Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

compound biuret, and therefore the reaction is called the biuret reaction. Amino
acids and dipeptides do not give this reaction, but tri- and polypeptides can react. In
biuret reaction, one copper ion is linked to 4–6 nearby peptide linkages by coordi-
nate bonds. The intensity of color produced is proportional to the number of peptide
bonds undergoing reaction.

NH 2

NH 2

NH 2

NH

NH

NH 2 NH 2

NH 2

NH 2

NH 2

NH 2

NH 2

NH 2

NH 2

HN

NH HN

O NH 3

OC

C

C

C

C

C

CC

C

C

C

O

O

O O

O

O

O

O

O

180 °C

Urea (in excess) Biuret

Cu Complex

+

Cu2+ Cu2+

10.3 Specimen Requirements

Both serum and plasma may be used for protein estimation, but serum is preferred
over plasma. A fasting specimen may be desired to decrease lipemia since
hemolyzed and lipemic samples strongly interfere with the protein measurement
by biuret method. Samples remain stable for 24 h if stored at room temperature.

10.4 Reagent

1.Biuret reagent–Dissolve 9 g of sodium-potassium tartrate in 500 ml of 0.2 N
NaOH solution. To this add 3 g of CuSO 4 previously dissolved in 100 ml distilled
water, gently with constant mixing. Then dissolve 5 g of potassium iodide, and
makefinal volume up to 1 liter with 0.2 N NaOH.
Sodium-potassium tartrate is used as a complexing agent to keep the copper in
Cu2+state in the solution. Potassium iodide is added to prevent auto-reduction.
NaOH provides alkaline medium.
2.Biuret blank: Prepare same as biuret reagent with all reagents except CuSO 4.
3.Protein standard (10% BSA):Dissolve 10 g BSA in 100 ml normal saline.

44 10 To Estimate the Amount of Total Protein and Albumin in Serum and to Find...