Denaturation:

1. Denaturation: • When a protein is heated,itsphysical,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 colourdevelops,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 coagulableproteins,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 coagulableproteins,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.