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Heat Coagulation Test for Protein

In this heat coagulation test for protein post we briefly summarises about: principle, reagents requirements, heat coagulation test procedure, result, application and limitations of heat coagulation test.

Heat Coagulation Test for Protein

Heat coagulation test of protein is a biochemical test that determines the presence of proteins such as albumin and globulin in protein. Protein coagulation in response to heat is a regular occurrence. 

Denaturation and agglutination, or the separation of the denatured protein in a specific form, are the two steps of heat coagulation of proteins. When coagulable proteins are heated to their isoelectric pH, they undergo a sequence of modifications, including dissociation of subunits or the quaternary structure, uncoiling of polypeptide chains, and finally matting together of uncoiled polypeptide chains. 

Coagulation and flocculation are examples of visual manifestations of changes in proteins that occur during the denaturation process.

Objectives

Heat coagulation test used to determine the presence of proteins in a sample, as well as the presence of albumin, globulin, and other proteins in a urine sample.

Principle

The heat coagulation test is based on the structure of proteins changing as a result of heat and pH changes. When a protein is heated in an acidic media, specific links important for the protein’s tertiary and quaternary structure are broken, resulting in denaturation. 

When coagulable proteins are heated to their isoelectric pH, the polypeptide chains become uncoiled and clump together, forming an insoluble mass. The resulting mass does not dissolve back into liquid. 

The isoelectric point is where the coagulation process reaches its peak, and the mass of the coagulum differs based on particle size and protein concentration in the sample. Chlorophenol red, which changes the pH of the sample to the isoelectric point of albumin, is employed in the heat coagulation test of albumin and globulin. 

Acetic acid is also incorporated in the reagent for this test, which aids in the breakdown of peptide bonds in the protein molecule, allowing for easier coagulation.

Requirements

Reagent

1. Chlorophenol red indicator

2. 1% acetic acid

3. Urine Sample

Materials

1. Test tubes

2. Test tube stand

3. Pipettes

Procedure

1. Fill 3/4th of the test tube with urine Heat the upper 1/3 rd of the urine column by a small flame, so that lower 2/3rd will serve as control. Add a drop of 30 % (v/v) acetic acid to it.

Interpretation

Heat coagulation test of proteins

Heat Coagulation Test for Protein

White turbidity if disappears on addition of acetic acid indicates the presence of phosphates or carbonates.

If the white turbidity formed remains or appears or intensifies on adding acetic acid points towards the presence of albumin. Addition of acetic improves the formation of turbidity since the acidification brings the pH of the medium towards 4.7 (IEP of albumin).

Points to Remember

There are chances to miss presence of albumin in the urine if the pH of urine is high and it is not brought down by adding acetic acid. Isoelectric point of human albumin is 4.7.

Normal urine contains less than 250 mg per 24 hours and it escape detection by the usually employed methods. Pathologically different proteins detected in urine-albumin, myoglobin, fibrin and oxyhemoglobin.

The proteinuria is most commonly seen due to leakage of serum albumin since it is the most abundant and smallest protein in the serum.

Albumin most often appears in urine due to altered structure of glomerulus in various kidney diseases. Albumin may appear in urine by entering below the kidneys (not by glomerular filtration) from blood, exudates or lymph is called false albuminuria.

Benign proteinuria: It is transient and not associated with any kidney disaease. Occurs with severe exercise and cold bath. Orthostatic albuminuria: Albumin appears in urine after prolonged standing

Albustix: Is a stiff cellulose strip impregnated at one end with indicator tetrabromphenol blue buffered at pH around 3 which has a yellow color at pH 3.0. Presence of protein turns it into green – blue. Buffer maintains the pH at 3 and hence pH of urine do not interfere. If protein is absent, the color will be yellow. In the presence of protein the color varies from green to blue. Highly alkaline urine and stale urine (due to the formation of ammonia) may overcome the buffering action of the strip and give a false positive response.

Uses

1. The presence of albumin and globulin protein in the urine sample is detected using a heat coagulation test. Because albumin and globulin in urine are seen in a variety of clinical situations.

2. Heat coagulation test can be used to confirm their presence and aid in disease diagnosis. One of the most widely used procedures for detecting proteins in urine is heat coagulation test.

Limitations

1. Other coagulable proteins that may be present in the urine may cause the heat coagulation test to come up positive in some situations.

2. The pH of the solution must be close to the isoelectric pH of the proteins suspected to be present in the sample.

Further Readings

Reference

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