Heat coagulation test of proteins definition
The test for heat coagulation of proteins is a test in biochemistry to find whether there are proteins such as albumin and globulin found in proteins. The coagulation of proteins in reaction the heat stimulus is an typical phenomenon. The process of coagulation by heat of proteins happens in two phases; denaturation or Agglutination, or the dissociation of the protein denatured in the form it is in. The coagulation process involves heating proteins at their isoelectrical pH, there are a variety of changes that occur to proteins, dissociation of subunits or Quaternary structure, the uncoiling of the polypeptide chains, eventually creating the matting of polypeptide chains that are not coiled. The processes of coagulation and flocculation are merely visible manifestations of the changes that happen within proteins during denaturation.
Protein coagulation is an irreversible process which occurs at the highest pH of proteins. Proteins that are coagulated by heat are an important test used in clinical trials to detect proteinuria. It is easy and less time-consuming. Both quantitative and qualitative evaluation of proteins is performed using the method of heat coagulation. The quantitative evaluation of coagulation may be done by measuring the amount of coagulum that forms in the tube.
Objectives of Heat coagulation test of proteins
- To identify the presence of proteins in a particular sample.
- To identify whether there is albumin and other proteins found in urine samples.
Principle of Heat coagulation test of proteins
The basic principle behind the heat coagulation is the modification in the protein’s structure because of temperature and change in pH. Heating a protein in an acidic environment causes the denature of proteins due to breaking certain bonds that are responsible for the tertiary and quaternary structures of proteins. In cases of proteins that are coagulable when they are heated to their isoelectric pH and the polypeptide chains break, they become uncoiling and adhere to each and form unsoluble masses. The mass that is formed does not dissolve back into the liquid. This process occurs maximal at the isoelectric point and the weight of the coagulum could vary depending on the size of the particle and the level of protein that are present in the test sample. In the test of heat coagulation of globulin and albumin chlorophenol red is employed, that adjusts the pH level of the test to that of the isoelectric point for albumin. The reagent used for this test also contains acetic acids, which aids in breaking of the peptide bonds in the protein molecules which facilitates the process of coagulation.
Requirements for the test
Reagent
- Chlorophenol red indicator
- 1% acetic acid
- Sample
Materials required
- Test tubes
- Test tube stand
- Pipettes
Procedure of Heat coagulation test of proteins
- Around two-thirds of the testing tube contains the sample.
- To the test tube 1 or 2 drops of red chlorophenol indicator is dropped drop by drop, and then mixed thoroughly.
- If a purple hue is detected on the test tube the acetic acid 1% will be added drops by drops until it shifts to a pale pink.
- It is tilted slightly to increase the temperature of the upper layer of the liquid.
- The tube is examined for the development that of the coagulum.
Result and Interpretation of Heat coagulation test of proteins
- Positive result: A positive outcome of the heat coagulation test can be manifested by the creation an extremely dense coagulum on the top of the mixture. The lower portion of the solution functions as a buffer.
- Negative result: Negative result of the heat coagulation test can be indicated by the absence of coagulum in the upper layer. This means that there is no albumin as well as other proteins in urine.
Uses of Heat coagulation test of proteins
- This test can be used to identify whether there is albumin as well as globulin proteins in the urine sample. Since albumin and globulin found in urine can be detected under various ailments and conditions, their presence can be definitively confirmed by this test. It assists in diagnosing ailments.
- This test is among the most frequently used methods to detect protein in urine.
Limitations
- In certain situations tests, it could yield a positive outcome for other proteins that could be found in urine.
- It is crucial to ensure that pH be within the range of the isoelectric pH of proteins that are suspected to be present within the specimen.
Precautions:
- Clean the equipment prior to and after conducting experiments.
- Be sure to handle all chemicals used during the experiment.
