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During purification, it is necessary to determine the concentration of proteins in the crude extract as well as in purified fractions. There are different methods to determine the concentration of proteins, but the only accurate method to determine the protein concentration is to hydrolyze the protein sample and then carry out the amino acid analysis on hydrolysate.
However, the total amino acid analysis is time-consuming particularly if multiple protein samples have to be analyzed. Therefore, in practice, there are some quicker methods available to determine the protein concentration that gives a reasonably accurate result. These methods are based on colorimetric analysis on which a certain portion of protein solution is reacted with a reagent producing a colored product.
By measuring the amount of colored product spectrophotometrically, the concentration of protein present in the solution can be determined. However, none of these methods is absolute because the development of color is dependent on the amino acid composition of the protein.
Aromatic amino acids like tyrosine and tryptophan present in the protein exhibit an absorption maximum at 280 nm wavelength. However, the extinction coefficient of individual protein may vary with the variation in the aromatic amino acid composition of protein and extinction coefficient of most of the protein lie in the range of 0.4-1.5.
This method is nondestructive unlike other colorimetric analysis and samples in the cuvette analyzed by ultraviolet absorption can be used for further analysis. This is an important tool while working with a small amount of protein with an intention not to waste any sample.
There are some biomolecules that also give absorbance in the range of 280 nm wavelength like nucleic acid. Nucleic acids give 10 times more absorbance than proteins at 280 nm hence presence of small amount of nucleic acid can influence the absorbance significantly. So, to remove this influence, two readings of absorbance can be taken; one at 260 nm and another at 280 nm wavelength.
Now, protein concentration can be calculated in terms of mg per cm3, using the formula protein concentration (mg/cm3)= 1.55A280-0.76A260
This method has been used widely in the past to determine the protein concentration, but now a day it is replaced by more sensitive methods. Folin-Ciocalteu method is reasonably sensible to detect protein concentration below 10 mg/cm3 with relatively constant sensitivity over the different types of proteins. When Folin reagent (a mixture of sodium tungstate, molybdate, and phosphate) together with a copper sulfate solution is mixed with the protein solution, a blue-purple color is developed.
By measuring the absorbance at 660 nm, the protein concentration can be quantified. However, some care must be taken because compounds like Tris, zwitterionic buffers, and EDTA can interfere with protein assay and, therefore, must not be present in the proteins. This method of protein quantification is based on both Biuret reaction (where the peptide bonds of proteins react with Cu++ ions under an alkaline condition to form Cu+ which further reacts with Folin reagent) and Folin-Ciocalteu reaction.
This folin-Ciocalteu reaction is poorly understood and is thought to involve the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by copper-catalyzed oxidation of aromatic amino acids. This method is partly dependent on the tyrosine and tryptophan content of the proteins.
The Bicinchoninic Acid method
This method is similar to lowry method and also involved in the conversion of Cu++ to Cu+ under an alkaline condition, but the amount of Cu+ is detected using bicinchoninic acid. The bicinchoninic acid reacts with Cu+ forming colored complex, the absorbance of which can be measured at 562 nm wavelength. Using this method, protein concentration below 0.5 µg/cm3 can be determined and, therefore, this method is more sensitive than lowry method.
In this method, protein concentration is determined based on the colored complex formed when coomassie brilliant blue dye binds to the protein. At low pH, the free dye has maximum absorption at 470 and 650 nm, but when it is bound to the protein, it shows the maximum absorbance at 595 nm wavelength. The practical advantages of this method are as; reagent is easy to prepare, color develops rapidly and more stable.
Although this method is sensitive to measure the protein concentration below 20 µg/cm3 , it is a relative method only because the amount of dye binding vary with the content of basic amino acids in protein samples like arginine and lysine.