ESTIMATION OF SERUM PROTEIN BY BIURET METHOD

AIM:
To estimate the serum total protein(Tp) 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.Standard protein solution
2.Dilute biuret reagent
(copper sulphate, sodium hydroxide, potassium iodide, sodium potassium tartarate)
3.Normal saline(0.9%)

PROCEDURE:
Colour development:
Set up 3 test tubes marked B for blank, S for standard, and Tp for total protein. To B
add 3 ml of water. To S add 3 ml of standard protein solution 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 TOTAL PROTEIN

Distilled water 3ml - 2.9ml

Standard protein - 3ml -

solution

Serum - - 0.1ml

Biuret reagent 3ml 3ml 3ml

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

REPORT:
The amount of total protein in the given sample of serum is ___________g/dl

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