Biochemistry of Proteins and Amino Acids.ppt

This is an explanation or slides regarding biochemistry of proteins and amino acids.

At first, the monomer of Proteins is explain the different Amino Acids particularly the different essential amino acids

1. CHAPTER 10
PROTEINS

2. Objective
At the end of this chapter, the student is expected to:
•Describe basic protein structure and composition
•Define amino acids
•State and describe the four stages of protein structure
•Show how protein properties can be used for their
identification and assay
•List the proteins synthesized by the liver
•Describe the physiological functions of proteins
•Describe how plasma protein abnormalities reflect
severity of hepatic dysfunction
•Describe the methods used for determination of protein
concentration in blood

3. Outline of protein lecture
•Introduction
•Classification of proteins
•Functions of proteins
•The plasma proteins
•Plasma proteins with clinical significance
•Properties of proteins
•Specific methods for the determination of proteins
•Serum protein electrophoresis
•Samples
•Interpretation
•Quality control
•summary

4. Introduction
Proteins are polymers of amino acids that are
linked covalently through peptide bonds.
Aminoacid: an organic cmpound containing both
amino and carboxyl functional groups; simplest
units of proteins
There are 20 different kinds of amino acids,
combined in different proportion and
arrangements to build all protein molecules
When only two amino acids combine by peptide
bond ,it is called dipeptide, when amino acids
involved in the bond formation become 3, 4, 5
they are named as tri-, tetra-, and penta-
peptides respectively.

5. Proteins, continued..
All proteins contain carbon, hydrogen,
oxygen, and nitrogen; some proteins may also
contain sulfur phosphorous, copper, iron, zinc,
iodine, and other elements.
 The presence of nitrogen in all proteins sets
them apart from carbohydrates and lipids.
The average nitrogen content of proteins is
approximately 16%.

6. Proteins, continued..
Comprises 50-70% of cell’s dry weight
Found in cells, as well as in all fluids,
secretions, and excretions
more than 300 different types of plasma
proteins are discovered

7. Classification
Protein classification
Proteins may classified based on what they are built from
as:
•simple proteins
•complex proteins :
- apoproteins,
- conjugated proteins

8. Protein classification, continued….
•Based on their shape proteins can be classified
as fibrous proteins and globular proteins
•Proteins have four levels of structure
-Primary structure
-Secondary structure
-Tertiary structure
-Quaternary structures.

9. Function of Proteins
•used to construct or build our body
•catalyze biochemical reactions as an enzyme
•regulate body metabolism as hormones
•protect our body from foreign body attack as an
antibody and components of complement
•maintain osmotic pressure in plasma
•Transport different lipids, minerals, hormones, vitamins
etc as hemoglobin, apolipoprotein, albumin etc
•assist to arrest bleeding and maintain homeostasis as
coagulation factor

10. Plasma proteins
•Many different proteins are present in the blood,
and collectively known as plasma proteins.
• They include Albumin, Alpha
1Acid
glycoproteins, ceruloplasmin, C-reactive protein,
complements, fibrinogen and immunoglobulins.
•Most of plasma proteins are synthesized and
catabolized in the liver.

11. Clinical significance of protein
•The two general causes of alteration of serum
total protein are:
change in volume of plasma water
change in concentration of protien
The relative hypoproteinemia --hemodilution.
The relative hyperproteinemia-hemoconcentration.

12.  
Plasma proteins with Clinical significance
Albumin
•the most abundant plasma protein extra vascular
body fluids, including CSF, Interstitial fluid, urine,
and amniotic fluid.
•accounts approximately one-half of the plasma
protein mass.
• a globular protein, with molecular mass of 66.3
KD.
•Because of its high net negative charge at
physiological pH, highly soluble in water, but
does not have carbohydrate side chain.

13. Functions of albumin
•maintaining the colloidal osmotic pressure in
both the vascular and extra vascular space with
continuous equilibration in between
•Binding and transportation of large number of
compounds, including free fatty acids,
phospholipids, metallic ions, amino acids, drugs,
hormones and bilirubin.

14. Clinical significance
Cause for an increase level of albumin
• acute dehydration and has no clinical
significance.

15. Decreased levels of albumin seen in..
•Edema and ascitis
•Analbuminemia
•Urinary loss
•Inflammatory conditions
•Gastrointestinal loss
•Hepatic disease
•Protein energy malnutrition

16. Alpha
1 –fetoprotein [AFP]
• one of the first α –globulin appear in mammalian
sera during development of the embryo
• Dominant serum protein in early embryonic life
• synthesized primarily by the fetal yolk sac and
liver..
• contains approximately 4% carbohydrate with a
molecular mass approximately 70KD.

17. Clinical significance
High AFP levels seen in:
•open neural tube or abdominal wall defect in
fetus.
•Multiple fetuses,
• fetal demise,
•fetomaternal bleeding, and
•incorrect estimation of gestational age
Hepatocellular and germ cell carcinomas in
childhood and adults

18. C-reactive protein
•The first APPs to become elevated in inflammatory
diseases
•consists of five identical subunits and is synthesized
primarily by liver.
•C-reactive protein (CRP) found in sera of acutely ill
individuals from S. pneumonia
•CRP activates the classic complement path way
starting at C1q and initiates opsonization,
phagocytosis, and lysis of invading organisms. such
as bacteria and viruses.

19. CRP continues…
•CRP can recognize potentially toxic autogenous
substances released from damaged tissue, to
bind them, and then detoxify or clear them from
the blood.

20. Clinical significance
CRP levels usually rise after
•myocardial infraction, stress, trauma, infection,
inflammation, surgery, or neoplastic proliferation.
CRP is clinically useful for
•Screening for organic disease
•Assessment of the activity of inflammatory
diseases

21. •Detection of inter-current infection in systemic lupus
erythematosus (ALE), in leukemia, or after surgery\
•management of neonatal septicemia and meningitis
• Cord blood normally has low CRP concentration,
but in intrauterian infection, the concentration will be
high.

22. properties protein
•Molecular size
•Differential solubility
•Electrical charge
•Adsorption on finely divided inert materials
•Specific binding to antibodies, coenzymes, or
hormone receptors

23. Specific methods for total protein determination.
• Biuret method
•Direct photometric methods.
•Dye-binding methods.
•Turbidimetric and nephelometric methods
 

24. Biuret method
Principle of the test
•peptide bonds react with Cu
2+
ions in alkaline
solutions to form a colored product
•absorbance is measured spectrophotometrically
at 540nm.

25. Biuret, continued….
•The biuret reaction occurs with other compounds
with structural similarity.
•One copper ion probably is linked to 6 nearby
peptide linkage by coordinate bonds.
• Amino acids and di peptides do not react, but tri
peptides, oligo peptides, and polypeptides react
to yield pink to reddish- violet products.
•The intensity of the color produced is
proportional to the amount of protein present in
the reaction system.
•Detect between 1 and 15 mg of protein in the
aliquot measured, an amount present in 15 to
200 μl of a serum containing protein at 7gm/dl.

26. Biuret, continued..
Specimen
type, source of errors, and preservation
Either serum or plasma, but serum is preferred.
A fasting specimen may be required to decrease the
risk of lipemia.
 Ammonium ions interfere
Hemolysis should be avoided.
Serum samples are stable for atleast 1week at room
temperature and for 1 month at 2 to 4
o
C.
Specimens that have been frozen and thawed should
be mixed thoroughly before assay.

27. Direct photometric methods.
Principle of the test.
Absorption of UV light at 200-225 nm and 272 –
290 nm is used
Absorption of UV light at 280 nm depend on the
aromatic rings of tyrosine and tryptophan
Peptide bonds are responsible for UV absorption
(70% at A
205) ;
Specific absorption by proteins at 200 to 225 nm
10 to 30 times greater than at 280 nm.

28. Limitations of the direct photometric methods
Accuracy & specificity suffer from
uneven distribution of tyrosine and tryptophan
among individual proteins
the presence of free tyrosine and tryptophan, uric
acid, and bilirubin, which also absorb light near
280nm.
interferences from free tyrosine and tryptophan is
significant at 200 to 225nm.
A 1:1000 or 1:2000 dilution of serum with sodium
chloride,0.15 mol/l ,circumvents this interferences.

29. Dye-binding methods
Principle of the test
Based on the ability of proteins to bind dyes
such as amido black 10B and Coomassie
Brilliant Blue.
The method is simple, easy, and linear up to 150
mg/dl.
assay of total protein in CSF and urine uses
CBB G-250

30. Limitation of dye binding methods
unequal affinities and binding capacities of
individual proteins for dyes
 inability to define a consistent material for use
as a calibrator.

31. Turbidimetric and nephelometric methods
Principle of the test
Protein in the sample is precipitated with
addition of sulfosalicylic acid alone, with
sulfosalicylic acid in combination with sodium
sulfate or trichloroacetic acid (TCA), or with TCA
alone to produce turbidity.
Degree of turbidity measured with
Turbidometeric or nephelometric methods

32. Assay Techniques for serum albumin
Dye-binding methods
Salt fractionation or the 'salting-out'
procedure
By difference
Electrophoresis
Immunochemical techniques.

33. Dye binding method
BCG
Method
Test
principle:
Albumin and BCG are allowed to bind
at pH 4.2, in succinate buffer,
absorption of the BCG-albumin complex is measured
at 628 nm.
At pH 4.2, albumin acts as a cation to bind the
anionic dye.
The reaction is extremely fast and goes to
completion in only a few seconds.

34. Reference Range
Adult serum albumin
Recumbent: 3.5 - 5.0 g/dl
In the upright position levels are about 0.3 g/dl
higher because of hemoconcentration.

35. Source of error and remedy
hyperlipemia
 hyperbilirubinemia
hemolysis
can generally be eliminated (minimized) by
dilution of serum 1:250

36. BCP
Method
Test
principle:
Yellow BCP dye, buffered at pH 5.2 with
acetate
turns green when complexed with albumin.
 Absorbance of the green complex is
measured at 603 nm.

37. Specimen
serum is recommended
Results tend to be erroneous if the overall
serum protein pattern is abnormal

38. Methods for the determination of total globulins
Methods for the quantitative determination of
total globulins
Colorimetric method
Globulin by difference
Electrophoresis
Immunochemical technique

39. Colorimetric method
Test
principle:
 glyoxylic acid reacts with tryptophan residues
of proteins to form a purple color.
Copper sulphate is added to enhance color
formation.
human globulins are known to contain 2 - 3%
tryptophan

40. Serum Protein Electrophoresis
•Electrophoresis is widely used in clinical
laboratories to study and measure the protein
content of biological fluids- serum, urine or csf.
•Screening tool for prtein abnormalities
•Electrophoresis techniques include:
–Cellulose acetate electrophoresis
–Gel and capillary electrophoresis
–Specialized techniques termed western
blotting, immunofixation, and two-dimensional
electrophoresis

41. Methodology for Protein Electrophoresis
Patient’s specimen is placed into a sample
trough within agarose gel, is placed in an
alkaline buffer solution
 a standardized voltage is applied and
allowed to run for 1hr
 the agarose gel is processed in acetic
acid and alcohol washes to fix the
proteins in the agarose.
 the protein fractions are stained with
Coomassie Brilliant Blue protein stain.

42. After a second wash, fixed protein bands
can be visualized and quantified with
densitometry.
In normal serum electrophoresis 5-6
bands are visible:
Albumin
Globulins: α1-, α2-, β-, and γ-

43. Materials and procedures of protein
electrophoresis
Buffer: barbital with an ionic strength of 0.05 and
pH 8.6
Sample volume: 3 to 5 µl
Power supply: 1.5 mA per 2-cm width of cellulose
acetate medium; 10mA per 1-cm width of agarose
medium
Run time:40 to 60 min producing a 5- to 6-cm
migration distance for aalbumin

44. Normal serum protein electrophoresis pattern
+ -
Albumin
1 
2  

45. Specimen for electrophoresis
•Serum
•CSF
•Concentrated urine

46. Interpretation of Results
Reference Range of total protein
–Serum---------------------------6-8 g/dl
–CSF----------------------------- 8-32 mg/dl
For electrophoresis
-serum: albumin-----------------3.9-5.1 g/dl
α1-globulin------------0.2-0.4 g/dl
α2-globulin------------0.4-0.8 g/dl
β-globulin--------------0.5-1.0 g/dl
γ-globulin---------------0.6-1.3 g/d
Compare the patient results with the reference range to
assess for hyper- or hypoglycemia

47. Quality Control
A normal & abnormal quality control sample should be
analyzed along with patient samples, using Westgard or
other quality control rules for acceptance or rejection of
the analytical run.
–Assayed known samples
–Commercially manufactured
Validate patient results
 Detects analytical errors.

48. Documentation of protein
Results
•Record patient results in result logbook
•Record QC results in QC logbook
•Retain records for recommended time

49. summary
Proteins are polymers of amino acids that are linked covalently
through peptide bonds.
The presence of nitrogen in all proteins sets them apart from
carbohydrates and lipids.
Proteins are classified based on the number of amino acid
molecules ,composition of amino acids.
Protein have four structural levels;1
0
,2
0
,3
0
,and 4
0
.
Properties of proteins include molecular size, differential
solubility, electrical charge, adsorption on finely divided inert
materials, and specific binding to antibodies, coenzymes, or
hormone receptors

50. Summary, continued…
Proteins function includes building our body , serving as
enzymes, as antibody. etc..’
Major plasma proteins include Albumin, Alpha1Acid
glycoproteins, ceruloplasmin, C-reactive protein, complements,
fibrinogen and immunoglobulins
Increase level of protein caused by acute dehydration and has
no clinical significance; decreased levels of proteins seen in
edema and ascitis, analbuminemia, urinary loss, inflammatory
conditions, gastrointestinal loss, hepatic disease, protein energy
malnutrition.
Specific methods for total protein determination include Biuret
method, direct photometric methods, dye-binding methods,
turbidimetric and nephelometric methods

 Serum protein electrophoresis used to fractionate proteins

51. Reference
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. Philadelphia,
2001.
2. Arneson, W and J Brickell: Clinical Chemistry:
A Laboratory Perspective 1st ed. 2007 FA Davis

3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
textbook of Clinical Chemistry. Philadelphia, 1999.

52. End of Clinical Chemistry I