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 |
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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