Thursday, December 17, 2015

Qualitative and qualitative estimation of Proteins



Proteins are the most abundant biomolecules, constituting more than 50% of the dry weight of the cell. They are known to perform many different biological functions.  Qualitative and quantitative estimation of proteins in biological fluids is based on the physico-chemical properties of proteins.

Denaturation of proteins

In each type of protein, the three dimensional structure is essential for its biological function.  A protein in the biologically active form is termed as the native protein and the native state is called “optimum” state. The disruption of the three dimensional structure is called denaturation.  On denaturation, the primary structure remains intact.  It is an all or none phenomenon and does not cause any alteration in the quantity of protein present.

Characteristics of denaturation

Following are the major changes involved in denaturation:
1.       Increase in the viscosity due to uncoiling
2.       Change in the isoelectric pH
3.       Changes in the properties of solubilization and crystallization
4.       Loss of biological activity.

Denaturing agents

1.       Heat
2.       Mineral acids or alkalis alter the ionisation of COOH- and NH2 groups,  destroying the salt bridges
3.       Vigorous shaking or grinding causes mechanical disruption of various bonds
4.       Ultraviolet radiation and Ultrasonic waves.
5.       Chemical agents like urea and certain acid and guanidine derivatives that    cleave H-bonds    

Heat Test


Based on principle of heat coagulation and precipitation of proteins

Procedure: Fill half the test tube with urine and heat the top 1/2 of the sample. Look for any turbidity at the upper part of the tube by comparing with the lower part of the tube. If any turbidity appears, add 2 drops of 33% acetic acid. (Acidification is necessary because in alkaline medium heating may precipitate phosphates).  If the precipitate is due to proteins, it will increase on acidification and if it is due to phosphates, it will dissolve again.       


Color reaction of proteins and amino acids

Proteins react with a variety of reagents to form coloured products, which can be measured colorimetrically. They are important for qualitative and quantitative estimation of protein and their constituent amino acids.

Ninhydrin reaction:  It is one of the most important reactions used for the qualitative detection of hydrolytic products of protein i.e. amino acids.  All amino acids give the ninhydrin reaction.                                                                

Principle: The amino acids are deaminated to CO2 , NH3 and an aldehyde under heat.  Ninhydrin reacts with NH3 and forms a purple coloured complex, which can be measured at 570 nm.  All amino acids, except proline, react with ninhydrin at ambient temperature to form a blue colored complex, which intensifies to purple on heating.  Proline forms a yellow coloured complex.
                                                                 
Ninhydrin  +  Amino acid ------------------------------>  Hydrindantin  +  Aldehyde + CO2 + NH3


Ninhydrin   +  Hydrindantin  +  NH3 ----------------------> Purple colored complex
                                                                                    (Ruhemann’s purple) 


Fig. Biruret test postive
Biuret reaction: When urea is heated to 180oC, it forms biuret which in the presence of strong alkali reacts with dilute solutions of copper sulphate to form a violet coloured complex.  Proteins and peptides give this reaction.  Presence of at least two peptide bonds is essential for the reaction.  Proteins give violet or purple colour while proteoses and peptones give light pink colour.  Histidine gives a positive Biuret reaction. Two or more - CONH2 groups joined directly or though a carbon or nitrogen atom gives this reaction.

Absorption maximum of the colored complex is 540 nm. Since the method is based on reaction with peptide bonds, it is an absolute one.  The main disadvantage is its lack of sensitivity.  It cannot be used to estimate protein less than 1 mg/ml.  Amino acids and dipeptides do not give this reaction.

Bradford Method: Coomassie Brilliant Blue complexes with protein (Absorption maxima - 595 nm). This method is simple and very sensitive.  Colour development is rapid but not very stable.  Sensitivity is 20 µg/ ml.

Lowry’s Method: This is also a sensitive method.  It involves the formation of a copper-protein complex in alkaline solution and the reduction of phosphotungstic and phosphomolybdic acids by the copper-protein complex into tungsten blue and molybdenum blue respectively.  λmax 660 nm.  Sensitivity is 10 µg/ ml.

Separation of amino acids:  The two most commonly used methods are:

Electrophoresis: Amino acids can be separated in an electric field, on the basis of the charge they carry.

Chromatography: This is a technique by which molecules are separated on the basis of their charge, molecular weight or specific ligand affinity.



   
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