for the students of MBBS , BDS & BSMT SUBJECT: medical biochem

1. Denaturation:
 When a protein is heated,its physical,chemical and
biological properties are changed due to breaking
up of certain bonds.the conformation of protein
will be changed.this process is known as
denaturation.
However,when the coagulable proteins are
heated at their isoelectric pH,a series of changes
occur involving dissociation of the protein
subunits(disruption of quaternary
structure),uncoiling of the polypeptide
chains(disruption of tertiary and secondary
structure) and matting together of the uncoiled
polypeptide chains(coagulation).

2.  While a denatured protein may be restored to its
original structure & function by certain
manipulations,coagulation is an irreversible process.
Coagulation is maximum at isoelectric pH.
 ISOELECTRIC pH:
the pH at which a protein possesses no net
charge is called its isoelectric point or Ph. At this pH
protein is electrically neutral.

3. Principle:
 When a protein is heated in acidic medium ,it
becomes denatured due to breaking up of certain
bonds. However when the coagulable proteins are
heated at their isoelectric pH,its polypeptide chains
becomes uncoiled and matted with each other to form
an insoluble mass.the process of coagulation is
maximum at the isoelectric pH of the protein. While a
denatured protein may be restored to its original
structure and function by certain manipulations,
coagulation is an irreversible process.

4.  Chlorophenol red is used to adjust the pH of solution
to the isoelectric pH of albumin which is about 5.4.
chlorophenol red gives a pink colour at this pH, a
purple red colour above it and a yellow colour below it.
 Acetic acid present in the reagents breaks down
peptide bonds on heating.

5. Reagents:
 Chlorophenol red indicator
 Acetic acid 1 %

6. Procedure:
 Fill two third of test tube with the given solution .
 Add 1_2 drops of chlorophenol red indicator drop by
drop and mix.
 When purple red colour develops,add 1 % acetic acid
drop by drop untill the colour changes to faint pink.
 Hold the test tube near its bottom,incline it slightly
and heat the upper portion of the fluid.

7. Interpretation:
• if heat coagulable proteins,albumin and globulin are
present,a dense coagulum will be formed in the upper
part of the solution.it may be compared with the lower
part of the solution which serves as control.
• In case of non coagulable proteins,gelatin,proteoses,
peptones and polypeptides, no coagulum will be
formed in the upper part of solution i.e they give
negative coagulation test.

8. Practical application:
 This is the most widely used test for the detection of
proteins in the urine.
 The proteins which appear in pathological conditions
in urine are albumin and globulin since both are heat
coagulable,their presence can be conclusively
established by this test.