Enzyme histochemistry

Enzyme histochemistry

1. Professor Emsley
Spring 2014

2. Definition of Enzymes
Enzymes are proteins that catalyse chemical reactions without them
being changed chemically.
E + S ES P +
(enzyme) (Substrate) (temporary combination) (Product)
E (Enzyme)
The primary use of enzyme histochemistry in the histopathology
laboratory is in the diagnosis of muscle disease.

3. A catalyst is a substance that changes the rate of the reaction of
chemicals without being consumed by the reaction.
A catalyst can either speed up ( positive catalyst) or slow down
(negative catalyst).
After the reaction, the catalyst is released to combine again with
other chemicals.
What is a Substrate?
A substrate is usually either an organic compound or ion becomes
more chemically active than it would normally be towards another
reactant.

4. Enzymes are usually very specific to their substrates.

5. 1. Enzymes are removed or destroyed by fixation, while others are sensitive
to freezing and thawing, so compromises have to be made.
2. Non-optimal substrate: sometimes optimal substrate concentration can’t
be obtained because of poor substrate solubility.
3. Non-optimal temperature: there is an optimal temperature for enzyme
activity, and sometimes, especially in azo dye simultaneous couple
techniques, a different temperature must be used. Enzyme activity is
usually destroyed at temperatures. greater than 56°C .
4. Non-optimal pH: most enzymes are best demonstrated at a pH near 7.0 ,
however, there are exceptions, as with acid and alkaline phosphatases.
5. Inhibitors: an excess of diazonium salts in the substrate, fixatives, heat
and some metallic ion may decrease or completely abolish enzyme activity.

6. Enzymes are specific as to the type of reaction they catalyze.
With the exception of ( trypsin and pepsin) enzymes are usually named by
adding “ase” to the specific substrate or molecule on which the enzyme acts.
There are 6 basic reaction classifications.
(a) Oxidoreductases, (b) transferases, (c) hydrolases, (d) lyases, (e)
isomerases, and (f) ligases.
Lyases add chemical groups to double bonds, isomerases rearranges
chemical groups on a substrate, ligases combine 2 substrates. These
enzymes are used in other reactions in the body, but are not enzymes for
which we stain in muscle system. The important enzymes in histopathology
and hematopathology are (A) Oxidoreductases, (B) hydrolases, and (C)
transferases.

7. A. Oxidoreductases: include I Oxidases:- use molecular oxygen
as the hydrogen acceptor with water forming.
II Peroxidases:- catalyze the oxidation of substrates by hydrogen
peroxide.
III Dehydrogenases:- remove hydrogen atoms ( a proton and
electron) from an organic substrate.
4H+ + 4e- + O2 2H2O

8. Hydrolases:- act on various substrates, usually through the addition of
water however, in some instances, water may be removed.
I. Esterases: - catalyze the hydrolyses of ester linkages; break the bond
between carboxylic acid and alcohol , phenols and napthols.
II. Phosphatases:- hydrolyze esters of phosphoric acid
III. Peptidases:- attack peptide bonds

9. Transferases:- transfer a functional group from one compound to another.
Phosphorylase:- transferases that transfer phosphate groups. Widely
distributed in animal and plant tissues and catalyze a number of reversible
reactions, leading to the synthesis and decomposition of saccharides.
Glycogen phosphorylase is the best known phosphorylase, catalyzing the
following reaction.
 glycogen + n ( H3PO4) n (glucose 1- phosphate)

10. In Vivo, phosphorylase is a cytoplasmic enzyme functioning only n
the degradation of glycogen.
In Vitro, muscle phosphorylase catalyze the synthesis and
hydrolysis of an unbranched polysaccharide of amylose type.

11. 1.For what purpose is enzyme immunochemistry techniques used?
2.What are the characteristics that distinguish skeletal muscles?
3.Can pathologic changes in muscle be seen in H&E stains?
4.Define Catalyst
5.Define Enzyme
6.What is meant by positive catalyst?
7.What is meant by negative catalyst?
8.What are enzymes?
9.What is a substrate?
10.List the 5 Properties that affect enzymes
11.Why is it necessary to fix tissue for enzyme demonstration?
12.How are muscle biopsies fixed?

12. 13.What is the usual way of classifying enzymes?
14.How many basic reaction classifications are there?
15.List the basic reaction classifications
16.What groups are of major importance in histopathology and
hematopathology?
17.Define hydrolases
18.What are the subclasses of hydrolases?
19.Define oxidoreductases
20.What are the subclasses of oxidoreductases?
21.Define Transferases
22.What is the subclass of Transferases?