen optode for direct determination of
dof heme-containing oxygen sensors.
We provide details on how to set up the assay, acquire and ﬁt data for accurate
ddetermination using
H-NOX (
d=
oxygen-binding proteins.
Key wordsMetalloproteins
1 Introduction
Oxygen gas is a key biological stimulus involved in a variety of
cellular functions [1]. The binding and transport of oxygen are
crucial to sustaining aerobic life. Furthermore, sensing and signal-
ing of oxygen regulate multiple physiological processes in all forms
of life [2]. For example,
strictly anaerobic microbes display a repellent chemotaxis response
upon sensing low nanomolar levels of oxygen [3]. Mammalian cells,
on the other hand, turn on200 transcription factors upon sensing
low micromolar oxygen concentrations [4]. Mechanistic insights
into these cellular processes require a quantitative understanding of
the oxygen sensing capacity of various oxygen binding proteins.
Depending on their biological functions, protein-based oxygen
sensors exhibit oxygen afﬁnity values ranging from low nanomolar
to high micromolar. In turn, how proteins utilize different metals,
Emily E. Weinert (ed.),
https://doi.org/10.1007/978-1-0716-3080-8_1,
©
1

cofactors, and complex macromolecular architectures to regulate
their oxygen afﬁnity (K
d) appropriate for their biological function is
an important question in protein chemistry and microbiology.
2 Anoop Rama Damodaran and Ambika Bhagi-Damodaran
Heme proteins form a major class of oxygen sensors in nature
and coordinate to oxygen via the Fe
2+
metal center [5]. The high
extinction coefﬁcient of heme (~100 mM
-1
cm
-1
) coupled with
distinct spectroscopic signatures for its oxygen-free and oxygen-
bound forms have been exploited forK
dmeasurements [6]. In this
regard, researchers have resorted to complex ﬂash-photolysis-based
kinetic approaches to characterize theK
dvalue of various heme
sensors [7 ]. However, this approach requires expensive instrumen-
tation including pulsed lasers and/or rapid-mixing stopped-ﬂow
apparatus that are generally inaccessible to biochemistry and molec-
ular biology labs. In order to make oxygen afﬁnity measurements
more accessible, we present a one-pot method that integrates a
UV-Vis spectrometer with an oxygen optode that is relatively inex-
pensive and capable of measuring oxygenK
dvalues from
the low nanomolar to high micromolar range. In this method, we
simultaneously record the protein’s UV-Vis spectrum along with
the free ligand concentration in the assay using an oxygen optode.
With a 750-fold [8] higher tendency to exist in its gaseous form
than dissolved in water, any dissolved oxygen in the assay can easily
escape into the headspace. Consequently,K
dcalculations that
employ total oxygen added as an input parameter are prone to
signiﬁcant errors and also require the use of protein concentrations
that are ten-fold lower than theK
dvalue. Our method circumvents
this challenge by directly measuring the free dissolved oxygen in the
protein solution, and allows an independent choice of starting
protein concentration. In this chapter, we provide details on setting
up this assay, using it to measure oxygen afﬁnities of various pro-
teins, ﬁtting the acquired data, and, ﬁnally, tips and tricks to make
the assay work. We employ this technique to measure the afﬁnity of
a well-studied heme-based oxygen sensor,CsH-NOX [7], with a
K
dvalue in the nanomolar range.
2 Materials
Prepare all solutions using Ultrapure deionized water and analytical
grade reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Diligently follow all waste disposal
regulations when disposing waste materials.
2.1 UV-Vis
Spectroscopy and
Integrated Oxygen-
Optode Sensor 1.Anaerobic glovebag/glovebox(seeNote 1).
2.
UV-V isspectrometer equipped withacuvette holder with
temperature control andmagnetic stirringcapabilities (see
Note 2).

Integrated Method for Measuring Protein Oxygen Afﬁnity 3
3.Dipping-probe oxygen optode, temperature probe (T-probe),
and controller with temperature compensation (seeNote 3).
4.Anaerobic cuvette with septum cap (seeNote 4).
5.Stir bar (see Note 5).
2.2 Oxygen
Calibration with
Chlorite/Chlorite
Dismutase (Cld) 1.Sodium chlorite (seeNote 6).
2.Puriﬁed and characterized Cld [9 ](seeNote 7).
3.20 mM MOPS buffer (see Note 8).
4.Microtubes and microtube holder.
5.0.2–10, 10–100, and 100–1000μL pipettes and
compatible tips.
6.Gastight syringe (10, 50μL).
2.3 Measuring the
Oxygen Afﬁnity of Cs
H-NOX 1.Puriﬁed and characterizedCsH-NOX (seeNote 9).
2.PD-10 column.
3.25 mM sodium dithionite solution.
4.Microtubes and microtube holder.
5.0.2–10, 10–100, and 100–1000μL pipettes and
compatible tips.
6.0.5 mL Centricons (centrifugal ﬁlter devices) with 10-kDa MW
cutoff.
7.Gastight syringe (10, 50μL).
8.50 mM Tris–HCl pH 8.
9.Aerated buffer (50 mM Tris–HCl pH 8) in gastight Reacti-
Vials with septum caps.
3 Methods
Carry out all procedures at room temperature unless otherwise
speciﬁed.
3.1 Assembling the
Spectroscope and
Oxygen Optode Sensor1.Equilibrate allcomponents in the anaerobic environment of
the glovebag for
at least 24 h and ensure sub-ppm reading in
the glovebag oxygen sensor. The oxygen optode when
immersed in the deoxygenated measurement buffer should
read zero (<±5 nM).
2.
Placethe anaerobic cuvette withtheseptum capinthecuvette
holder ofthespectrophotometer (Fig.1a).Setthetemperature
oftheholder toavalue atwhich youwishtomeasure the
oxygen afﬁnity ofyourprotein system. Forallexperiments
performedinthischapter ,thetemperature ofthecuvette
holder wassetto20°C.

4 Anoop Rama Damodaran and Ambika Bhagi-Damodaran
02550
10
1
10
3
10
5
Time (min)
Free O
2
(nM)
1
2
3
4
5
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
ClO
2added (nM)
slope = 0.98
R
2
=0.99
UV-Vis spectrometer
Cuvette with
septum cap
O
2optode
T-probe
ClO
2 Cl +
2
Cld
ab
--
75
Fig. 1 a
UV-Vis spectroscope and oxygen optode sensor along with the T-probe. Inset shows the optode sensor
inserted in the cuvette. (b) Calibration of oxygen optode sensor with Cld-chlorite system. The plot shows free
oxygen generated upon addition of different amounts of chlorite to Cld solution. Inset shows that the release of
free oxygen upon chlorite addition is stoichiometric in low nanomolar to high micromolar range
3.Unscrew the septum cap and punch two tight-ﬁtting holes, one
for the oxygen optode and another for the compensating
T-probe. Insert the oxygen optode and T-probe through
these holes such that they would be well submerged in the
assay buffer upon tightening the cap onto the anaerobic
cuvette (inset, Fig.1a).
3.2 Calibration of
Oxygen Optode Sensor
with Chlorite/Chlorite
Dismutase
1.Deoxygenate MOPS buffer using a Schlenk line following a
protocol that includes three sets of 15 min deoxygenation
cycles, with each cycle alternating between vacuum and argon
gas as described previously [10]. Transfer deoxygenated buffer
to the anaerobic glovebag and let it stir at 500 rpm for at least
48 h before experiment.
2.Deoxygenate 300M Cld via threefreeze-pump-thaw cycles
usinga Schlenk line and transfer it to the glovebag for over-
night storage at 4
13,000 rpm, before actual use to remove unfolded protein
precipitates. Remeasure Cld stock concentration using
UV-Vis spectroscopy. The Soret maxima of Cld at 392 nm
has an extinction coefﬁcient of 99,000 M
-
cm
-
and enables
easy protein quantiﬁcation [11].
3.Transfer ~100 mg of sodium chlorite to the glovebag in a glass
vial with a slightly loosened cap so that the vial and its contents
are effectively deoxygenated during the pump/purge cycles of
the glovebag antechamber. Leave the vial cap loosened for
overnight equilibration with the glovebag environment.
4.Dissolve chlorite salt in deoxygenated MOPS buffer to prepare
a 500 mM stock solution which can then be serially diluted to

Integrated Method for Measuring Protein Oxygen Afﬁnity 5
obtain various concentrations of chlorite. For our measure-
ments (Fig.1b), we prepared a stock solution of 442 mM
chlorite with serial dilutions to 4.422 mM, 442M, 44M,
8.8M, and 4.4M concentrations.
5.Assemble the oxygen optode-spectrometer system as described
in Subheading3.1. Pipette out 4.2 mL of MOPS buffer in the
anaerobic cuvette. Add Cld to the cuvette such that the ﬁnal
Cld concentration in the cuvette is 200 nM. Drop a stir bar into
the cuvette and tighten the septum cap onto it such that the
oxygen optode and T-probe are well submerged. Turn on
stirring and let the cuvette and its contents stabilize to the
set temperature of 20
6.Start recording the oxygen concentration proﬁle using the
optode sensor. Verify that the deoxygenated buffer-Cld solu-
tion reads near-zero (±5 nM).
7.For measurement 1, inject 10L of the lowest dilution of
chlorite (4.4M) into the cuvette using a gastight syringe.
This results in an increase in the oxygen concentration as
measured by the optode and corresponds to the dismutation of chlorite by Cld. Note the reading as the oxygen concentra-
tion proﬁle plateaus to a stable value. For subsequent measure-
ments, we added 10L each of 4.4, 8.8, 44, and 442M
chlorite and noted down corresponding readings of free oxy-
gen concentration using the optode sensor (Fig.
1b). To get a
stable free oxygen concentration in the assay solution, it is
important to minimize the headspace volume in the cuvette
and also ensure a tight seal of the cuvette cap.
8.Plot the concentration of chlorite added on the x-axis and
oxygen measured by the optode on the y-axis. A linear ﬁt of
the data with the
the oxygen optode. We obtained a slope of 0.98 with an
2
value of 0.99, suggesting excellent stoichiometry between the
chlorite added and oxygen generated/measured over nanomo-
lar to micromolar concentration regimes (inset, Fig.
1b).
3.3 Measuring the
Oxygen Afﬁnity of Cs
H-NOX
1.Transfer ultrapure deionized water and Tris
glovebag using techniques discussed in Subheading3.2,
1
techniques discussed in Subheading3.2,
sodium dithionite in deoxygenated water to prepare 25 mM
dithionite solution.
2.Discard thestorage solution from the PD-10 column, chip off
the bottom, and transfer the column inside the glovebag after
three pump/purge cycles in the antechamber. Run 5 mL of
25 mM dithionite solution through the PD-10 column and
allow it to pass completely. Discard the ﬂow-through. Wash the

column with 25 mL of deoxygenated water followed by 25 mL
of deoxygenated assay buffer, and discard the ﬂow-through.
The PD-10 column can be stored in the glovebag with the
assay buffer in it for long-term use.
6 Anoop Rama Damodaran and Ambika Bhagi-Damodaran
3.Bring in 100μL, 500μMCsH-NOX in a microtube inside the
glovebag and store at 4°C until ready to use. Add 10 equiva-
lents of dithionite to the protein and run it through the
PD-10 column. Collect the colored fractions eluting from the PD-10 column.
4.Wash a Centricon with 0.5 mL Tris–HCl buffer by centrifuging
the tubes for 5 min at 13,000 rpm. Discard the ﬂow-through
and pipette in 0.5 mL ofCsH-NOX over the cellulose mem-
brane of the Centricon. Centrifuge the tubes for 5 min at
13,000 rpm, 4°C. Repeat centrifuging cycles till the protein
volume is ~0.1 mL corresponding to ~0.4 mM protein con-
centration. Pipette out the concentrated protein in a micro- tube, and record the absorbance spectra of the protein to
ensure that it is fully reduced with a Soret band maxima at
431 nm.
5.Assemble the oxygen optode-spectrometer system as described
in Subheading3.1. Pipette out 4 mL of Tris–HCl buffer in the
anaerobic cuvette, and wait for the temperature to stabilize to 20°C. Take a blank spectrum. Add 50μL of reducedCs
H-NOX to the cuvette, turn on the stirrer, and let the protein
mix with the buffer. Record the UV-Vis spectra of the reduced
protein and conﬁrm the Soret band maxima at 431 nM and a protein concentration of ~5μM (see initial spectrum, dark blue
data, Fig.2a). Tighten the septum cap with the oxygen optode
and T-probe onto the anaerobic cuvette such that the probes
are well submerged. Start recording the free oxygen concentra-
tion using the optode sensor and verify that it reads zero (< ±5 nM) for the reducedCsH-NOX sample in the cuvette.
6.Bring into the glovebag a sealed Reacti-Vial ﬁlled with 4 mL
Tris–HCl buffer that was aerated externally under ambient
conditions. This sealed, aerated buffer is anticipated to possess ~250μM dissolved oxygen, but can lose signiﬁcant amounts of
oxygen (~25%) during the glovebag transfer process. Given
that our approach of determining oxygen afﬁnity involves
direct measurement of free oxygen concentration (using the
oxygen optode) in the assay, our measurements are not
impacted by errors in estimations of dissolved oxygen concen-
tration in the Reacti-Vial.
7.We begin oxygen titration by adding 10μL of aerated buffer
using a gastight syringe to the cuvette with stirring turned
on. The optode reading for the free oxygen concentration

Integrated Method for Measuring Protein Oxygen Afﬁnity 7
300 400 500 600
-0.2
-0.1
0.0
0.1
409
435
Wavelength (nm)
D
400 500 600
0.1
0.3
0.5
550 600
432
416
Absorbance
Wavelength (nm)
0 500 1000 1500
0.0
0.1
0.2
0.3
DD
Free O
2(nM)
c
K
d= 23 ± 2 nM
R
2
=0.99
Cs
2
556
592
ab
Fig. 2
spectroscopy and oxygen optode system. (a
(inset) bands of
form (red). (c
afﬁnity plot for
and
dvalue for
the protein
increases initially and then plateaus to a stable value. In order to
achieve a stable free oxygen concentration in the assay solution,
it is important to minimize the headspace volume and also
ensure a tight seal of the cuvette cap. Record the stabilized
free oxygen concentration and the corresponding UV-Vis spec-
tra of the protein sample.
8.Add 5
for each dose record the free oxygen concentration andcorresponding protein spectra. Upon binding oxygen, the
Soret maxima ofshift from 431 nm (100% reduced) to 416 nm (100%
oxy-bound), the magnitude of which depends upon the free
oxygen concentration in the assay and the oxygen afﬁnity of theprotein (Fig.
2a). The ﬁnal dose of aerated buffer should be
such that a completely oxy-bound spectra is obtained and theSoret maxima does not shift any further with subsequent oxy-
gen additions. In all, for our measurements, we added a total of
ﬁve 10L shots of aerated buffer, followed by a 25L and a
ﬁnal 50L shot.

8 Anoop Rama Damodaran and Ambika Bhagi-Damodaran
9.For data analysis, each UV-Vis spectrum recorded is corrected
for dilution by multiplying the ratio of the total assay volume
after aerated
buffer
addition to the original assay volume in the
cuvette. For example, when 10μL aerated buffer is added to
the cuvette containing 4050μL protein solution, the
corresponding UV-Vis spectrum is multiplied by (4060/
4050) for dilution correction.
10.Next, obtain the difference spectrum (Δabsorbance, Fig.2b)
for each oxygen titration step by subtracting the spectrum of
the reduced protein from the corresponding dilution-
corrected protein spectra. The difference spectra exhibit a
maxima and a minima corresponding to the Soret band at
409 and 435 nm, respectively. Now, for each recorded free
oxygen concentration, computeΔΔabsorbance by subtracting
theΔabsorbance at 435 nm from theΔabsorbance at 409 nm.
Generate a plot ofΔΔabsorbance vs free oxygen concentration
(x) and ﬁt the data to Hill’s equation (ΔΔabsorbance=const.
*
x
xþK
d
) to obtain theK dofCsH-NOX. Our measurements
reveal aK
d=23±2 nM with anR
2
value of 0.99 for the ﬁt.R
2
represents the goodness of the Hill’s ﬁt and should typically be
between 0.9 and 1 for a reliableK
dmeasurement.
11.Instead of using an aerated buffer as the oxygen source, one
can also utilize the Cld-chlorite method of generating oxygen
in situ as described in Subheading3.2. This can offer the
advantage of generating a broad range of oxygen concentra-
tions while simultaneously minimizing the headspace in the
cuvette. This method, however, would be unsuitable for pro-
teins that cross-react either with the ferric form of Cld or with
chlorite.
4 Notes
1.The humidity level in the glovebag/glovebox should be con-
trolled to prevent any condensation
on the
cuvette surface
(especially for measurements at 4°C) and associated artifacts
in the UV-Vis measurements.
2.We used the Cary 60 UV-Vis spectrometer, and a custom-built
stirrer and temperature controller.
3.We used the oxygen dipping probe DP-PSt6 sensor (4-mm
shaft diameter), T-probe (2-mm shaft diameter), and the
OXY-1 SMA trace controller from PreSens GmbH.
4.
Weused3.5-mL screw-cap quartzcellswithseptum (1-Q-10-
GL14-S) fromStarnaCellsastheanaerobic cuvette. Thecuv-
ettesareﬁlled upsuchthattheheadspace afteroptode and
T-probe immersion isminimal.

Integrated Method for Measuring Protein Oxygen Afﬁnity 9
5.The Teﬂon coating of the stir bar can absorb oxygen. Leave the
stir bar overnight in the glovebag and let it stir in 25 mM
dithionite solution for an hour to remove any
residual oxygen
from
the Teﬂon coating.
6.Analytical grade sodium chlorite is 80% pure. While determin-
ing chlorite concentration, adjust the active chlorite based on
manufacturer’s instructions.
7.Methods for expression and puriﬁcation of Cld are detailed
previously [9,11]. Cld should be well characterized before
use. This includes characterization via protein gel electropho-
resis, mass spectrometry, and hemochromogen assay.
8.It is important to use a chloride-free buffer for running
Cld-based assays. Chloride is a product of chlorite dismutase
reaction and can inhibit the enzyme’s activity.
9.Heme proteins used for these studies should be well character-
ized via protein gel electrophoresis, mass spectrometry, and
hemochromogen assay. The spectral features of the reduced
(ferrous), oxidized (ferric), and oxy-bound form of proteins
should be recorded before performing afﬁnity experiments. We
recommend choosing a protein concentration such that Soret
maxima absorbance is ~0.5.
Acknowledgements
This work was supported by the Regents of the University of
Minnesota and NIH NIGMS grant # R35GM138277. The authors
thank Profs. Bollinger and Krebs (Penn State) for the Cld plasmid
and Prof. Marletta (UC Berkeley) for theCsH-NOX protein. We
thank Grant Larson for help with purifying the Cld enzyme.
Author ContributionsARD conceived, designed, and performed
the assay. ARD and ABD wrote the paper.
References
1.van Dongen JT, Licausi F (2015) Oxygen sens-
ing and signaling. Annu Rev Plant
Biol 66:
345–
367
2.Bhagi-Damodaran A, Lu Y (2019) The peri-
odic table’s impact on bioinorganic chemistry
and biology’s selective use of metal ions. In:
Mingos DMP (ed) The periodic table II: cata-
lytic, materials, biological and medical applica-
tions, Structure and bonding. Springer, Cham,
pp 153–173
3.Tran R, Boon EM, Marletta MA, Mathies RA
(2009) Resonance Raman spectra of an
O
2-binding H-NOX domain reveal heme
relaxation upon mutation. Biochemistry 48:
8568–8577
4.Chan M
olt-Martyn JP, Schoﬁeld CJ, Rat-
cliffe PJ (2016) Pharmacological targeting of
the HIF hydroxylases–a new ﬁeld in medicine
development. Mol Asp Med 47–48:54–75

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5.Girvan HM, Munro AW (2013) Heme
sensor
proteins. J Biol Chem 288:13194–13203
6.Bhagi-Damodaran A, Michael MA, Zhu Q,
ReedJ,
Sandoval BA,
Mirts EN,
Chakraborty S, Moe¨nne-Loccoz P, Zhang Y,
Lu Y (2017) Why copper is preferred over iron
for oxygen activation and reduction in haem-
copper oxidases. Nat Chem 9:257–263
7.Hespen CW, Bruegger JJ, Guo Y, Marletta MA
(2018) Native alanine substitution in the gly-
cine hinge modulates conformational ﬂexibility
of heme nitric oxide/oxygen (H-NOX) sens-
ing proteins. ACS Chem Biol 13:1631–1639
Haynes WM (2014) CRC handbook of chem-
istry and physics. CRC Press, Boca Raton
9.Sanyal R, Bhagi-Damodaran A (2020) An
enzymatic method for precise oxygen afﬁnity
measurements over nanomolar-to-millimolar
concentration regime. J Biol Inorg Chem 25:
181–186
10.Bhagi-Damodaran A, Petrik ID, Marshall NM,
Robinson H, Lu Y (2014) Systematic tuning of
heme redox potentials and its effects on O
2
reduction rates in a designed oxidase in myo-
globin. J Am Chem Soc 136:11882–11885
11.Streit B
D uBois JL (2008) Chemical and
steady-state kinetic analyses of a heterologously
expressed heme dependent chlorite dismutase.
Biochemistry 47:5271–5280

Chapter2
Measurement of O
2Binding by Sensory Hemeproteins
Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
Abstract
The discovery of an increasing number of proteins that function in the detoxiﬁcation and sensing of gaseous
ligands has renewed interest in hemeproteins. It is critical to measure the afﬁnities of these proteins for
ligands like O
2, CO, and NO, know with conﬁdence when a protein is fully saturated with a speciﬁc ligand,
and be able to estimate how well a ligand will compete against other ligands for a speciﬁc protein. Below we
describe how to obtain an intact O
2-binding hemeprotein with a full complement of heme, how to evaluate
the factors that can impact its afﬁnity for O
2, and how to determine accurately the equilibrium and kinetic
parametersK
d,k
on, andk
offfor O
2binding.
Key wordsHeme proteins, O
2afﬁnity, Equilibrium binding, Competition binding, Stopped ﬂow
spectroscopy
1 Introduction
In all kingdoms of life, O
2-binding hemeproteins carry out essential
catalytic, transport/storage, and sensory roles. The latter function,
the most recent one to be discovered, is manifested in a superfamily
of hemeproteins [1–4]. They can harbor the heme cofactor in at
least eight distinct domain folds (PAS, GAF, globin, LBD [Holi],
HNOB, SCHIC, CooA, FIST) and couple ligand binding at the
heme iron to a functional domain’s activity (e.g., histidine kinase,
nucleotide or dinucleotide cyclase, cyclic nucleotide phosphodies-
terase, DNA binding) (Fig.1). Consequently, the ligation of a
signal molecule at the heme iron can activate or inactivate the
functional domain. Since nearly all these proteins bind, not only
O
2, but also CO and NO, for any given protein, the assignment of a
signal ligand depends on the physiological environment and chem-
ical properties. Below we present approaches to measure the equi-
librium and kinetic parameters for O
2binding. Many of these
approaches were developed to study mammalian hemoglobins
and myoglobins [5]. They have been adapted to the more recently
discovered proteins, which are extremely varied in their properties.
Emily E. Weinert (ed.),Oxygen Sensing: Methods and Protocols, Methods in Molecular Biology, vol. 2648,
https://doi.org/10.1007/978-1-0716-3080-8_2,
©The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
11

12 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
Fig. 1Heme-based sensors as modular proteins containing a sensory heme domain coupled to a functional
domain (e.g., histidine protein kinase) output (A ) and their broad families (B ) organized by the heme domain
folds along with some biological functions they regulate
2 Materials
2.1 Proteins Whether an oxygen-binding hemeprotein is in the unliganded Fe
II
(deoxy-), Fe
II
O
2(oxy-), or Fe
III
(met-) state, it is intensely colored.
This is because hemeproteins absorb strongly at around
414–437 nm. The position and width of this peak, called the
Soret band, depends on the heme iron ligation state. In particular,
for the oxy-state of a protein having a single heme per monomer, an
extinction,ε~140 mM
-1
cm
-1
is typical at the peak of absorption,
which is usually around 416– 419 nm. In other words, the Soret
band would show an absorbance of 1 for ~7μM of the hemepro-
tein, and its solution would look blood red.
An a
f handling of recombinant hemeproteins that may
strongly impact success in obtaining them is the rate of their
expression. High-copy vectors like pUC, for replication inE. coli,
which maintain the relevant genes undertac-promoter regulation,
typically work well. In pilot experiments to decide on a strain or the
appropriate time course of induction by isopropylthio–βgalactoside
(IPTG), expression can usually be glimpsed, even before the cells
are lysed, by a reddish hue of the harvested cell pellets.
Itisquite unnecessar ytoappend atagtoahemeprotein.
Indeed, theroutine addition ofa6xHis-tag isstrongly discouraged.
Exposure ofhemeproteins tohighconcentrations ofimidazole
(200 mM) mayleadtotheaccumulation ofanimidazole-met
(Fe
III
Imid) species. Ifso,thenthisbuildup isoften followed by
anirreversible extraction oftheheme, which yields bis-imidazole
heme andtheapoprotein asitsproducts. Most hemeproteins
behave wellinclassical puriﬁcat
ions,which canbemonitored by
eye,orbyachromatography system withamultiwavelength
UV/V isdetector setto415nmand280nm(BioLogic QuadT ech,
Bio-Rad). Strategies thatwehavesuccessfully usedforprotein

puriﬁcations include an initial precipitation of a crude lysate with
ammonium sulfate to recover a red precipitate. The dissolved pre-
cipitate may be directly fractionated by hydrophobic interaction
chromatography (e.g., on phenyl Sepharose) and then a size-
exclusion step (Superdex S-200). Alternatively, the precipitated
proteins may be desalted (e.g., Sephadex G-25), fractionated by
anion-exchange chromatography (DEAE Sephacel), and then a
size-exclusion step (e.g., Superdex S-200) [6 –9].
Measurement of O2Binding by Sensory Hemeproteins 13
If you must tag a hemeprotein because you want to use it to
purify a complex or to ﬁsh for one or more interacting proteins, a
strategy different from a 6xHis-tag is advised. We have, for exam-
ple, fused themalEgene (MBP fusion) to one protein in such a
case [9].
2.2 Solutions and
Reagents –4.2 M pyridine in 0.20 M NaOH.
–Deoxygenated water.
–Sodium dithionite.
–O
2tank.
–CO tank.
–Potassium ferricyanide.
2.3 Equipment –UV-visible spectrophotometer.
–Anaerobic chamber.
–G-25 desalting columns (2).
–High-accuracy gastight syringe.
–UV-transparent plastic cuvettes.
–Sealable cuvettes and stopper/septa.
–Stopped ﬂow apparatus (seeNote 1).
–Laser ﬂash photolysis instrument (seeNote 1).
–Nonlinear ﬁtting software (Excel, Igor, Prism,
MATLAB, etc.).
3 Methods
3.1 Pyridine
Hemochromogen
Assay of Heme ContentThis assay provides a measure of the heme content of a hemepro-
tein. This is not only an important intrinsic aspect of a protein but
also a good measure of potential damage. The assay relies on a
pyridine extraction of the heme from a puriﬁed protein and a
comparison of the recovered heme to a set of hemin standards [10].
1.Measure protein concentration independently by a protein
assay (e.g., Micro BCA).
2.
Prepare a stockofhemin (~0.50 mM)in0.10MNaOH; ﬁlter,
andstore inadarkcontainer fornomore than24h.To

14 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
calculate the hemin concentration, an aliquot is diluted 50-fold
in 2% (w/v) sodium borate, pH 9.23, and scanned at
350–700 nm, assuming anε390
nm=50 mM
-1
cm
-1
.
3.Mix 800μL of freshly prepared 4.2 M pyridine in 0.20 M
NaOH with
200μLo
f10–50μM protein and add to
UV-transparent plastic cuvettes (seeNote 2).
4.Similarly treat solutions of 10–50μM hemin in 10 mM NaOH
with pyridine as the standards.
5.Assuming anε556
nm=33.9 mM
-1
cm
-1
, the 350- to 700 nm
spectra of the protein solutions are compared to similarly trea-
ted hemin solutions to quantify the amount of heme.
3.2 Basis Spectra Basis spectra of a hemeprotein homogeneously in one state are
invaluable in studies that involve conversion of the protein from one
state to another, or studies that require knowledge of the exact
protein composition. Record all the spectra in the same buffer and
at 25°C. For part of this work, you will need an anaerobic chamber
(Coy Laboratory Products, Inc., Grass Lake, MI, USA) to serve as an
O
2-free bench. Without cluttering the chamber, keep inside it the
following: solutions of completely deoxygenated water and buffers,
Pipetman and tips, an Eppendorf rack, a cuvette rack, cuvettes that
can be sealed and the stoppers (e.g., silicone) for sealing them,
aliquots of premeasured solid dithionite in Eppendorf tubes, and
two G-25 columns (~4 mL) pre-equilibrated with anoxic buffer. It
is useful to bubble anoxic bag gas continuously through a 250 mL
graduated cylinder of water for preparation of future anaerobic
solutions.
3.2.1 Deoxy-State, Fe
II
1.Inside the anaerobic chamber, treat a stock of the puriﬁed
protein sample with
2 molar
equivalents of sodium dithionite
for 2 min;immediatelypass the solution through a G-25 col-
umn pre-equilibrated with deoxygenated buffer. Ideally, the
eluted hemeprotein concentration should be 0.5μM or higher.
Base your buffer on your protein’s preferences (seeNote 3).
2.Fill a 1 mL cuvette with 0.80 mL of the deoxy-hemeprotein
solution and tightly seal the cuvette.
3.
Remove the sealed cuvette fromtheglove bagandrecord the
sample’s 260–750nmabsorption spectr umat25°Cwith
aUV-visible spectrophotometer (e.g., Cary4000 UV-Vis,
Agilent, Santa Clara, CA,USA). Inthedeoxy-state ofmost
hemeproteins, theheme ironispentacoordinate; asaresult, the
electronic spectra showabroad alpha/beta bandnear556nm
andaSoret bandaround 437nm[7](Fig.2).When theiron
atom inadeoxy-hemeprotein ishexacoordinate, theprotein
displays sharp alpha andbetabands near560and530nm,
respectively ,andaSoret bandnear426nm[11].

Measurement of O2Binding by Sensory Hemeproteins 15
Fig. 2Example of basis spectra for deoxy-DevS (blue) and oxy-DevS (red) (inset:
picture of oxy-DevS during puriﬁcation using an anion-exchange column (DEAE
resin)
3.2.2 Oxy-State, Fe
II
O2 1.After recording the spectra for the deoxy-state, as described in
Subheading3.2.1, open the cuvette to air and pipet the protein
solution in and out
to mix
it with the air.
2.Record the air-saturated 250– 750 nm absorption spectrum at
25°C. Air-saturated aqueous solutions contain 256μMO
2:a
concentration sufﬁcient to saturate most, but not all,
O
2-binding hemeproteins. Oxy-hemeproteins typically display
an alpha band near 578 nm, a beta band near 546 nm, and a
Soret band around 416 nm (Fig.2). Record the UV-vis
spectrum.
3.Prepare a solution of O
2-saturated buffer (1280μMO
2).
(a)Fill a BD vacutainer tube with ~8 mL of buffer and ﬁt the
rubber septum with two syringe needles.
(b)Gently bubble pure O
2into the liquid via one syringe
while keeping the other syringe needle open, above
the gas.
(c)After 15 min of bubbling at 1 atm and 23°C, the O
2
concentration in the buffer should be 1280μM. Remove
both syringe needles and keep the solution at 23°C.
(d)In a sealed cuvette, mix 0.16 mL of protein with 0.64 mL
(i.e., four volumes) of the O
2-saturated buffer, for a ﬁnal
concentration of ~1024μMO
2.
(e)
Record the 250–750nmabsorption. Ifthisleaves the
spectr umunchanged, compared tothespectr uminair,
butonlydilutes theprotein, thenitisfullysaturated with
O
2inair.Ifthespectr umchanges, in>1000 μMO
2,to
show anincreased propor tionoftheoxy-state, thenyou
havegotalow-O
2afﬁnity hemeprotein (seeautoxidation
ratebelow).

16 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
3.2.3 Met-State, Fe
III
1.Add to an open cuvette with oxy-protein 2 molar equivalents of
potassium ferricyanide and then remove this oxidizing agent
with a Bio-Spin column.
2.Record the 250– 750 nm absorption
spectrum
of the oxidized
protein.
3.Repeat the same procedure after treating the protein with
5 molar equivalents of potassium ferricyanide. If the two spec-
tra are identical, then they represent the met-state. The absorp-
tion spectra of met-hemeproteins differ greatly from each
other: depending on the polarity of the heme pocket and
proximity of potential ligating side chains, the spectra may
represent an aquo-met (Fe
II
H
2O), pentacoordinate-met, or
hexacoordinate-met (bis-His, His-Met, etc.) state. Indeed,
met-state spectra can provide a great deal of information
about the chemical environment of the heme iron.
3.3 Autoxidation
Rate Constant,k
ox
For many O
2-binding hemeproteins, the oxy-state oxidizes to the
met-state at some ﬁnite and measurable rate. This oxidation rate
varies greatly, however, and it is most rapid for proteins with low O
2
afﬁnity [12].
1.Prepare air-saturated protein as described in Subheading3.2.2.
2.Set the spectrophotometer jacket for the cuvettes to 37°C.
3.Collect spectra every 20 min for a total of 300 min.
4.For a met-protein endpoint, add 5 equivalents of potassium
ferricyanide and record the spectrum (subtracting a blank that
consists of buffer and the same quantity of ferricyanide). The
data should give clear isosbestics for the conversion from oxy-
to met-hemeprotein.
5.To determine the fraction of met-hemeprotein at each time
point, ﬁt the whole spectra to proportions of the basis spectra
for the oxy-state and the met-state. Plot time (min) versus Ln
[1-(fraction met-hemeprotein)], to get the autoxidation rate
constant,k
ox,in min
-1
, from the slope. Given the broad range
of observedk
oxvalues for hemeproteins, you may need to reﬁne
your measurements to a shorter or longer time range. If the
protein’s autoxidation proves to be rapid, then you might
investigate whether reducing agents likeß-mercaptoethanol,
dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), or
ascorbic acid will stabilize the oxy-state for your studies. Alter-
natively, you might correct for effects of the met-state on your
measurements.
3.4 Equilibrium
Dissociation Constant,
K
d, for Binding of O2
Thisisclearly animpor tantparameter tomeasure foranO
2-bind-
ingrotein.orensorhisilliveouonsiderablenfora-
tionboutheangefensingnd,fhissultimericrotein,
revealnyooperativity

Measurement of O2Binding by Sensory Hemeproteins 17
3.4.1 Direct Titration
1.Prepare O
2-saturated buffer (1280μMO
2) at 1 atm and 23°C
as described above (under oxy-state, Subheading3.2.2,
step 3).
2.Prepare O
2-saturated water (1280μMO
2) according to the
same method.
3.From these stock solutions, prepare solutions of 256μM and
128μMO
2by diluting the O
2-saturated buffer in deoxygenate
buffer.
4.Inside an anaerobic chamber, mix 560μL of deoxygenated
buffer with 40μLof15μM deoxy-protein.
5.Dispense 150μL of this solution of 1μM deoxy-hemeprotein
into four identical Eppendorf tubes. Seal
all the
tubes.
6.One by one, transfer each solution to a sealed 200μL
quartz cuvette, and remove it from the chamber to the
spectrophotometer.
7.With a gastight syringe add 2μL aliquots of 128μMO
2
(adding up to no more than 8μL) to the ﬁrst 150μL sample
of deoxy-hemeprotein in the sealed cuvette at 25°C.
8.Record the spectrum with each addition; when ﬁnished, open
the cuvette to air and record the 350– 750 nm spectrum in air.
9.Starting with another fresh solution of deoxy-hemeprotein,
repeat the same process, adding 2μL aliquots of 256μMO
2
and recording the spectra; again, follow with exposure to air to
get a spectrum for air-saturated protein.
10.Repeat this process again, with fresh protein and 2μL aliquots
of the 1280μMO
2, and then follow with exposure to air. In
case of a mistake, you have got an additional tube of deoxy-
protein in reserve.
11.Prepare protein in a synthetic air-saturated (~256μMO
2)
solution at 25°C, by mixing 10μL of deoxy-protein, 110μL
of deoxygenated buffer, and 30μL of 1280μMO
2buffer.
Record the 350– 750 nm spectrum in a sealed cuvette.
12.Prepare a completely O
2-saturated protein sample at 25°Cby
mixing 10μL deoxy-protein with 140μLO
2-saturatedbuffer,
so that the ﬁnal O
2concentration is ~1182μM. Record the
350–750 nm spectrum in a sealed cuvette.
13.
Thisstrategy should giveyou,attheend,spectra foryour
protein at1.7,3.3,4.9,6.5,3.4,6.6,9.8,13,17,33,49,
65,256,and1182 μMO
2.Afteryouexamine yourdata,this
range ofO
2concentrations might prove tobetoohighortoo
lowforyourprotein. Forinstance, ifyourprotein issaturated
atnearly allthepoints, youmight wanttorepeat thisexperi-
mentwithamoredilute solution ofprotein andstocksolutions
of64,32,and16μMO
2.Alternatively ,ifyoufailtoseem
uch

18 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
Fig. 3Example of a typical titration curve for O
2binding, as measured for the
heme-based sensor DosT
O
2binding to your protein, except above 50μMO
2, for
example, then you should repeat this experiment, combining
your protein with greater fractions of O
2-saturated buffer.
When you are satisﬁed that you have collected distinct spectra
for some 10–15 O
2concentrations, calculate the saturation at
each concentration of O
2from linear combinations ofwhole
deoxy-state and oxy-state basis spectra.
14.Fit the data to a quadratic single-binding equation or Hill plot
equation (Fig.3).
3.5 Binding by
Competition In some cases, an extraordinarily high ligand afﬁnity may prevent
any accurate measurement of the K
dby a direct titration of the
deoxy-hemeprotein with O
2[7,13]. A strategy of competition of
the O
2against another ligand of known afﬁnity can instead be
employed. In this type of study, CO offers advantages that include
a reasonably high afﬁnity for heme proteins, stability in solution,
and a capacity to minimize the oxidation of the heme iron when
bound.
The formal equilibrium may be described as a combination of
two independent processes, where P is the pentacoordinate heme
iron in the protein, and O
2-P and CO-P are the protein bound to
O
2and CO, respectively.
CO‐P⇋PþCO;K
d
CO
O2þPO 2‐P;1=K d
O2
Global equilibrium:CO‐PþO 2⇋O2‐PþCO
Mathematically, this equilibrium can be described as follows:
K
d
CO=Kd
O2=CO?O 2‐P?ðÞ =O 2?CO‐P?ðÞ ,

β
þ? ð Þ
if
Measurement of O2Binding by Sensory Hemeproteins 19
This experiment requires that we know the CO afﬁnity of the
hemeprotein, i.e.,K
d
CO. This may be calculated fromk off/konfor
CO binding and requires the CO-state basis spectra.
3.5.1 Obtain an Apparent
App
K
d
To minimize labor, before you carry out a full titration, ﬁrst, get an
apparent
App
K
dfrom Eq.1and then plug this value into Eq.2to get
an initial estimate of your K
d
O2:
O
2‐P%ðÞ=O 2?=
App
KdþO 2?
μβμ
β100 ð1Þ
App
Kd
O2=Kd
O21 CO=K d
CO
μβ
2
1.Determine the basis spectrum for the Fe
II
CO (carbonmonoxy)
species at 350–500 nm. Starting with the Fe
II
(deoxy) state,
add 10μM CO by diluting a stock of CO-saturated buffer.
These manipulations are entirely analogous to the determina-
tion of the Fe
II
O
2(oxy) basis spectrum (Subheading3.2.2).
2.Add 10μM of CO to your Fe
II
protein and measure the
spectra.
3.Add 50μMofO
2and record the spectra.
(a)Suppose for example these additions result in 65% of the
CO being displaced by the O
2. From Eq.1,
O
2-P=65%, [CO]=10μM, and [O 2]=50μM), then
we get an apparent
App
K
dof 26.9μM. This value, if
plugged into Eq.2, gives aK
d
O2estimate of 54 nM.
Keeping this estimate in mind, we can now design O
2
titrations in distinct backgrounds of CO concentrations
that will yield full saturation curves.
4.Repeat this procedure with at least three different concentra-
tions of CO, resulting in three distinct apparent
App
K
d.
5.
(a)
Plot
App
K
dversus the concentration of CO, according to Eq.2,
to yieldK
d
O2as the graphical intercept.
An example of this procedure was carried out to measure
the O
2afﬁnity of HGbI, an oxygen-avid hemeprotein,
whoseK
dfor CO was 0.67 nM [13]. Titration curves
were obtained by using micromolar concentrations of O
2
to displace the CO (Fig.4), and then single binding curves
yielded the
App
K
ds. Further treatment of these data with
Eq.2provided aK
dof 0.53 nM for O
2at 25°C.
3.6 Dissociation Rate
Constant,k
off, for
Binding of O
2
ForO
2binding hemeproteins, theO
2dissociation ratek
offistypi-
callyﬁrstorder.Theexactrates,however ,arestrongly inﬂuenced by
factors suchasstabilizing hydrogen bond donation tothebound
O
2.Asarule,theoff-rate dataﬁtasingle exponential, butwehave
observedcaseswhere theydidnot,probably because ofalternative
conform ations ofsidechains neartheligand intheheme pocket.

A determination of O2dissociation rate requires knowledge of the
deoxy-state and oxy-state difference spectrum at 350– 500 nm. The
peaks ofdifferenceare the best wavelengths at which to measure
such a rate. There are numerous ways to approach this experiment.
It sufﬁces to trap the O
2or change the heme status by a process that
is faster than the dissociation of O
2from the Fe
II
O
2species. A
simple method is the rapid addition, with a stopped-ﬂow apparatus,
of sodium dithionite to a sample of oxy-hemeprotein. The strategy
here is to saturate the protein completely with O
2and then quickly
remove any of the free O
2being produced, by its reaction with a
large excess of sodium dithionite. An important consideration is
that the reaction of O
2with dithionite generates strong acids;
it is therefore important to buffer the solution strongly at your
desired pH.
20 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
Fig. 4Example of a competition binding experiment using CO with HGbI to obtain
theK
dfor binding of O2
1.Studies of O
2dissociation rate require the use of a stopped-ﬂow
apparatus.
2.First prepare ~1.5 mL of a 0.5μM solution of O
2-saturated
hemeprotein in more than 50 times its K
dvalue in the O
2
concentration and a buffer such as 100 mM Tris–HCl,
50 mM NaCl, pH 8.0.
3.Place this solution of oxy-hemeprotein in one chamber of the stopped ﬂow.
4.In the other chamber, place an equal volume of 2.5 mM of
sodium dithionite, freshly prepared in anaerobic buffer.
5.Equilibrate both solutions to 25°C.
6.Using the stopped ﬂow, aliquots of the two solutions are mixed
in the apparatus, and the kinetic traces are rapidly collected at a single wavelength. For example, the rate of conversion to the
deoxy-state may be monitored at 436 nm.
7.The b
uration for the traces must be determined
empirically.

Measurement of O2Binding by Sensory Hemeproteins 21
(a)Start with collecting one-second traces and shorten or
lengthen this data collection time.
(b)The instrument can be programmed to ﬁt every
trace
immediately to
a single exponential.
If the ﬁt is poor, use a double exponential, but repeat the
measurements with different preparations of the pro-
tein and try to rule out the possibility of experimental
errors.
(c)It is important during such experiments to collect 10–12
measurements (shots) and, for each one, take notes on
(1) the observed rate,k
obsin s
-1
; (2) the change in absor-
bance over the entire kinetics trace,ΔA; and the estimated
error,E. Except in rare cases, thek
offwill be thek
obsvalues
one directly averages from those measurements.
3.7 Association Rate
Constant,k
on, for
Binding of O
2
The association rate constants for O2binding to hemeproteins are
often estimated from the equilibrium dissociation constant,K
d,
and the dissociation rate constant,k
off, based on the relation
K
d=koff/kon. Such a relation assumes a classic bimolecular reaction
with a singlek
onandk
off. One can also directly determine the
observed on rates,k
obs, for O
2binding by ﬂash photolysis, or by
stopped-ﬂow mixing. Both kinds of measurements may be done by
a stopped-ﬂow/ﬂash photolysis spectrometer ﬁtted with a Pi-star
stopped-ﬂow drive unit, like the LKS-60 instrument (Applied
Photophysics Ltd., Leatherhead, UK). For sample excitation by
light, the spectrometer is coupled to a laser (Quantel Brilliant B
Nd/YAG with second-harmonic generation). A 12-bit ADC card
within the instrument workstation provides for data acquisition
from slow measurements and a digital oscilloscope (Agilent
54830B, Santa Clara, CA, USA) provided for data acquisition
from faster measurements.
3.7.1 Measurement by
Flash Photolysis In ﬂash photolysis, one measures the rates of O
2binding,k
obs, from
solutions of oxy-hemeprotein. The oxy-protein is prepared in vary-
ing concentrations of O
2, and then the O
2is photolyzed by an
intense laser ﬂash, and the rate of O
2rebinding is measured. Since
the quantum yield for photolyzing off the O
2is typically quite small
(~0.3%), this experiment requires hemeprotein of relatively high
concentration. On the other hand, the method is not destructive,
and the same protein may be ﬂashed multiple times.
1.Start with a stock of 25μM deoxy-hemeprotein in any reducing
agent you require (e.g., 1 mM DTT), deoxygenated buffer,
and O
2-saturated buffer (1182μMO 2), and prepare, in 1 mL
sealed cuvettes, sequentially, 0.80 mL of each of the following:
(a)5μM
in 1024μMO
2
(b)
5μM hemeprotein in512μMO 2

22 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
Fig. 5A typical example of a ﬂash-photolysis experiment for measuring an observed association rate (k obs) for
O
2with DosT (a ) and the ﬁnal data analysis to yield the on-rate constant (k
on)(b)
(c)5μM hemeprotein in 256μMO
2
(d)5μM hemeprotein in 128μMO
2
(e)5μM hemeprotein in 64μMO
2
(f)5μM hemeprotein in 32μMO
2
2.For each of these solutions, after a ﬂash, monitor the rate of
decrease of deoxy-species at ~436 nm or increase of the
oxy-species at ~414 nm (Fig.5a).
3.Repeat
the ﬂash
and measurement 10–20 times. Each time,
note the observed rate,k
obsin s
-1
; change in absorbance,ΔA;
and error in theΔA.
3.7.2 Measurement by
Stopped Flow In the stopped-ﬂow approach, one measures the rates of O2bind-
ing,k
obs, after rapid stopped-ﬂow mixing of a solution of the deoxy-
hemeprotein with solutions of O
2of varying concentrations.
1.Prepare ~1.5 mL of a 0.5μM solution of deoxy-hemeprotein in
buffer containing any reducing agent you might require (e.g.,
0.4 mM DTT).
2.In the other chamber, put an equal volume of a buffered O
2
solution of known concentration, starting with 64μMO
2.
(a)Additional O
2solutions to measure in this case would be
128μMO
2, 256μMO
2, 512μMO
2, 1024μMO
2, and
1182μMO
2in buffer.
3.For each O
2concentration, do 10–20 1:1 shots, mixing an
aliquot of deoxy-hemeprotein with an equal volume of the
O
2solution.
4.Each t
ote theﬁnalO
2concentration after mixing; the
observed rate,k
obsin s
-1
; change in absorbance,ΔA; and error
in theΔA.

Measurement of O2Binding by Sensory Hemeproteins 23
3.7.3 Data Analysis
Subsequent analyses and considerations are the same for stopped-
ﬂow and ﬂash photolysis.
1.When ﬁnished with all the runs, average each set ofk
obsvalues
for a given O
2concentration.
2.Plot [O
2]μM versus averagek
obs(s
-1
) (Fig.5b), to get thek
on
from the slope inμM
-1
s
-1
.
(a)You will have to determine empirically the best collection
times for your kinetic traces. Typically, these are millise-
conds to seconds.
3.You might reﬁne subsequent repeats of these studies by collect-
ing more points in the low or the high O
2concentration range,
depending the approximatek
onvalue for your protein.
4 Conclusions and General Considerations
The measurements presented above should be repeated in their
entirety at least three times, ideally with different preparations of
protein, for veriﬁcation of their reproducibility. With regard to the
study of sensors that couple O
2binding to another activity, the
above measurements provide an important foundation. A key prop-
erty of any sensor is its dose response, i.e., its activity, as a function
of its saturation with ligand. Different O
2sensors will certainly
present their own peculiar challenges, but in all cases, measure-
ments of the dose response should become vastly more accessible
with knowledge of the protein’s stability to O
2and its parameters
for binding of O
2—and perhaps other ligands, like CO [8 ,14]. For
example, theBradyrhizobium japonicumFixL (BjFixL) paradoxi-
cally exhibited an exceptionally low O
2afﬁnity (K
d=140μM),
despite stringent regulation of its kinase by quite low concentra-
tions of O
2[14]. In the course of studying its dose response, a
hysteresis phenomenon was uncovered. This hysteresis ﬁts a mem-
ory effect involving a slow relaxation of thekinaseto an active state
subsequent to its inhibition by the O
2-bound heme. These studies
required minimizing the oxidation of the heme iron as well as
precisely controlling its saturation with O
2throughout the enzy-
matic assays. With knowledge of the protein’s responses to O
2and
CO, this became achievable with controlled mixtures of these two
ligands.
5 Notes
1.
Integrated stopped ﬂow-laser ﬂashphotolysis instruments are
available and
willallowformeasurement ofO
2association and
dissociationates.

24 Marie A. Gilles-Gonzalez and Eduardo H. S. Sousa
2.To avoid damage to glass or quartz cuvettes, the spectra of
solutions with NaOH or pyridine should be collected in
UV-transparent plastic cuvettes.
3.Typical buffers that we have used are 50 mM Tris–HCl, 50 mM
KCl, and 5% (v/v)
ethylene glycol,
pH 8.0; or 50 mM sodium
phosphate, 50 mM NaCl, and 5% (v/v) glycerol.
Acknowledgment
EHSS is thankful to CNPq (EHSS 308383/2018-4) and the
National Institute of Science and Technology on Tuberculosis
(INCT-TB, ﬁnanced by CNPq/FAPERGS/CAPES/BNDES),
and MAGG is grateful to the US National Science Foundation
(grant no. MCB620531) for providing ﬁnancial support.
References
1.Gilles-Gonzalez MA, Gonzalez G (2005)
Heme-based sensors: deﬁning characteristics,
recent developments, and regulatory hypoth-
eses. J Inorg Biochem 99(1):1–22.https://
doi.org/10.1016/j.jinorgbio.2004.11.006
2.Lopes LGF, Gouveia
Ju´nior FS, Holanda
AKM, de Car
valho IMM, Longhinotti E,
Paulo TF, Abreu DS, Bernhardt PV, Gilles-
Gonzalez M-A, Dio´genes ICN, Sousa EHS
(2021) Bioinorganic systems responsive to the
diatomic gases O
2, NO, and CO: from
biological sensors to therapy. Coord Chem
Rev 445.https://doi.org/10.1016/j.ccr.
2021.214096
3.Shimizu T, Huang D, Yan F, Stranava M,
Bartosova M, Fojtikova V, Martinkova M
(2015) Gaseous O
2, NO, and CO in signal
transduction: structure and function relation-
ships of heme-based gas sensors and heme-
redox sensors. Chem Rev 115(13):
6491– 6533.https://doi.org/10.1021/acs.
chemrev.5b00018
4.Gondim ACS, Guimara˜es WG, Sousa EHS
(2022) Heme-based gas sensors in nature and
their chemical and biotechnological applica-
tions. Biochemist 2:43–63.https://doi.org/
10.3390/biochem2010004
5.Antonini E, Brunori M (1971) Hemoglobin
and myoglobin in their reactions with ligands.
Elsevier, New York
6.Scopes RK (2000) Protein puriﬁcation: princi-
ples and practice, 3rd edn. Springer, New York
7.Sousa EHS, Tuckerman JR, Gonzalez G,
Gilles-Gonzalez M-A (2007) DosT and DevS
are oxygen-switched kinases in Mycobacterium
tuberculosis. Protein Sci 16(8):1708–1719.
https://doi.org/10.1110/ps.072897707
8.Tuckerman JR, Gonzalez G, Sousa EH, Wan X,
Saito JA, Alam M, Gilles-Gonzalez MA (2009)
An oxygen-sensing diguanylate cyclase and
phosphodiesterase couple for c-di-GMP con-
trol. Biochemistry 48(41):9764–9774.
https://doi.org/10.1021/bi901409g
9.Tuckerman JR, Gonzalez G, Gilles-Gonzalez
MA (2011) Cyclic di-GMP activation of poly-
nucleotide phosphorylase signal-dependent
RNA processing. J Mol Biol 407(5):633–639.
https://doi.org/10.1016/j.jmb.2011.02.019
10.Appleby CA, Bergersen FJ (1980) Preparation
and experimental use of leghaemoglobin. In:
Bergersen FJ (ed) Methods for evaluating
biological nitrogen ﬁxating. Wiley, New York,
pp 315–335
11.Delgado-Nixon VM,
Gonzalez G, Gilles-
Gonzalez MA (2000) Dos, a heme-binding
PAS protein from Escherichia coli, is a direct
oxygen sensor. Biochemistry 39(10):
2685– 2691.https://doi.org/10.1021/
bi991911s
12.
Gonzalez G, Gilles-Gonzalez MA, Rybak-
Akimova EV,Buchalova M,Busch DH
(1998) Mechanisms ofautoxidation oftheoxy-
gensensor FixLandAplysia myoglobin: impli-
cations foroxygen-binding heme proteins.

Measurement of O2Binding by Sensory Hemeproteins 25
Biochemistry 37(28):10188–10194. https://
doi.org/10.1021/bi980529x
13.T
eh AH, Saito JA, Baharuddin A, Tuckerman
JR, Newhouse JS, Kanbe M, Newhouse EI,
Rahim RA, Favier F, Didierjean C, Sousa EH,
Stott MB, Dunﬁeld PF, Gonzalez G, Gilles-
Gonzalez MA, Najimudin N, Alam M (2011)
Hell’s Gate globin I: an acid and thermostable
bacterial hemoglobin resembling mammalian
neuroglobin. FEBS Lett 585(20):3250–3258.
https://doi.org/10.1016/j.febslet.2011.
09.002
14.
Sousa EH, TuckermanJR,Gonzalez G,Gilles-
Gonzalez MA(2007) Amemor yofoxygen
binding explains thedose response ofthe
heme-based sensor FixL. Biochemistr y
46(21):6249–6257.https:/ /doi.org/10 .
1021/bi7003334

Chapter3
Resonance Raman Characterization of O
2-Binding Heme
Proteins
Samuel N. Snyder, Tapiwa Chiura, and Piotr J. Mak
Abstract
A vast array of critical in vivo processes and pathways are dependent on a multitude of O2-binding heme
proteins which contain a diverse range of functions. Resonance Raman (rR) spectroscopy is an ideal
technique for structural investigation of these proteins, providing information about the geometry of the
Fe-O-O fragment and its electrostatic interactions with the distal active site. Characterization of these oxy
adducts is an endeavor that is complicated by their instability for many heme proteins in solution, an
obstacle which can be overcome by applying the rR technique to cryogenically frozen samples. We describe
here how to measure rR spectra of heme proteins with stable oxy forms, as well as the technical adaptations
required to measure unstable samples at 77 K.
Key wordsResonance Raman, Raman spectroscopy, Heme, Heme proteins, Protein chemistry, Oxy
adducts, Oxy heme, Oxygen binding, Cryogenic, Myoglobin
1 Introduction
A variety of O2-binding heme proteins play vital roles in many
important cellular processes, with functions including, but not
limited to, oxygen storage/transport (globins) [1,2], catalytic
monooxygenation (cytochrome P450, heme oxygenases) or dioxy-
genation (indoleamine-2,3-dioxygenase, tryptophan-2,3-dioxy-
genase) [3 –6], chemosensing, and signaling (DOS, FixL,
HemAT) [7–10]. Investigation of the interplay between the pro-
tein’s active site environment and the structure of the heme pros-
thetic group and its endogenous and exogenous axial ligands is
critical for understanding the structure– function relationship in
these important enzymes, an undertaking for which resonance
Raman (rR) spectroscopy is an ideal technique [11,12]. Three
vibrational normal modes associated with the inherently bent Fe-
O-O fragment can be theoretically detected in rR spectra of heme
proteins, namely, two stretching modes,ν(Fe-O) andν(O-O), and
Emily E. Weinert (ed.),Oxygen Sensing: Methods and Protocols, Methods in Molecular Biology, vol. 2648,
https://doi.org/10.1007/978-1-0716-3080-8_3,
©The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
27

oneδ(FeOO) bending mode. These vibrational frequencies can be
exploited to illustrate the changes in the associated Fe-O and O-O
bond strengths or modulation of the Fe-O-O angle. Protein archi-
tecture and conformational changes dictate the structure and reac-
tivity of the heme oxy forms. The geometry of the Fe-O-O
fragment can be affected by H-bonding or electric ﬁelds of amino
acid side chains on the distal side of the heme pocket, as well as
steric contacts that can distort the electronic structure of the heme
macrocycle.Fur
thermore, the reactivity of the exogenous O
2
ligand depends on the nature of the proximal residue that serves
as a heme trans axial ligand. rR spectroscopy is particularly well-
suited for studying the oxy adducts of heme proteins, especially
since their comprehensive characterization is often unattainable by
other spectroscopic methods; e.g., these diamagnetic adducts are
EPR silent, and in infrared spectra, theν(Fe-O)stretching
mode is
not observed andν(O-O) occurs in an area convoluted with many
overlapping modes that complicate its assignment [13].
28 Samuel N. Snyder et al.
Detection and assignment of the vibrational modes associated
with the Fe-O-O fragment has been a subject of many rR spectro-
scopic and computational studies which have established certain
trends [11,12,14]. Theν(Fe-O),ν(O-O), andδ(FeOO) modes
are generally well-enhanced for heme proteins where the heme
proximal ligand is the thiolate group of cysteine. However, the rR
enhancement of theν(O-O) mode is not very effective for
imidazole-ligated proteins and can be observed only when the Fe-
O-O fragment experiences strong H-bonding interactions with
distal active site amino acid residues [14 ], or when using cobalt-
substituted derivatives, as was shown for oxyCoMb and oxyCoHb
[15]. The frequency of theν(Fe-O) stretching mode falls within a
certain range depending on the strength of the proximal heme
ligand. Histidine-ligated proteins like globins and heme oxygenases
have a weaker imidazole ligand and theν(Fe-O) frequency is typi-
cally within the 554– 576 cm
-1
range [13,14,16]. For cysteine-
ligated proteins, the strong electron donating capabilities of the
proximal thiolate ligand lowers the frequency ofν(Fe-O) to around
517–541 cm
-1
[3,1 ]. F ν(O-O)
stretching mode is observed in the 1125–1140 cm
-1
region. The
low frequency of theν(O-O) mode, together with a Mo¨ssbauer
quadrupole splitting for the iron, indicates that the correct formu-
lation of the oxy adduct is as a ferric superoxide species, instead of a
ferrous dioxygen form [3,17].
Distal active siteelectrostatic interactions withtheFe-O
P
-O
T
fragment (where O
P
indicates theproximal, oriron-adjacent atom,
andO
T
stands forterminal, orouter oxygen atom) canalsobe
determined astheyshiftν(Fe-O) andν(O-O) inwell-established
ways. Combined computational density functional theory(DFT)
andexperimental rRstudies ofheme proteins andmodel heme
compounds showed forboth thiolate- andimidazole-ligated

species that H-bonding to O
P
results in a positiveν(Fe-O)/ν(O-O)
correlation, while H-bonding to O
T
generally results in a negative/
inverse relationship of these modes [14,18]. The correlation plots
for Fe-O and O-O bond distances have larger slopes for thiolate
than imidazole-ligated oxy adducts, indicating the former has
greater sensitivity to distal active site electrostatic interactions
[18]. H-bonding to the inner O
P
atom downshifts theν(Fe-O)
andν(O-O) frequencies simultaneously by increasing antibonding
of the Fe-O and O-O bonds to give greater sp
2
character to O
P
.On
the other hand, donation of H-bonds to the outer O
T
atom
increases backbonding which weakens the O-O bond and strength-
ens the Fe-O bond, resulting in shifts of theν(O-O) andν(Fe-O)
modes to lower and higher frequencies, respectively. The geometry
of the Fe-O-O fragment can also be implicated by unusually large
isotopic
shifts ofν(Fe-O) which can indicate a greater extent of Fe-
O-O bending that causes deviation from the Fe-O
2harmonic
oscillator model [16,19].
Resonance Raman Characterization of O2-Binding Heme Proteins 29
While O 2adducts ofsome heme proteins likemyoglobin
(Mb) andhemoglobin (Hb)arestable forhours atroom tempera-
ture,oxyformsofmany other proteins areunstable andhavea
propensity toautoxidize atphysiological conditions inﬂuid
solution. Oxyadducts ofcytochromes P450 (CYPs) haveahalf-
life(t
1/2)ontheorder oftensofmilliseconds at37°Ctohundreds
ofseconds at-30°C,andnoautoxidation isobservedattempera-
turesbelow 200K[20].Thus, necessity dictates thatrRcharacteri-
zation ofsome O
2-binding heme proteins mustbeperformedat
cryogenic temperatures. Thefrequencies ofheme modes inthe
spectra collected onfrozen samples donotchange substantially
fromthose atroom temperature (≤2–3cm
-1
)[
21],asshown in
Fig.1aforoxymyoglobin. Low-temperature rRmeasurements
often produce sharper ,narrower peaks andprovide better resolu-
tionandisolation ofvibrational modes [22].Inconducting rR
studies onfrozen samples, somecomplications maybeencountered
andspecial procedures needtobeapplied, e.g.,caution regarding
thelaserpower (<1.0 mW) mustbeexercised toavoid uninten-
tional sample warming. Itisalsopossible thatsomefrozen samples
mayexhibit highﬂuorescence thatobscures therRsignal. Insuch
cases, additional puriﬁcation ofprotein isneeded toremove these
ﬂuorescent impurities. Additionally ,concer nsregarding pH–tem-
perature dependence forbuffersystems aretypically resolved by
utilizing amixture ofaqueous bufferwith10–40%ofanorganic
solvent suchasglycerol which wasshown todramatically mitigate
suchchanges inpHuponfreezing [23].Itshould benoted thatthe
presence ofcryoprotectants canincrease thesample ﬂuorescence
upon freezing, andinsuchcasesadditional puriﬁcation ofthese
solvents maybeneeded. Thedetection andidentiﬁcation ofmodes
associated withtheFe-O-O fragment intheabsolute spectra can
sometimes bedifﬁcult when theoxygen-sensitive modes areweak,

overlap with heme modes, or are obscured by ﬂuorescence. Such
complications can be easily overcome by generation of samples
using
16
O
2and
18
O
2gases (Fig.1b). The
16
O
2–
18
O
2difference
traces clearly reveal the isolated vibrational modes associated
with the Fe-O-O fragment while all other modes and spectral
features are canceled (non-shifting). The methods used to generate
rR spectra of high quality and resolution for oxy heme proteins at
ambient and cryogenic conditions are outlined below.
30 Samuel N. Snyder et al.
2 Materials
2.1 Sample
Preparation 1.Pure protein (>95%) in an inorganic buffer like potassium
phosphate
(100 mM, pH 7.4). Use UltraPure deionized
water for making buffers. Other additives that improve the
stability of some proteins may also be included in the buffer
such as dithiothreitol (DTT) or ethylenediaminetetraacetic
acid (EDTA) (seeNote 1). If intending to measure frozen
protein sample and wishing to minimize pH change upon
freezing, use 5–30% glycerol in sample.
2.Glycerol used as an additive for rR measurements at lower
temperature, such as 77 K, should be puriﬁed to avoid an
increase in the spectral background. We tested several commer-
cially available glycerol samples of various levels of purity (syn-
thetic 99%,>95.5% ACS Reagent, UltraPure HPLS, UltraPure
Bioreagent, redistilled for molecular biology or 95.5% spectro-
photometric grade), and all of them exhibited ﬂuorescence
when measured at 77 K. We found that vacuum distillation
with charcoal pellets removes the majority of the ﬂuorescence
background. A volume of 10 mL of glycerol (99.5% spectro-
photometric grade) containing activated carbon (0.5% w/v
activated charcoal, decolorizing) is placed in a 50 mL round-
bottom ﬂask which is then connected to a vacuum system
through the condenser. The mixture is ﬁrst degassed, and
then the temperature is increased by heating in a silicone oil
bath slowly until reaching ~150°C when glycerol starts to
distill off.
3.NMR tubes (our lab uses glass, 7 inches long, 5 mm OD,
Economy) ﬁtted with rubber/silicone septa for a gastight envi-
ronment of protein sample. Use paraﬁlm to wrap the septa
every time a new puncture is made with a needle.
4.Argon gas tank with oxygen scrubber ﬁxed with ﬂexible PVC
tubing and male Luer-Lock adapter for attaching needles.
5.
>99% pure
16
O
2and
18
O
2gastanks ﬁxedwithﬂexible PVC
tubing andmaleLuer-Lock adapters forattaching needles.

Resonance Raman Characterization of O2-Binding Heme Proteins 31
Fig. 1The high-frequency rR spectra of equine heart myoglobin
16
O2adducts at
room temperature (i) and cryogenic temperature (ii) in 100 mM potassium
phosphate, pH 7.4 (a ). The asterisk on spectrum ii at 1356 cm
-1
indicates the

32 Samuel N. Snyder et al.
6.50 mM sodium dithionite (hydrosulﬁte) in 100 mM potassium
phosphate, pH 7.4, prepared anaerobically (seeNote 2): In a
4 mL glass vial sealed with a r
ubber/silicone septum,
add
23.21 mg of solid sodium dithionite. In a separate 4 mL
septum-sealed glass vial, add 2.00 mL of 100 mM potassium
phosphate buffer, pH 7.4. Degas each vial separately by con-
necting a needle from the argon gas tank tubing to the vial and
then inserting a small-gauge exit needle to allow the ﬂow of
argon at a pressure of ~5–10 PSI for roughly 15 min. For the
vial containing the buffer, the argon needle should be inserted
into the liquid to bubble the buffer for more efﬁcient gas
displacement. After 15 min, remove the exit needle from the
vial ﬁrst and then the argon needle and wrap the lid/septum of
the vial with paraﬁlm. After degassing, connect both vials with a
double-tipped transfer needle (12″long) (Fig.2). Next, insert
the argon needle into the buffer vial and then the exit needle
into the solid dithionite vial. Position the end of the double-
tipped transfer needle in the bottom of the buffer to enable the
complete transfer of the liquid to the solid dithionite vial.
Finally, remove the exit needle ﬁrst and then the transfer needle
from the vial containing the 50 mM dithionite solution and
wrap the lid/septum with paraﬁlm.
7.10μL gastight syringe with long (9″), 22-gauge needle.
8.5 mL gastight Luer-Lock tip syringe afﬁxed with an airtight
stopcock adapter and a short (1.5″), 25-gauge needle with
Luer-Lock tip.
9.Vacuum ﬁxed with PVC wire helix reinforced tubing connected
to a short (1.5″) 18-gauge needle.
10.Three-neck round-bottom ﬂask (10 mL) ﬁtted with three rub-
ber septa.
11.Liquid nitrogen.
12.Dewar for storing frozen samples.
2.2 Resonance
Raman Setup 1.Continuous wavegas laser that produces a line of a certain
wavelength which is
in resonance with the Soret band of the
oxy form of the heme protein. For example, Kr
+
ion lasers can
operate at a series of wavelengths in the visible region and
1(continued)ν 4mode of the photodissociated oxy sample that contributes
the appearance of some additional modes in this spectrum. The
frequency rR spectra of Mb-
16
O
2(i), Mb-
18
O
2(ii) and their
16
O
2-
18
O
2
rence trace (iii) measured at room temperature (b). The labeled frequencies
″
Fig.
to
low-
diffe
of theo
xygen sensitive modes in spectra i and ii are inaccurate due to overlap
with heme modes. All spectra were measured with 413.1 nm excitation line

Resonance Raman Characterization of O2-Binding Heme Proteins 33
Fig. 2Schematic illustration of the anaerobic preparation of sodium dithionite solution
produce 406.7 nm and 413.1 nm lines which, collectively,
provide effective rR enhancement for the majority of oxy
adducts for cysteine- and histidine-ligated heme proteins that
have Soret maxima around 410– 420 nm. Most commercially
available Kr
+
lasers will also require a laser cooling system, such
as open- or closed-loop heat exchangers.
2.Nonreﬂective neutral density ﬁlters (gray ﬁlters) and laser
power meter for ﬁne-tuning the laser power on the sample.
3.Long focal length spectrometer equipped with appropriate
grating (1200 g/mm or 2400 g/mm) to provide desired spec-
tral resolution of Raman bands.
4.Preferably cryogenically cooled CCD detector with broad
wavelength coverage,>95% quantum efﬁciency, enhanced sen-
sitivity, and appropriate operating software.
5.Software program for processing rR spectra, which can include
calibrating, subtracting, deconvoluting/curve ﬁtting, etc.
(such as GRAMS, Origin, etc.).
6.Standard s
or calibrating rR spectra (such as indene,
fenchone, toluene).

34 Samuel N. Snyder et al.
Fig. 3Schematic of rR setup for room temperature (a ) and cryogenic (b ) measurements
7.Various optical elements for ﬁltering, directing, focusing, and
polarizing excitation
and scattered
light. For brevity, only the
kinds of optics used in our laboratory will be discussed (Fig.3),
which includes the following: premonochromator to ﬁlter out
laser plasma from the laser beam, mirrors to direct laser beam, a
cylindrical lens to focus laser beam as a line image on the sample
(seeNote 3), a prism to direct the light to the sample (for 180°
backscattering geometry), a collimating lens to collect and
collimate the scattered light, a focusing lens to focus the scat-
tered light on the entrance slit of the spectrometer, either a
notch ﬁlter or low-pass ﬁlter to reject Rayleigh scattered light
(and also anti-Stokes shifted light for the latter), and a scram-
bler to elliptically polarize the scattered light.
8.Sample h
ith spinners for ambient and cryogenic tem-
perature rR measurements (Fig.3). Both sample holders need
to have a chamber that holds the NMR tube containing the
protein sample with entry and exit valves for gas (N
2or air) to
pass through and spin the sample. Alternatively, samples can be
spun using magnetic stirrers. The cryogenic sample holder
should contain a low-temperature cell made of either glass or
quartz (preferably) with inner and outer walls separated by
vacuum.

Resonance Raman Characterization of O2-Binding Heme Proteins 35
3 Methods
3.1 Preparing Oxy
Adducts of Protein
Samples 1.Prepare the 50 mM sodium dithionite solution anaerobically,
wrap it with paraﬁlm, and set aside
for later
use.
2.Prepare the ferric protein sample and place in an NMR tube.
The preferred protein concentration is 50–300μM, with the
higher end of the range being more favorable for frozen sam-
ples (seeNote 4). Fix the rubber/silicone septum into the
opening of the NMR tube and wrap it with paraﬁlm.
3.Remove oxygen from the NMR tube under ﬂow of argon (see
Note 5). Attach a needle (with standard hub or female Luer-
Lock) to the argon tank. Insert needle from the argon tank into
the septum of the NMR tube, followed by an exit needle. Flow
argon over the sample at a pressure of 5–10 PSI for
~15–20 min. Then remove the exit needle from the NMR
tube ﬁrst, followed by the argon needle, and immediately
wrap the septum with paraﬁlm.
4.Reduce the ferric protein sample to the ferrous state with
dithionite. Using a 10μL gastight syringe ﬁtted with a 9″
needle, draw up 10μL of the 50 mM sodium dithionite solu-
tion. Insert the needle into the NMR tube such that the point is
in the liquid sample and add the calculated volume of dithionite
to the protein (seeNote 6). Remove the needle and wrap the
septum with paraﬁlm.
5.
Prepare
16
O
2and
18
O
2isotope gasesinaseparate three-neck
round-bottom ﬂask(seeNote7).First,ﬁteachneckoftheﬂask
witharubber septum oftheproper sizeandwrapwithparaﬁlm.
Fixthereinforced tubing ofavacuum withashort(1.5″)
needle andconnect ittooneneckoftheﬂask. Inaseparate
neckoftheﬂask,inserta5mLLuer-Lock tipgastight syringe
ﬁtted withanairtightstopcock adapter (with stopcock open)
andashort(1.5″)needle. Intheﬁnalunoccupied neckofthe
round-bottom ﬂask, insertshort(1.5″)needles attached to
argon andeither
16
O
2or
18
O
2gastanks. Toprevent leaks,
wrapwithparaﬁlm around alltheneedles andtherespective
septatheyareconnected to.Turnonthevacuum toevacuate
thegasfromtheﬂask,andthenremove thevacuum needle and
opentheargon gastanktoﬁlltheﬂaskwithargon. Close the
argon tank, insertthevacuum needle again, andrepeat this
process 2–3times, rewrapping withparaﬁlm everytimeanee-
d
leisremoved orinserted.Then remove theargon needle,
evacuate thegasfromtheﬂaskunder vacuum onelasttime,
andremove thevacuum needle. Thetubing ontheO
2tank
shouldtilleollapsedfheacuumealsaintained.r
slowlypenhe
2tank,andoncetheplunger onthesyringe is
pushed outtoindicate itisﬁlledwiththeO
2gasisotope, close

36 Samuel N. Snyder et al.
both the gas tank and the stopcock on the syringe and remove
from the ﬂask. This process will be repeated from the beginning
for the other isotope of oxygen gas,
16
O
2or
18
O
2.
6.Form the oxy adduct of the heme protein by introducing
16
O
2
or
18
O
2gas to the NMR tube containing the sample. Open the
stopcock on the syringe containing the appropriate isotope of
oxygen gas and begin pushing down on the plunger immedi-
ately prior to and during insertion of the needle into the
septum of the NMR tube to evacuate any atmospheric gases
out of the tip of the needle. Once the needle is in the NMR
tube, continually push down on the plunger of the syringe (the
pressure in the tube should keep pushing the plunger back to
the original position) to mix the O
2gas with the argon in the
tube. Hold the NMR tube relatively horizontally while gently
shaking the sample to maximize the surface area of the liquid
exposed to the O
2in the tube. The amount of time the sample
will need to be exposed to O
2in order to form the oxy adduct
will vary depending on the protein, although most globins and
cytochromes P450 form oxy adducts relatively instantaneously.
Then remove the needle from the sample and rewrap the
septum with paraﬁlm. If preparing the oxy adduct at lower
temperature, keep the bottom part of NMR tube in cooling
bath at desired temperature. (0°C, ice bath;-10°C, 70% ice
bath with 30% calcium chloride;-15°C, 80% ice bath with
20% ammonium chloride;-20°C, 75% ice bath with 25%
sodium chloride;-23°C, carbon tetrachloride with dry ice;
-25°C, dry ice with 1,3-dichlorobenzene; etc.).
7.If the oxy adduct needs to be frozen, the sample will then be
dipped into a cryogenic container ﬁlled with liquid nitrogen
(immediately after forming the adduct of unstable oxy forms).
Preferably, freeze the sample incrementally, dipping only the
very bottom tip of the NMR tube in the liquid nitrogen at ﬁrst.
This allows the sample to expand upwards as it freezes, whereas
ﬂash freezing the entire sample at once might cause expansion
of the liquid in all directions and break the NMR tube. Upon
freezing the entire sample, remove the septum from the NMR
tube so that once the sample is brought back to room temper-
ature, the rapidly expanding gases do not cause the tube to
explode. Samples can then be transferred to a loosely capped
dewar ﬁlled with liquid nitrogen to be stored indeﬁnitely with-
out autoxidation.
3.2 Resonance
Raman Measurements 1.
Tooptimize thesignal, thelaserlineandscattered lightneedto
bealigned
andfocused onthesample andentrance slitofthe
spectrometer ,respectively ,byadjusting thedifferent optics
composing therRsetup (Fig.3).Thealignment should be
performedandoptimized ﬁrstonanNMR tubecontaining
thecalibration standard (such asindene orfenchone).

Resonance Raman Characterization of O2-Binding Heme Proteins 37
2.Determine the position of the grating that will span a certain
frequency range using the calibration standards. The grating
will need
to be
adjusted such that the following regions can be
measured: the 200– 800 cm
-1
range (includesν(Fe-O),ν
7, and
peripheral group modes), the ~900–1200 cm
-1
region (which
includes theν(O-O) stretching mode), and the
~1350–1700 cm
-1
range (which will encompassν
4,ν
3,ν
2,
ν
10, and vinyl stretching modes).
3.Turn on the gas (can use N
2or air) to begin rotating the
spinner in the sample holder. Measure rR spectra on the cali-
bration standard(s) at room temperature in the aforemen-
tioned frequency regions. Any desired laser power can be
used for the calibration standards.
4.Adjust the laser power to that used for measurements of the
oxy protein samples. The laser power is typically ~1 mW (see
Note 8).
5.For room temperature measurements, put the oxy sample in
the sample holder while spinning (seeNote 9) and ﬁne-tune the
optics to optimize the Raman signal (focusing length will be
slightly different for the colored translucent sample than the
transparent calibration standards). Then measure the rR spec-
tra of the
16
O
2and
18
O
2adducts in each of the designated
frequency regions without changing anything about the rR
setup that could cause differences in the background/baseline
between the two isotopes (e.g., there should be no need for
readjusting the low-pass ﬁlter and other optics). The measure-
ment time required will depend on the signal to noise ratio, but
anywhere between 20 and 60 min in each frequency region is
typical, with longer acquisition times being necessary for reso-
lution of modes of relatively weak intensity.
6.For cryogenic measurements, direct a stream of room temper-
ature gas (N
2or air) on the outside of the low-temperature cell
to prevent ice from forming on the outer wall that would
obscure the path the laser line must travel to reach the sample.
Fill the cryogenic cell with liquid nitrogen and wait for the
temperature to equilibrate for ~10 min (seeNote 10).
7.
Turnonthegastobegin rotating thesample spinner inthe
cryogenic cell.TheNMR tubecontaining thefrozen oxysam-
pleshould betransferr edfromthestorage dewar tothecryo-
genic sample holder asquickly aspossible sothatitdoesnot
heatupand/or formiceontheoutside ofthetubefrom
condensation ofmoisture intheair.Once thesample is
submerged intheliquid nitrogen ofthecryogenic cell,ensure
thatthattubeisspinning atanacceptable ratebyadjusting the
gaspriortopositioning thesample furtherdown inthecryo-
genic cellintothepathofthelaserline(seeNote 11).

38 Samuel N. Snyder et al.
8.Fine-tune the optics to optimize the signal for the frozen
sample (focusing length will
be slightly different for opaque
sample than that of the
transparent, liquid calibration stan-
dard). Then measure the rR spectra of the frozen
16
O
2and
18
O2adducts in each designated frequency region without
changing anything about the rR setup that could cause differ-
ences in the background/baseline between the two isotopes.
Reﬁll the cryogenic sample holder with liquid nitrogen when
needed.
9.After measuring the rR spectra, the frozen samples should be
transferred to a storage dewar ﬁlled with liquid nitrogen for
storing in case they will be needed for further use.
10.Using a suitable software, the rR spectra should then be pro-
cessed by removing cosmic rays, averaging, calibrating, sub-
tracting difference spectra, and deconvoluting/curve ﬁtting.
The difference traces should be constructed from individual
spectra that were not baseline corrected to prevent uninten-
tional modiﬁcation of the resulting trace.
4 Notes
1.Certain reagents/additives and the impurities therein can ﬂuo-
resce, raise the baseline, lower
the signal
to noise ratio, or
otherwise decrease the quality of the rR spectra. Therefore,
ensure the reagents used in the buffers of protein samples are of
the highest purity attainable. When possible, avoid using addi-
tives containing aromatic compounds as they tend to ﬂuoresce.
2.Sodium dithionite (hydrosulﬁte) must be made fresh immedi-
ately before use as it decomposes when exposed to molecular
oxygen, increasingly so when in aqueous solution or in the
presence of moisture. Because of its instability and the purity
limitations of commercially available dithionite, a fraction of
the dithionite will be inactive immediately upon dissolving.
The assumption is made that 75% of the dithionite added to
the solution will be chemically active and this is accounted for
when calculating the mass of the solid to be dissolved. In cases
requiring an exact molarity for a dithionite solution, the con-
centration can be determined via UV-Vis spectroscopy using
the extinction coefﬁcient,ε
315 nm=8043 M
-1
cm
-1
[24].
3.Focusing the laser as a line image, rather than a point on the
sample helps to avoid localized heating and photodissociation
of the sample by dispersing the light energy over a larger
area [25].
4.
Forcr yogenically frozen samples, thesolution willfreeze ina
fashion thatwillmake theconcentration ofprotein lower on
thelateral/exterior surfaceofthesample andgreater inthe

Resonance Raman Characterization of O2-Binding Heme Proteins 39
medial/interior region. Unlike liquid samples, the laser will
only be able to access the exterior surface of the frozen samples.
Therefore, a greater initial concentration of protein should be
used for frozen samples to accommodate for this effect of
freezing to ensure a favorable signal in rR measurements.
5.Degassing of samples can be performed using a Schlenk line/
vacuum line, but if this is not readily available, then ﬂowing
argon over the sample for a longer period will remove oxygen
sufﬁciently.
6.When reducing the sample to the ferrous state, use the mini-
mum amount of dithionite necessary. The ferric protein sample
can be titrated with dithionite and conversion to ferrous state
can be monitored by UV-Vis or rR spectroscopy. If using
UV-Vis, the conversion must be monitored by changes in the
Q-band region as the concentration of the sample will exceed
the acceptable range for Beer’s law at the Soret band. Addition-
ally, an adapter that accommodates an NMR tube would have
to be used in the cuvette holder. If monitoring by rR spectros-
copy, look for the shift ofν
4in the high-frequency region to
indicate conversion from the ferric to ferrous state of the pro-
tein sample.
7.Again, adding
16
O
2and
18
O
2isotope gases can be performed
using a Schlenk line/vacuum line, but if one is unavailable,
then the technique with a three-neck round-bottom ﬂask can
be used as a suitable alternative.
8.The laser power for oxy protein samples should be kept very
low, around 1 mW or below, because oxy adducts are very easily
photodissociated. Furthermore, if the samples are measured at
77 K, higher laser power can unintentionally warm up the
sample and cause protein autoxidation to the ferric state.
9.We do not designate a value of rpm at which the sample should
be spun. Qualitatively, however, the room temperature samples
should be spun at the maximum speed possible that does not
cause the liquid to “climb” up the walls of the NMR tube. A
greater rate of spinning will enable better mixing of the sample
to prevent localized heating and photodissociation. If the sam-
ple solution “climbs” up the wall, it will photodissociate more
easily because it creates a thin ﬁlm of liquid of smaller volume in
the path of the laser line that is unable to distribute and dissi-
pate the heat energy as effectively.
10.
Theliquid nitrogen usedinthecryogenic sample holder cell
should befreshly collected inadewar withalidprotecting it
fromairtoprevent formation oficecrystals fromcondensing
moisture intheatmosphere. Iftheliquid nitrogen inthecryo-
genic cellcontains toomuch ice,itwillproduce scattering that
willobscure thesignal from thesample intherR
measurements.

40 Samuel N. Snyder et al.
11.Oxy adducts of heme proteins are easily photodissociated or
warmed up, even more readily so when they are frozen and
incapable of mixing because the laser hits the same circumfer-
ential line around
the sample.
It is still important to spin the
sample because if the tube is stationary, it will take a very short
amount of time for the laser beam to photodissociate or warm
up a spot on the sample. If the situation is encountered where a
spot or circular line of the frozen sample has been photodisso-
ciated, the tube can be repositioned slightly higher or lower in
the sample holder such that the laser hits a different part of the
sample where the oxy adduct is intact.
Acknowledgments
We gratefully acknowledge Dr. J. R. Kincaid of Marquette Univer-
sity and the members of his research group for the contribution
into development of these methods.
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— Ei ollut onnea minun nimelläni, sanoi Joutsian isäntä sisarelleen.
— Muistatko, kuinka sinä toivoit, että minun nimeni tekisi lapsen
minun kaltaisekseni?
He istuivat kiikkulaudalla Haimalan puutarhassa, ja heidän
edessään oli sininen järvenselkä. Aurinko paistoi lämpöisesti ja
laineet kimmelsivät ja välkkyivät. Oli sunnuntai, ja koko talon väki,
yksin lapsenhoitajakin, oli kirkossa.
Helena istui kädet ristissä ja seurasi silmillään pikku Juhaa, joka oli
istumassa ruusupensaan alla nurmella. Lapsen kädessä oli kaksi
puista palikkaa ja niitä se sommitteli päälletysten. Siinä se taas oli
istunut puolen tuntia kumminkin, liikkumattomana. Äidin kasvoissa
kuvastui sanomaton tuska. Hän oli kalpea ja laihtunut, otsaan
silmien välille oli syntynyt kaksi syvää piirtoa. Kyynel valui poskea
alas. Lapsen sininen kolttu loisti iloisesti nurmelta, tuulenpuuska
liehutteli sen pellavankarvaista tukkaa ja varisteli ruusupensaista
valkoisia lehtiä sen päälaelle ja hartioille.

X
Nainen se pitkin matkaa oli ollut viittana Jakob Sandin, Keihäsjärven
papin tiellä. Harvoin se oli kulkenut lyhty kädessä ja johtanut
valoisille ylängöille. Jos sillä olikin ollut lyhty, niin Jakob Sand oli
puhaltanut sen sammuksiin ja sitten oli riennetty kuumiin horhiin…
Nainen se oli, joka vihdoin tuli Jakob Sandin kompastuskiveksi. Mutta
ei ylhäinen Editha-rouva eikä liioin Haimalan Helena. Vaan se oli
Joutsian pieni piikatyttö, sen hurskaan äidin lapsi, Susanna.
Muisti pastori varsin hyvin sen syksyisen illan, jolloin hän hänet
otti haltuunsa. Se tapahtui Tulettänessä ja siellä oli silloin paljon
miehiä koolla ja elämä oli iloista, kuten aivan vanhoina, hyvinä
aikoina. Oo, elämä Keihäsjärvellä oli viime aikoina käynyt niin
ikäväksi ja siivoksi että inhotti! Vapaus oli vanhana talluksena
poljettu jalkain alle ja erotus hyvän ja pahan välillä oli käynyt
ihmisten lapsille niin epäselväksi, että he pelkäsivät kaikkea eivätkä
uskaltaneet mennä minnekään — ei edes kirkkoon, sillä siellähän
hallitsi Jakob Sand. No niin… kuka tuo tyttö lienee ollutkaan, se oli
joka tapauksessa nuori, verevä, veikeä lapsi. Hän tuntui kyllä tutulta,
pastori muisteli jossakin nähneensä huivin alta pilkottavan paksun,
pellavankarvaisen palmikon. Olisiko tyttö toissa vuonna ollut
rippikoulussa? Ei pastori muistanut varmaan.

Ihmeellinen taika on tuollaisessa nuoressa, koskemattomassa
tytössä, sen pienessä kiemailussa ja sitten sen pelossa… Tämä oli
pelännyt häntä, sillä se oli todella ollut kokematon ja koskematon.
Sellainen vanha, tottunut naisten kesyttäjä kuin Jaakko Sand sen
tunsi käsiinsä, heti kun hän kiersi kätensä naisruumiin ympäri. Tyttö
ei tahtonut taipua. Hänet oli lähetetty asialle… Isäntä oli käskenyt
joutua… Tyttö oli itkenyt, hän oli luvannut huutaa, hän oli rukoillut
sekä Jumalaa että sitä miestä, joka hänen edessään huohotti ja jota
ei hän lehdon pimeydessä voinut nähdä… Onneton lapsi, ei se
ymmärtänyt, että pelastus kävi sitä mahdottomammaksi, jota
enemmän hän itki ja rukoili… Sittemmin unohtui koko tyttö, kuten
kymmenet ennen häntä, kunnes pastori eräänä sunnuntaina keskellä
saarnaansa huomasi hänet kirkon penkissä, vaipuneena itkemään.
Mistä hän hänet tunsi? Siitä, että huivi oli valunut päästä ja näkyviin
oli tullut paksu, vaalea palmikko, jollaista ei ollut kenelläkään muulla
hänen seurakunnassaan. Hän oli silloin illalla jo punninnut palmikkoa
kädessään… Tyttö-raukka itki katkerasti. Mikähän hänen oikein oli?
Pastori miltei suli, kun hän ajatteli tämän elämän kovuutta, ja kun
hän luki saarnanjälkeisiä rukouksia, hän teki sen hartaana ja ajatteli
kaiken aikaa tuota tyttöä.
Mutta kävi vaikeammaksi, kun hän huomasi, että hän tuli rippiväen
joukkoon. Jos tyttö katsahtaa häneen, jos tyttö tuntee hänet, jos
tämä karkaa ylös penkistä… Pastori pelkäsi nuoren tytön silmiä,
hänen kätensä vapisivat niin ettei hän tahtonut saada rippileipää
hänen hampaisiinsa, ja kalkista läikkyi viiniä maahan, kun hän muutti
sitä suusta suuhun. Jos… jos tyttö luo häneen silmänsä, niin hän…
niin hän pyörtyy tai hän kivettyy siihen paikkaan…! Ei hän ollut
ensinkään kaunis: kasvot olivat täynnä äkämiä, leveät kasvot,
poskipäät pystyssä. Ainoastaan otsa oli puhdas ja siitä nousi
pehmoinen pellavanvalkea tukka, joka katosi huivin alle ja valui

paksuna palmikkona pitkin selkää. Kädet olivat puserruksissa
nenäliinan ympärillä alttaripöydällä, karkeat, veripunaiset kädet
suuren, kotikutoisen nenäliinan ympärillä, joka lisäksi oli itketty
likomäräksi. Niissä kasvoissa oli ääretön avuttomuus, ääretön
hyljättyys, ne hytkivät ja vääntyivät ja kurkussa kulahti vähän väliä,
kun tyttö nieli kyyneliään. Ihmiset veisata vetelivät: Oo Jumalan
karitsa, joka pois otat maailman synnit… Silmät turvonneina nousi
tyttö alttaripöydän äärestä ja hoippui penkkiin. Pastori pakeni
sakaristoon, haki kaapista pullonsa ja kulautti kurkkuunsa. Ja sen
tiensä meni pahoinvointi ja hän ainoastaan ihmetteli, kuinka tyttö
silloin illalla oli voinut tuntua niin viekoittelevalta. Mutta ainahan asiat
päivänvalossa näyttävät toisilta kuin pimeässä.
Myöhemmin pastori näki kerran vilahdukselta tutun vaalean
palmikon. Silloin hän kysyi, kuka tyttö oli, ja sai tietää, että hän
palveli Joutsiassa. Hän ei ollut käynyt rippikoulua Keihäsjärvellä,
vaan naapuripitäjässä, koska sinne oli lyhyempi matka sieltä
metsäkulmalta, missä hän asui.
Ikävä asia, että hän palveli Joutsiassa. Joutsian mies oli käynyt
pastorille yhä vastenmielisemmäksi, sillä hän juuri hiljaisella,
näennäisesti vaatimattomalla käytöksellään täällä villitsi ihmisiä
tekopyhyyteen ja itsevanhurskauteen. Niin juuri… Ja hän sai aikaan
enemmän kuin olisi luullut yhden miehen ehtivän saada. Se yksi
mies vaikutti kuin kahle sekä pastoriin että Hallbomiin, nimismieheen
ja henkikirjuri Nylanderiin.
Eräänä aamuna kertoi Amanda, pastorin lihava taloudenhoitajatar,
sellaista juttua, että Joutsian piika, se sellainen nuori Sanna-niminen
tyttö, oli kadonnut. Sitä ei löydetty mistään.
— No, onko haettu? kysyi pastori ja ikään kuin hätkähti.

— On.
— Kai se on mennyt kotiinsa.
— Kun ei se ole mennyt kotiinsa. Kuuluvat pelkäävän, että se on
hukuttanut itsensä.
Pastorin mieleen juolahti puheet Helenasta. Kuinka monta kertaa
hänen hukuttautumisestaan puhuttiinkaan! Ei ihminen sentään
niinkään hukuta itseään! Hukuttiko Helena? Mitä vielä! Lastaan
lellittelee par'aikaa Haimalassa! Niin kai tekee se Joutsian
piikatyttökin! Tätä ei pastori kumminkaan sanonut.
— Ovat hakeneet kaivot ja avannot, jatkoi Amanda. — Eikä ole
löytynyt.
— Kyllä routa porsaan kotiin ajaa, sanoi pastori. — No, mitä sinä
katselet?
Amanda katseli pastoriin pitkään.
— No mitä sinä katselet! ärjäisi pastori uudelleen.
— En mitään, sanoi Amanda, veti alahuultaan pitemmälle ja läksi
keittiöön.
Pastori nielaisi kirouksen, nousi, astui pariin kertaan lattian poikki
ja meni sitten kaapille, jossa hän säilytti juomiaan. Hähhäh sentään!
Hän tunsi olevansa kuin tervassa. Jos toista jalkaa nosti, niin toinen
takertui. Jota kiihkeämmin koetti siirtää ajatusta muuhun, sitä
pahemmin se sotkeutui siihen samaan. Kunhan ei tyttö sittenkin
tekisi jotakin hullutusta! Hohhoh, Jakob Sand, mihin kaikkiin sinä
sotkeudutkin!

Iltapäivällä, kun pastori seisoi ruokasalin ikkunassa, näki hän, että
Joutsian hevosella ajettiin sivuitse tavatonta kyytiä.
— Mihinkähän nyt on niin kiire? sanoi Amanda, joka pesi astioita
keittiössä.
Pastori kuuli selvästi hänen puheensa ja aikoi jo ärjäistä, että
"eivätkö joutsialaiset enää saisi ajaakaan ilman sinun lupaasi?" Mutta
hän päästi ainoastaan pienen kirouksen ja meni huoneeseensa.
— Kun eivät olisi löytäneet Sannaa, jatkoi Amanda taasen
keittiössä, — ja menisi hakemaan vallesmannia…
— Herra siunatkoon kuitenkin! sanoi karjapiika siihen.
Pastori nousi tuimasti ja paiskasi kiinni keittiöön vievän oven, jotta
ikkunat helisivät. Kyllä ne akat osaavat rämpättää! Ettei niitä olisi
tässä maailmassa .. Eikös tuo mokomakin, tuo Amanda, lähtenytkin
pihalle tähystelemään!… Kaivelee tuossa nyt hampaitaan ja katsoo ja
katsoo… Pastori oli jo ikkunan ääressä ja koputtamaisillaan ruutuun,
mutta malttoi mielensä. Ties mitä Amanda olisi ruvennut
ajattelemaan! Hän katseli äsken muutenkin jo niin hävyttömästi…
Jos lähtisi tästä ulos… Mutta mihin? Tulettäne oli pahassa paikassa,
itse Joutsian vieressä… No, jopa hän nyt oli tulemaisillaan lapseksi
uudestaan, kun niin pelästyi sitä, että Amanda tähysteli maantielle!…
Mutta kas, nytpä tämä näkee jotakin, koska karkaa portille. Siihen
pysähtyy huivitta päin, lämmittelee käsiä esiliinassaan ja hyppii
vuorotellen toisella, vuorotellen toisella jalalla. Hullun näköinen se
on, mokomakin!… Voi sinuas, sinä pullea Amanda, sinuakin minä
olen viitsinyt katsella!…

Totta tosiaan, vallesmannia siellä tuodaan Joutsian hevosella.
Amanda pysäyttää hevosen ja pastori näkee, kuinka hän huojuttaa
päätään ja huitoo käsillään.
Pastorin päähän syöksyy veri niin tuimasti, että tuntuu siltä, kuin
se pursuisi esiin ihon alta. Hetkisen perästä puistattaa kylmä häntä
ja kädet käyvät likomäriksi. Nojaten kirjoituspöytäänsä hän
kuuntelee sydämensä lyöntejä, raskaita, epätasaisia lyöntejä kuin
vasaran iskut… Kuolema tulee, ajattelee hän itsekseen. Hän koettaa
liikkua, mutta ei pääse paikaltaan. Silmissä on kaikki mustaa,
liikkuvaa.
Kuolema! ajattelee hän taasen ja outo lamaus lyö hänet. Mutta
hetkisen perästä hän näkee pimeyden haihtuvan kuin mustan
verhon, jota hiljaa vedetään sivu, ja samassa hän pääsee liikkeelle.
Kuinka jalat painavatkin, kuinka huonoksi hän on käynyt!
Editha! parkaisee hän ilman ääntä ja hänen silmiensä ohitse
välähtää jotakin valkoista. Seuraavassa hetkessä tulee se musta taas
takaisin, se, joka on kuin kuolemaa, ja hänessä on tilaa vain yhdelle
ajatukselle: kun pääsisikin kaapille! Ja hän panee kaikki voimansa
liikkeelle saavuttaakseen tämän päämäärän, viimeisen elämässä:
päästäksensä kaapille.
Pullo, pullo! Ei tarvitse lasiakaan! Kunhan hän vain löytää pullon…
Kaapille! Kunhan pääsee keinutuolille ensin… Sitten on vain pari
askelta! Ei lasiakaan… kunhan vain saa pullon!
Hää! Hän pääsee, hän pääsee! Siunattu pullo! Elämä palaa,
lämmin, suloinen elämä. Se karkaa kuin tuli läpi luitten ja ytimien.
Mies pääsee pystyyn, esineet pysyvät paikoillaan silmien edessä,

permanto on taasen lujana jalkojen alla. Mies ojentuu suoraksi, eikä
mikään muu todista mennyttä myrskyä kuin sinertävä puna poskilla.
— Mitä sinä taas älmennät! tokaisee hän Amandalle vasten
kasvoja, kun tämä juosta lönköttää sisään ja jo tullessaan huutaa ja
toimittaa:
— No, Herran tieten, kun se tyttö on hukuttanut itsensä! tulee
Amandan suusta kuin koskesta. — Kun Joutsian miehet tänä aamuna
menivät ruoppakuopalle, niin sieltä löysivät huivin, joka oli jäänyt
lammikon pinnalle. Sitten rupesivat etsimään ja eivätkös löytäneetkin
tyttöä!… Se oli niin kovasti pelännyt vanhempiansa… Arvelevat, että
se siksi… Kun se oli, raukka, ollut niin kovasti jäätyneenä ruoppaan,
etteivät tahtoneet irti saada…
— Älä nyt huuda, keskeyttää pastori hänet vihdoin — Kuuleehan
sitä nyt vähemmälläkin.
Amanda luo häneen vihaisen katseen ja katoaa keittiöön. Ja
hetkisen perästä kuuluu vain etenevä ryty, kun piikojen askeleet ja
äänet häviävät pirttiin.
Pastori koetti ottaa avukseen järkensä. Ehkei se asia ensinkään
ollut sillä lailla. Tiesihän sen jokainen, mitenkä huhut muuttuivat
kulkiessaan. Jos olisi lähtenyt Joutsiaan tiedustelemaan? Mutta ei
maittanut se esitys… Olisiko talonpoikaisihmisellä ollut niin paljon
häpeäntuntoa ja hienotunteisuutta? Olisiko sellaisessa piikatytössä
ollut niin paljon rohkeutta — niin, suorastaan rohkeutta? Ei hän
saattanut sitä uskoa! Ei ole leikintekoa ottaa itseltään henki.
Pastori pääsi pian epäilyksistään, sillä Joutsiasta palatessa poikkesi
nimismies Liljeblad pappilaan. Kaikki, kaikki oli totta.

Ystävykset istuivat pastorin kamarissa kuten niin monena
monituisena iltana ennenkin, samassa nahkasohvassa, jonka
yläpuolella riippui Lutherin kuva ja pastorin ampuma-aseet. Heidän
edessään olivat totivehkeet kuten niin monena monituisena iltana
ennen, mutta puhe ei tahtonut luistaa.
— Hyi, sanoi nimismies ja sylkäisi. — On se koiran virka se minun
virkani: nuuskin kaikkia haaskoja ja… Hyi saakeli sentään!
Ja sitten hän kertoi minkä näköinen ruumis oli ollut. Se oli ollut
niin kiinni liejussa, että oli rautakangilla irroitettava. Eikä siitä
osannut muuta eroittaa kuin silmien valkuaiset ja hiukset, jotka
pääsivät irti ja laahasivat reen perässä kuin mikäkin kellertävä harja.
Tavattomat hiukset sillä ihmisellä olikin!
Pastori istui äänetönnä ja kuunteli, kasvot kankeina.
Nimismies loi häneen äkkiä katseensa. Molempien posket
punoittivat juomisesta.
— Kunnon veliseni, virkkoi hän, — ikävintä tässä jutussa on se,
että sekoittavat sinut siihen…
Pastori koetti olla hämmästyvinään.
— Minut…!
— Niin, että sinä Tulettänessä…
— Tiedän, tiedän.
— No, älä nyt noin pelästy! Kaipa sen helposti saa todistetuksi,
ettet sinä sinä iltana ollut Tulettänessä.

— Oliko… oliko tyttö sanonut mitään?
— Ei, eivät olleet saaneet häntä sanomaan, vaikka emäntäkin
monta kertaa oli koettanut pyytää oikein kauniisti, kun tyttö oli niin
onneton.
Pastori sivaltaa kädellä otsaansa ja hengittää helpommin. Mutta
samalla kiihtyy tuska hänen sielussaan: hyvä tyttö se on ollut,
arkaluontoinen tyttö. Kuinka kauhean onneton hän onkaan mahtanut
olla!
— Vai niin, sanoo hän hajamielisesti.
— Niin, jatkaa Liljeblad. — Hallbomin väki sen helposti todistaa,
sinä olet niin monet palvelukset tehnyt heille.
— Tiesi niitä Hallbomeja!
Nimismies purskahti nauruun.
— Kyllä minä ne takaan! Sinun pitää ennen kaikkea ottaa tämä
asia järkevästi. Mitä sinä nyt tämmöisestä noin sydännyt? On niitä
nähty pahempiakin! Ja kuulepas vielä! Olisi hyvä, jos sinä jollakin
lailla voisit hieroa sovintoa rengin, sen Epramin kanssa. Hän se on
pahin. Kas, hän oli tytön sukulainen ja kaikesta päättäen pikiintynyt
tyttöön. Hän murjotti niin mustasti, että luuli hänen aikovan syödä
joka miehen. Hän se juuri käräjiin tahtoo. Minusta voisivat peittää
unheeseen koko jutun: eihän tyttö enää siitä kostu, mitä hänelle nyt
tehdään.
Mutta pastori oli jo niin humalassa, ettei hän tietänyt mistään. Hän
rallatteli itsekseen ja näpäytti sormillaan ikään kuin ajaakseen

lentoon kärpäsiä. Kun Liljeblad yritti lähteä pois, takertui hän tämän
kaulaan ja rupesi itkemään.
— Älä jätä minua, veli. Minä kuolen tänä yönä.
Nimismies nouti Amandan ja yhdessä he johdattivat pastorin
sänkyyn.
Tuli kauhea yö. Pastori makasi kuin paasien alla, ei hän voinut
huutaa eikä valittaa, ei hän saanut edes hengitystä kulkemaan. Hän
oli joka hetki tukehtumaisillaan ja hänen täytyi ponnistaa voimiaan
äärimmilleen, ennen kuin rinnasta läksi ääntä.
— Aaa… Auttakaa…!
Amanda tölmäsi ylös unestaan ja sytytti tulta. Pastori oli
kasvoiltaan ihrankarvainen ja hiki pisaroi hänen otsaltaan. Amanda
kostutti hänen ohimoltaan kylmällä vedellä, antoi hänen haistella
etikkaa ja murisi äreästi: "Kun ei anna ihmisten maata."
Pastori virkistyi hiukan ja meni uudelleen uneen. Hän painaa
jotakin rintaansa vasten ja etsii sen niskaa. Siitä lähtee jotakin
paksua, mustaa… Se on ruoppaa, se on kylmää… Jäätyneen
lammikon pinnalla pilkottaa jokin vaate… Näkyy punakirjavaa röijyä,
näkyy pellavankeltaista tukkaa… Tukka aukeaa ja alkaa laahata reen
perässä… Ruopan alta katselevat avonaiset silmät, nurin päässä…
Kaikkea tätä pitää pastori sylissään ja painaa sitä rintaansa vasten…
Amanda herää taasen pahaan parahdukseen ja sytyttää tulta. Ei
tässä tänä yönä näy saavan nukkua! Sellaista se on, kun juo kuin
sika eikä tiedä määrää. Palakoon nyt tuli koko yön.

Yö on pitkä. Hiljaisuus hymisee, kynttilään tulee pitkä karsi, joka
rupeaa rätisemään ja käryttämään. Amanda kuorsaa jonkinlaisella
vuoteella, jonka hän on tehnyt lattialle ovensuuhun.
Hiiretkö ne nakertelevat uuninnurkassa? Jyrsivät, jyrsivät,
juoksentelevat vinnillä, että jyskyy, toisesta päästä toiseen! Pastori
käskee silmiänsä aukenemaan, hän tahtoo avata silmänsä ja nähdä:
hän ei saa niitä auki! Kannet ovat kuin juotetut kiinni! Eivät aukea!
Mutta kansien läpi hän on näkevinään pienten hiirien hissuttavan
esiin nurkista, pysähtyvän vuoteen ääreen katselemaan häntä… Niillä
on mustat, liikkumattomat silmät kuin helmet… Niiden silmät
menevät nurin!
Pastori karkaa ylös vuoteesta. Karsi kynttilässä on tuumaa pitkä ja
käryää ja rätisee. Liekki leimuaa sen ympärillä, koko huone liikkuu,
kiikkuu ja huojuu… Henget leijailevat ja liehuttavat harmaita
vaippoja.
— Auttakaa! parahtaa pastori ja putoaa takaisin vuoteelleen.
— No mikä nyt taas on? ärisee Amanda. — Kuka käskee sillä lailla
juomaan! Nooh… tuonko viinaa vai aarakkia, vai…?
Ja hän sylkäisee hyppysiinsä, niistää kynttilän, viskaa karren
nurkkaan ja lähtee toiseen huoneeseen.
— Aijai, aijai, puhelee pastori itsekseen. — Minä en uskalla lausua
sinun nimeäsi, sinä… ylhäisin. Olen sitä niin paljon turhaan lausunut.
Jumala…!
Aamupuoleen yötä hän vihdoin menee uneen, joka antaa voimia ja
virkistää. Hämärissä herättää Amanda hänet ilmoittamalla, että

Joutsian isäntä jo kauan on ollut täällä odottamassa. Hän tulee
kirjoituttamaan ruumista.
Sitä ei Amandan laisinkaan tarvitsisi ilmoittaa, sen pastori kyllä
arvaa muutenkin… On hän sentään tänään aika paljon parempi. Se
oli kammottava se viime yö. Miten voikaan mielikuvitus päästä
sellaiseen valtaan?
Hän on tullut vanhaksi ja raihnaaksi. Äää sentään, mikä rytö
ihminen on!
Nyt on edessä ilkeä tehtävä: istua kuuntelemassa itsevanhurskaan
miehen selityksiä. Tai ehkäpä se nyt tulee se suursiivous… Äää
sentään…! Tämä elämä on roskaa!
Juha oli lammasnahkaturkissa, musta kaulus jonkin verran auki,
niin että paljas kaula näkyi. Hän istuutui tuolille ovensuuhun ja
kierteli lakkia käsissään. Hänen hymynsä oli vähän väkinäistä ja
odotus oli nostanut malttamattomuuden punan hänen poskilleen.
— Tottahan pastori jo on kuullut siitä kuolemantapauksesta, alkoi
hän.
— Johan minä siitä vähän kuulin, vastasi pastori huolettomasti.
Hän istui kirjoituspöytänsä ääressä ja kaiveli paperiveitsen kärjellä
hampaitaan. — Onhan se aika ikävä tapaus.
— On. Kyllähän me sitä kauan pelättiin, että tyttö jotakin tekee,
kun se oli niin onnettomana. Ei se saanut lohdutusta Jumalan
sanasta eikä mistään.
— Vainaja ei muuten taitanut olla kirjoissa tässä pitäjässä?
huomautti pastori.

Juha kertoo kaikki mitä tietää ja pappi tekee muistiinpanoja
hopeavartisella kynällä, jonka vihdoin laskee kädestään
tomuttuneelle, helmillä ommellulle kirjoitusmatolle… Mitähän tuo
isäntä nyt aikoo? miettii hän itsekseen katsellessaan Juhaa. Juha on
todella sen näköinen kuin hän aikoisi sanoa jotakin painavaa ja
juhlallista. Hän näyttää itse kärsivän sanottavastaan eikä pastori
suinkaan aavista mitään hyvää. Kai hän nyt vihdoinkin aikoo käydä
siihen puhdistustyöhönsä!
— Amanda! huutaa pastori ruokasaliin päin, — etkö sinä nyt jo tuo
sitä?
Vaikeapa isännän todella on päästä puheen alkuun! Hän asettaa
käsivarret polvilleen, kiertelee lakkia käsissään, oikaisee selkänsä
suoraksi, katselee Lutheruksen kuvaa ja tarkastaa ampumavehkeitä
seinällä. Mutta sanoja ei hän saa suustaan.
Kuinka merkillisesti kaikki kelpo ihmiset ovat toistensa näköisiä!
ajattelee pastori itsekseen katsellessaan Juhaa. Magnus Ståhle
esimerkiksi ja tuo talonpoikainen isäntä tuossa muistuttavat suuresti
toisiaan. Ties mistä syystä. Ne ovat sitä tulevaisuuden kansaa, ne
ovat ne, jotka perivät maan…
Amanda toi sisään pullon ja kaksi lasia. Ilman tarjotinta läiskäyttää
hän ne kirjoituspöydälle, jolla ennestään on kaikenlaista romua.
— Ikäviä ne sellaiset tapaukset ovat! saa Juha vihdoin suustaan.
— Ovat, vastaa pastori vakavasti.
— Onko pastori koskaan ajatellut syytä siihen, että tämä meidän
pitäjä on kuin pakanain maata?

— Lieneekö tämä huonompaa kuin muutkaan pitäjät! sanoo
pastori nauraen.
— Mutta eivät nämä tällaiset tapaukset todista kristillistä elämää.
Ja kun niitä sattuu niin usein. Ei ole siitä kauan, kun Immolan renki
tappoi oman veljensä ja Peräkulman kylän miehet rankkitiellä
heittivät yhden tovereistaan hankeen paleltumaan…
— No niin, hyvä naapuri, keskeyttää pappi. — Seurakunta on
sekalainen. "Antakaa molempain kasvaa elonaikaan asti, ja elonajalla
sanon minä elomiehille: kootkaa ohdakkeet lyhteisiin poltettavaksi,
mutta nisut korjatkaa minun aittaani", sanotaan Herran omassa
sanassa.
Pastori sai kun saikin ne sanat suustaan. Hänen mielenmalttinsa ja
kristillisyytensä palasivat vaistomaisesti paikalla, kun hän joutui
rahvaan kanssa tekemisiin. Joutsia vaikeni, nojasi käsivarret polviinsa
ja katseli saappaitaan.
— Mutta kun ei meidän seurakunnassa tunnu nisuja olevankaan,
puhui hän taas. — Kun kaikki onkin vain ohdakkeita.
— Noo, eiköhän siellä Joutsiassa liene nisujakin.
Pastori pani sanoihinsa aika paljon kärkeä ja Juha ymmärsi hänet
paikalla. Hän vilkaisi pastoriin ja tuli ihan punaiseksi.
— Mutta kyllä siihen kyllästyy, sanoi hän tiukemmin, — kun joka
päivä katselee sitäkin elämää Tulettänessä. Siellähän tämäkin
tyttöraukka tuhottiin.
Vai niin, ajatteli pastori, sinne sinä pyrit! Mutta ei pidä sinun
ainakaan sitä voittoa saada, että minä sinun edessäsi seisoisin

nolona.
— Jaa, jaa, sanoi hän. — Maailman pahuus on suuri. Mutta
kenenkä syy se on, että Tulettäne Joutsian maalle joutui? Vanhan
isännän! Minä kuulin omin korvin, kuinka hän rukoilemalla rukoili
kauppias Hallbomia ottamaan kestikievarinpidon haltuunsa. Ei se
kauppias ensinkään ollut tulossa, sillä ei se hollinpito kenellekään
huvia ole… No niin, ja kuka käskee ihmisiä räyhäämään
Tulettänessä?
Juha ei puhunut mitään. Keskustelu meni jo toiseen suuntaan kuin
hän oli tarkoittanut eikä hän tahtonut saada kiinni siitä kohdasta,
jossa se häneltä hämääntyi.
— Mutta, sanoa tokaisi hän äkkiä, — kyllä sentään papin pitäisi
koettaa olla esikuvaksi seurakunnalle.
Pastori katsoi häneen pitkään.
— Vai niinkös isäntä arvelee? Mutta pappikin on kai ihminen.
— Mutta ainaisen kanssakäymisen Jumalan kanssa pitäisi antaa
hänelle voimia seisoa kiusauksia vastaan.
— Mutta jollei kanssakäyminenkään auta?
— Silloin ei rukous ole rukoiltu hengessä eikä totuudessa…
Nyt se oli sanottu. Juuri tätä Juha niin kauan oli hautonut
mielessään ja vaikea sitä oli ollut sanoa. Hän loi pastoriin tutkivan
katseen ja odotti jännittyneenä, mitä tämä sanoisi. Mutta pastori ei
sanonut mitään, purskahti vain nauruun.

Ja nauroi kauan. Nauroi! Se hävetti Juhaa paljon enemmän kuin
jos hän olisi torunut. Sillä se todisti, ettei hän välittänyt hänen
sanoistaan vähääkään ja että hän vain teki hänestä pilkkaa.
— Seurakunta elää niinkuin pappi elää, jatkoi isäntä. — Vaikeata
se on, kun pitää taistella luontoaan vastaan. Se on niin, että
hammasta puree ja nyrkkiä pui. Mutta jollei sitä tee, niin sittenhän
menee kaikki, menee käsistä, menee talo, koti, menevät lapset…
Kaikki menee niinkuin Simolakin meni…
Äkkiä leimahti pastorin silmissä ja hän ojentui suoraksi ja kiivastui:
— Ihmisen ei koskaan pidä taistella luontoaan vastaan! Juuri siitä
tulevat kaikki erehdykset ja harha-askeleet. Se on suurin rikos,
minkä ihminen voi tehdä…
Isäntä tuijotti pastoriin suurin silmin ja hänen kasvonsa kalpenivat.
— Mitä pastori nyt…? Tietäähän sen mitä ihmisen paha luonto
tahtoo: se tahtoo juoda ja myllätä ja murhata… Mutta ihmisessä on
hyväkin luonto. Pitää taistella sen hyvän luonnon puolesta…
— Juominen ja myllääminen ja murha ei ole mitään
itsevanhurskauden ja tekopyhyyden ja lähimmäisen tuomitsemisen
rinnalla…
Väri palasi isännän kasvoille ja hän tunsi pastorin sanojen
tähtäävän suoraan itseensä. Pastorista henki tavaton uhma ja kosto.
He katselivat toisiaan hetkisen silmästä silmään ja hetki tuntui
pitkältä, vaikkei se ollut kuin silmänräpäys.
— Mutta, virkkoi isäntä vihdoin ikään kuin rukoillen, — jos ihminen
elää niinkuin pastori nyt sanoo oikeaksi, niin hänhän tuhoaa niin

paljon muita ihmisiä. Niinkuin nyt sen meidän piikatyttömmekin
kävi… Ja entä omaisten…! Kuinka…?
Juha vaikeni ja jäi tuijottamaan pastoriin. Tämä oli äkkiä
muuttunut ikään kuin häntä olisi lyöty kasvoihin.
Pastori istui kirjoituspöytänsä ääressä, kasvot vihertävää, likaista
lasiruutua vastaan. Lihava leuka riippui kappaleen matkaa alas
rintaa, kasvot olivat pöhöttyneet, tuuheissa mustissa hiuksissa
erottautui harmaita hapsia. Miehen yllä oli likainen paita, ylhäältä
puuttui nappi, takin rintapielet olivat tahroja täynnä. Jopa oli mennyt
surkeaksi Keihäsjärven ennen niin muhkea pappi!
Oli jo suuri päivä eikä tulta olisi enää tarvittu. Mutta kynttilä palaa
käryytti yhä kirjoituspöydällä kaikkinaisen kaman joukossa: siinä oli
likainen kaulus, olkaimet, koiran kaulahihna rautavitjoineen, kirjoja,
papereita, tupakantuhkaa ja pullo sekä kaksi lasia.
Pastori äänsi jotakin niin hiljaa, ettei Juha saattanut sitä kuulla.
Hänen silmänsä tuijottivat ikään kuin tyhjyyteen ja niiden reunat
olivat punaiset ja silmälaudat turvonneet.
Juhan tuli paha olla. Että hän olikin ruvennut lyömään noin lyötyä
miestä.
— Ei pastori pahastu, sanoi hän ja nousi. — Pastori taitaa olla
kipeä. Ei pastori pahastu. En minä ole tarkoittanut pahaa…
Pastorin outo, pingoittunut katse kääntyi isäntään, jonka kasvoilla
oli hyväntahtoinen hymy.
Kuinka sillä miehellä oli kirkas otsa! Kuinka sen kasvoihin oli
turvallista katsoa! Tuon hymyn lämmössä pastorin kasvot ikään kuin

sulivat.
— En minäkään, sanoi pastori vihdoin vastaukseksi isännän viime
sanoihin.
Juha yritti jo tulla ihan ymmälle, sillä ei hän käsittänyt pastoria.
Mutta samassa tuli pastorin kasvoihin iloa ja hän muuttui äkkiä aivan
ennalleen ja nousi reippaasti seisomaan. Ihmeellinen mies oli
pastori: vasta vallan lamassa ja äkkiä taasen pystyssä!… Isäntä oli jo
lähtemäisillään, mutta muisti samassa:
— Niin, minunhan vielä piti puhua hautaamisesta. Olemme
aikoneet haudata vainajan jonakin päivänä ensi viikolla. Tottahan
siihen taasen saa haudata kirkon aidan taakse, minne ennenkin on
haudattu tällaisia kuolleita. Kaipa ne nyt siksi saavat asian tutkituksi.
Huomenna pitäisi tohtorin tulla kaupungista. Kun vain saisi ruumiin
sulamaan siksi ja vähän siivotuksi sitä ruopasta. Jos pastori sitten
olisi hyvä ja tulisi pitämään sitä toimitusta. Jos sopisi vähän
puheessa lohduttaa niitä omaisia. Kyllä minä sitten pastorin vaivat
palkitsen. Kai niitä huomenna tulee tänne katsomaan. On tämä niille
kova kolaus, jos on kauhea sivullisillekin.
Nyt tulee kaikki takaisin, palaa koko se yöllinen painajainen!… Vai
pitää hänen tyttö vielä haudatakin… Taikka kuopata, sillä eihän
itsemurhaajia haudata… Hänen täytyy nähdä musta arkku
upotettavan kuoppaan kirkon aidan taa. Häpeässä täytyy toimituksen
tapahtua, ei sille raukalle lueta edes samoja lukuja kuin muille eikä
sen arkun päälle panna edes vihittyä maata.
Tässä toimituksessa ei pastori saata olla osallisena! Jumaliste ei!
Hän kuolee ja kaatuu siihen kuoppaan, jollei hän kuole jo
ennemmin… Vaikka kuolisi nyt heti… Hän kuulee kummallista ääntä.

On kuin mustat siivet taikka yölepakkojen nahkaiset evät kohisisivat
ja räpyttelisivät hänen ympärillään. Kuoleman musta enkeli varmaan
painuu hänen päällensä, katsoo häntä silmiin ja ojentaa kätensä
ottamaan hänen sydäntään… Eikä hänen tarvitse muuta kuin
puhaltaa, niin elämän liekki sammuu.
Isäntä on aikoja sitten mennyt. Mutta yhä istuu pastori tuolissaan,
pää painuneena korkeaa selkänojaa vasten.
— Mitäpä siitä, jos ne valallaan puhdistavatkin hänet epäluuloista
ja rangaistuksesta! Sen ne kyllä tekevät, sen hän kyllä uskoo: ovat
ne ennenkin tehneet vääriä valoja! Mutta jos tätä tällaista sieluntilaa
jatkuu, niin tämähän on helvetti maan päällä. Nyt hän näkee tällaisia
jo valoisana päivänä — entäs sitten kun tulee yö!
— Aamiainen on pöydässä, ilmoittaa Amanda ja tuo muassaan
paistetun sianlihan hajua.
Hän oli taasen kuullut uusia juttuja kuolemantapauksesta ja
latelee niitä nyt pastorille. Manu siellä Joutsiassa on sentään
merkillinen mies, kun hän tietää ihmisten kuolemankin edeltäkäsin.
Eräänä iltana olivat väet kaikki istuneet pirtissä puhdetöidensä
ääressä ja isäntäkin oli ollut siellä, mutta Sanna ei ollut — niin Manu
tuli äkkiä sisään, puisteli ruumistaan ikään kuin olisi palellut ja sanoi:
"Kuka taas kuolee, kun niin veistelee kirstunlautoja!" Miehet
rupesivat kaikki nauramaan, mutta tytöt kysyivät pelästyksissään:
"Missä?" "Tuolla riihen takana!" sanoi Manu. Siihen oli sitten Eprami
nauraa tirskunut, että "kaikkia turhia!" Mutta eipäs naura enää
kukaan. Kun se ruoppakuoppakin on riihen takana.
Pastori seisoi ruokakaapin ääressä, pullo kädessä ja otti ryypyn
toisensa perästä. Kun Amanda sen huomasi, karkasi hän kiinni

pulloon.
— Heretkää nyt jo! huusi hän. — Olette taas juovuksissa kun
pitäjäläisiä tulee. On sitä viidessäkin ryypyssä…
— Menetkö hiiteen, akka! huusi pastori takaisin ja uhkasi häntä
pullolla. — Minä juon niin paljon kuin tahdon.
Hän karkotti Amandan huoneesta ja söi ateriansa suurella melulla
ja kolinalla. Sitten hän pani maata ja nukkui päivälliseen asti.
Illansuussa kävi hän pirtit, navetat, hevostallit ja kyseli ja määräili,
niin että palvelijat hämmästyivät. Mutta se oli sellaista, ettei hän
tahtonut jäädä yksin. Ja yötä hän pelkäsi jo ennen kuin se oli
tullutkaan. Tulettänessä oli moni ilta tähän asti kulunut rattoisasti ja
hauskasti — sinne ei nyt ollut yrittäminenkään. Liljebladille — kuka
sinne olisi uskaltanut lähteä! Siellä tietysti olisi saanut kuulla kaikki
historiat uudelleen.
Mutta tottakin; tänäänhän on saunapäivä. Se on oikein
erinomaista, se.
Saunassa tulee ihminen terveeksi.
Pastori valmistautui erityisellä varovaisuudella vastaanottamaan
yötä. Hän ei illallisekseen syönyt siansorkkiakaan, joista hän niin
paljon piti, sillä hän tahtoi välttämättömästi saada unta. Ei hän
sentään ollut terve. Hän tunsi sen koko ruumiissaan. Varmaankaan ei
viikatemies ole kaukana…
Sillä Joutsian miehellä oli niin kirkas otsa. Niin, niin… Hän ja hänen
kaltaisensa ne lopultakin voittavat. Ne ovat niitä, jotka eivät ole
osanneet elää, mutta jotka sen sijaan osaavat kuolla. Ei niille
miekkosille koskaan tule sellaista painajaista kuin niille, jotka ovat

osanneet elämisen taidon. Hohoijaa, kuinka tämä Keihäsjärvi tulee
hyväntapaiseksi, jahka tästä viikatemies korjaa pois vanhan papin,
Jakob Sandin… Äää sentään. Mutta hän ei vielä ole korjattu, hän
elää vielä ja aikoo elää! Hän aikoo nukkua ja unen loppumattomasta
lähteestä ammentaa voimia elämiseen.
Uni, mustasiipinen, ja mustasilmäinen, joka kulkee hiipimällä ja
helistelee hiljaisuuden tiukua yössä, kuinka tervetullut se onkaan
onnettomien vuoteiden ääreen! Mutta se menee vain onnellisten luo,
niiden ylle levittää se laheat siipensä, niihin katselee syvine,
rauhallisine silmineen, niiden korvan juuressa helistelee se
hiljaisuuden tiukua. Yö, mustasiipinen, mustasilmäinen yö… ei se
armahda niitä, jotka sitä huutavat luoksensa! Ei se sinäkään kovana
yönä armahtanut Jaakko Sandia, Keihäsjärven pappia.
Hän väänteli, käänteli ja hikoili vuoteessaan.
— Kun en minä uskalla lausua sinun nimeäsi, sinä kaikkein
korkein! ähki hän.
— Editha, Editha! Sinäkin hylkäsit minut — tule edes sinä
luokseni…!
— Helena! Minä olin kova sinulle, mutta sinä annoit aina anteeksi
— armahda minua tällä hetkellä!
— Jos minä uskaltaisin huutaa sinua avukseni, sinä kaikkein
korkein!… Mutta minä en uskalla…!
Ei ääntä, ei hiiskahdusta vanhassa pappilassa. Jo alkaa kuulua
jotakin… On kuin joku kehräisi… kaukana… hyrisee, hymisee,
sihisee… Ääni kasvaa. On kuin puitaisiin jossakin hyvin etäällä…

Sahataan. Ja taas hyrisee, hymisee, jyrisee. Jo kuuluu selvästi, että
höylätään… Kirkas terä karkaa pitkin laudan pintaa ja lastukiehkura
lennähtää ilmaan. Terä kulkee itsestään, näkymätön käsi sitä vie…
Kirstunlautoja! Paljon kirstunlautoja! Tyttönen, joka
ruoppakuoppaan hyppäsi, ei paljoa tarvitse. Pieni oli tyttönen! Mutta
sillä oli suuri tukka. Koko sen pienen tyttösen saattaa peittää niihin
pellavankarvaisiin hiuksiin. Niin, ja entä sen lapsi…! Kirstunlautoja,
kirstunlautoja!… Höylä käy, höylä käy — hei! Lastukiehkurat lentävät
— hei! Kirstunlautoja suurelle miehelle! Suuri mies viedään kunnialla
hautaan — hei hei! hautaan! Vihittyä multaa kirstun kannelle — hei!
Kauniita puheita haudan partaalla — hei!… Tyttönen kuopataan
aidan taakse, missä siat tonkii ja vasikat tanssaa! Kirstunlautoja
isälle, äidille, lapselle! Kirstunlautoja kaikille kolmelle! Nimeen isän,
pojan, pyhän hengen, amen…!
Höylä käy vimmatusti, laulu kohisee kuin myrskyn pauhina: hei,
hei, hih, hei!
— Kun minä uskaltaisin huutaa avukseni kaikkein pyhintä nimeä!
lennähtää pastorin päähän kuin salaman välähdys. — Sitten minä
olisin pelastettu… Auta…!
Höylä käy yhä. Kirstunlautoja, kirstunlautoja! On kiire. Tyttönen
odottaa laudalla riihessä…! Suurta miestä ei lauta kannata, suuri
mies pannaan suoraan arkkuun. Hihhihhih-hei!
Tulikirjaimilla kirjoitettuna välähtää pastorin silmien edessä nimi,
jota ei hän ole uskaltanut lausua: Jeesus Kristus. Oi, jos hän saisi
sen vaikkapa kuiskatuksi, niin se auttaisi häntä ja hän pelastuisi!

Mustasiipinen enkeli katselee häneen… Sen sulat ovat laheat ja
suuret. Enkeli ojentaa kättään, ojentaa toista kättään, laskee ne
Jakob Sandin ohimoille… Ne ovat kylmät kuin jää ja ne hivelevät
polttavaa päätä. Enkeli katselee syvältä… Hiussuortuva valuu
olkapään yli… hiuksista valuu ruoppaa ja vettä…
Jakob Sand kokoaa viimeiset voimansa ja huutaa äärimmäisessä
hädässä:
— Jeesus Kristus!
Aamulla tavattiin Jakob Sand, Keihäsjärven pappi, hengettömänä
vuoteestaan.

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Oxygen Sensing Methods And Protocols Emily E Weinert

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Oxygen Sensing Methods And Protocols Emily E Weinert

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