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Construction of Protein Standard Curve using Folin’s Lowry Method
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Objective: 

 

To construct a standard curve to determine the specific activity of the beta amylase enzyme.

 

 

Theory :


Lowry’s assay for total protein is one of the most commonly performed colorimetric assays. This procedure is sensitive because it employs two colour forming reactions. It uses the Biuret reactions in which Cu2+ in presence of a base reacts with a peptide bond  of protein under alkaline conditions resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+),  and Lowry’s reaction in which the Folin Ciocaltaeu reagent which contains phosphomolybdic complex which is a mixture of sodium tungstate, sodium molybdate and phosphate,  along with copper sulphate solution and the protein, a blue purple colour is produced which can be assessed by measuring the absorbance at 650-700nm.

 

 The blue purple color is formed due to the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper catalysed oxidation of aromatic amino acids tryptophan and tyrosine. Thus the  color intensity depends on the amount of these aromatic amino acids present and will thus vary for different proteins.

 

The measurement of protein with  Folin's reagent has certain advantages. Firstly,  it is a sensitive assay which  requires no digestion.  Secondly, It is 10 or 20 times more sensitive than measurement of the ultraviolet absorption at  280 nm and is much more specific and much less liable to disturbance by turbidities, thirdly,  it is several fold more sensitive than the ninhydrin reaction  and is  simpler, as well as much easier to adapt for small scale analyses. Also it is 100 times more sensitive than the biuret reaction.

There are  disadvantages also for the Folin's reaction, like the amount of color varies with different proteins,  it is lessconstant than the biuret reaction, but more constant than the absorption at 280 nm.  The color is not always  proportional to concentration.Inspite of all these concerns, folin's method can be used in measurement of protein during enzyme fractionations, mixed tissue proteins, measurement of very small absolute amounts of protein, or  highly diluted protein  and  analyses of large numbers of similar protein samples.

 

Most protein estimation techniques use Bovine Serum Albumin (BSA) as a standard protein, because of its easy availability and  low cost with improved purity.  Constructing a  protein standard curve is of prime importance in studying the activity of an enzyme as this analysis relies on accurate quantitation of protein concentration.
 

 

 

 


 

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