ESTIMATION OF SERUM PROTEIN BY BIURET METHOD

AIM:
To estimate the serum total protein(Tp), Total Albumin(Ta), Total Globulin(Tg) & A:G
ratio by biuret method.
OTHER METHODS OF ESTIMATION:
Total protein estimation:
1.lowrys method
2.kjeldahls method
Total albumin estimation:
1.Dye binding method
PRINCIPLE:
The peptide bonds present in the protein react with copper sulfate in an alkaline
medium to form a purple/violet coloured complex. The intensity of this colour is
proportional to the number of peptide linkages present and thus is a measure of the
concentration of proteins.
REAGENTS:
1.28% Sodium sulfite
2.Standard protein solution
3.Dilute biuret reagent
(copper sulphate, sodium hydroxide, potassium iodide, sodium potassium tartarate)
4.Normal saline(0.9%)

PROCEDURE:
Preparation of Globulin free filtrate:
Pipette 0.2 ml of serum into a test tube. Add 5.8 ml of 28% sodium sulfite solution.
Allow to stand for 5 mins. Filter through Whatman No:44 dry filter paper. Use the
clear filtrate for estimation of albumin.
Albumin present in the sample is estimated by precipitate of globulin with 28%
sodium sulphite. The filtrate obtained contains only albumin which reacts with biuret
reagent to form violet colour complex.
The difference in protein content of whole serum and serum filtrate (albumin) after
sodium sulphite treatment give value of globulin. Measure the OD @ 540nm.
 Colour development:
Set up 4 test tubes marked B for blank, S for standard, A for albumin and Tp for total
protein. To B add 3 ml of water. To S add 3 ml of standard protein solution. To A add 3
ml of globulin free filtrate and for Tp add 0.1 ml of serum and 2.9 ml of distilled
water(D/W).
To all the four tubes add 3 ml of biuret reagent and mix well. After 10 mins read the
Optical Density using a green filter(540 nm).

REAGENTS BLANK STANDARD ALBUMIN TOTAL PROTEIN

Distilled water 3ml - - 2.9ml

Standard protein - 3ml - -

solution
- - 3ml -
Globulin free filtrate
- - - 0.1ml
Serum
3ml 3ml 3ml 3ml
Biuret reagent
Mix and after 10 mins read the OD values using green filter( 540nm)

OD @ 540 nm

CALCULATION:
1.Total protein in 100 ml of serum
𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
= X X 100
𝑂𝐷 𝑜𝑑 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑−𝑂𝐷 𝑜𝑑 𝑏𝑙𝑎𝑛𝑘 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝑂𝐷 𝑜𝑓 𝑇𝑝−𝑂𝐷 𝑜𝑓 𝐵 𝐶𝑜𝑛𝑐.𝑜𝑓 .𝑠𝑡𝑑

= X X 100 mg%
𝑂𝐷 𝑜𝑓 𝑆−𝑂𝐷 𝑜𝑓 𝐵 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝑂𝐷 𝑜𝑓 𝑇𝑝−𝑂𝐷 𝑜𝑓 𝐵 𝐶𝑜𝑛𝑐.𝑜𝑓 .𝑠𝑡𝑑 100

= X X g%
𝑂𝐷 𝑜𝑓 𝑆−𝑂𝐷 𝑜𝑓 𝐵 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 1000

2.Albumin in 100 ml of serum

𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
= X X 100
𝑂𝐷 𝑜𝑑 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑−𝑂𝐷 𝑜𝑑 𝑏𝑙𝑎𝑛𝑘 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝑂𝐷 𝑜𝑓 𝐴𝑙𝑏−𝑂𝐷 𝑜𝑓 𝐵 𝐶𝑜𝑛𝑐.𝑜𝑓 .𝑠𝑡𝑑

= X X 100 mg%
𝑂𝐷 𝑜𝑓 𝑆−𝑂𝐷 𝑜𝑓 𝐵 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

𝑂𝐷 𝑜𝑓 𝑇𝑝−𝑂𝐷 𝑜𝑓 𝐵 𝐶𝑠 100
= X X g%
𝑂𝐷 𝑜𝑓 𝑆−𝑂𝐷 𝑜𝑓 𝐵 𝑉𝑠 1000
3.Globulin in 100 ml of serum= Total protein – Albumin
=_____________g%
4.Albumin:Globulin ratio-

REPORT:
The amount of total protein in the given sample of serum is ___________g/dl
The amount of albumin in the given sample of serum is ___________g/dl
The amount of globulin in the given sample is ______________g/dl
A : G Ratio ________

CLINICAL SIGNIFICANCE:
Normal value:
Total serum protein = 6-8 g/dl
Albumin = 3.5-5.5 g/dl
Globulin =2-3.5 g/dl
A :G ratio = 1.2:1 – 1.5:1

CAUSES OF INCREASED SERUM PROTEINS:

 Dehydration
 Multiple myeloma (mainly globulin increases, albumin remains constant)
 Chronic infection
 Exercise
CAUSES OF DECREASED SERUM PROTEINS:
 Main cause of decreased serum protein is due to decreased albumin level.
 Protein energy malnutrition
 Increased catabolism of proteins
 Reduced protein synthesis
CAUSES OF DECREASED ALBUMIN:
1.Loss of albumin.
 Nephrotic syndrome
 Protein losing enteropathy
 Severe burns
 Severe hemorrhage
2.Defective anabolism.
 Liver disorders
 Malnutrition
 Carcinoma of stomach or pancreas
3.Increased catabolism.
 Thyrotoxicosis
 Trauma
 Uncontrolled diabetes
 Toxaemia of pregnancy
4.Defective absorption.
 Congenital malabsorption syndrome
 Malignancy of GIT
 Intestinal fistula
 Severe tuberculosis