Subject: Practical biochemistry

Telafer University – Collage of Nursing

Experiment NO: 3
Name of EXP : Estimation of Total Serum Protein .
Theory :
Proteins are nitrogenous compounds , have
high molecular weight ,the consist of a long
chains of amino acids (there are 20 L-alpha-
amino acids commonly found in protein) linked
by peptide bonds formed between the carboxyl
group of one amino acid and the amino group of
the other , which contain from 50 to many
thousands of amino acid.
proteins may be classified as simple and
conjugated proteins , according to their
structure , simple proteins such as albumins and
collagen . conjugated proteins such as
phosphoproteins and glycoproteins .
Also, proteins can be divided according to
the physical characteristics, to the fibrous
proteins such as keratin and globular proteins
such as hemoglobin .

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 Subject: Practical biochemistry

Telafer University – Collage of Nursing

Human body contains soluble and nonsoluble

proteins , clinical biochemistry deals with soluble
proteins which found in plasma , urine and
cerebrospinal fluids .
Sources of protein
1- Dietary: egg, milk, meat
2- Synthesized in the liver exept globulin
Classification of serum total protein
There are three major types of plasma
proteins , (albumin , globulin and fibrinogen) ,
globulin have four types (alpha 1- glob , alpha
2- glob , beta- glob and gama - glob) .
Hyperproteinemia:( increase the
concentration of total serum Protein).
 That most cases of high protein in the blood,
due to the dehydration, which occurs because of:
1- Decrease in water intake about 10 - 15 %
2- Loss of water as in increase vomiting.
 diarrhea .
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 Subject: Practical biochemistry

Telafer University – Collage of Nursing

 addisons disease.
 diabetic acidosis .
Hypoproteinemia: (decrease the concentration
of total serum Protein).
1- Nephrotic syndrome .
2- Malnutrition.
3- Chronic liver diseases.
4- Gastro - intestinal tract diseases.
5- Burns.
6- Extensive bleeding.
Normal range of plasma proteins:
Albumin (4.0 - 5.5) g / 100 ml serum
Globulin (2.2 - 2.7) g / 100 ml serum
Total Serum Protein (6.2 - 8.2) g / 100 ml serum
Methods for protein estimation:
1- Kieldahl method.
2- direct spectrophotmetric method.
3- Folin method .
4- Biuret method : Alkaline copper solution
reacts to give a blue to purple color with

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 Subject: Practical biochemistry

Telafer University – Collage of Nursing

substances containing two or more peptide

links. The color given by this reaction
varies appreciably for different proteins.

Biuret copper ion

The intensity of which being measured by

spectrophotometer at (550 nm) .
Working solution : Sodium hydroxide 3.8
mmol/l, potassium sodium tartarate 21 mmol/L,
potassium iodide 30 mmol/l, and copper sulfate
6 mmol/l .
Standard Solution : Consists of (6 gm/dL)
Protein .
Procedure : We take three test tubes, and follow
the following steps:

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 Subject: Practical biochemistry

Telafer University – Collage of Nursing

test tube

Protein in Standard Blank tube

the blood tube
(sample)
Material used

Reagent (Kit) 1 ml 1 ml
Serum 20 μl
Standard 20 μl
solution
Distilled 1ml
water

-After the additions, mix well and let stand for

10 minutes at room temperature .
-After that, the absorbance is measured by
spectrophotometer at (550 nm) .
Calculations :
Protein level gm/dl , calculated by the following
equation:
Protein concentration (gm/dl) = (A)sample  standard concentration
(A)standard (6 gm/dl)