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1. Biuret method, in which a copper ion (Cu + 2) complexes with peptide bonds of
protein and peptide and under appropriate alkaline conditions is reduced to Cu+,
which reacts with Folin reagent, and
2. Folin-Ciocalteau reaction, which involves the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper-catalysed oxidation of
aromatic amino acids tyrosine and tryptophan (Kolakowski 2005)
This blue color can be quantified by its absorbance at 660 nm. The intensity of blue
color is partly dependent on amount of tyrosine and tryptophan amino acids present
in protein sample. This technique is more sensitive to low concentrations of proteins
than the Biuret method detecting down to 10 μg cm
−3
of protein. However, interference by an exhaustive number of compounds such as Tris, zwitterionic buffers such
as Pipes and Hepes, and EDTA affects the effectiveness of this method. These interferences are eliminated by number of modifications including heating sample before
and after treating it with Folin reagent, addition of perhydrate, SDS, chloramines-T
and lipid removal by extraction with organic solvents (Kolakowski 2001).
Bradford Method (CB Dye-Binding Assay)
Bradford assay is a colorimetric method that relies on electrostatic interactions
between basic amino acid molecules such as arginine, lysine and histidine with
Coomassie brilliant blue G-250 (CBB) in an acidic matrix which results in a dyeprotein complex with srectral shift from reddish to bluish form of dye (RedmileGordon et al. 2013). At low PH free dye is protonated with an absorption spectrum
maximum at 465 nm. On binding to protein, a metachromatic response is observed
due to formation of bluish dye-protein complex with maximum absorbance at
595 nm. At this wavelength the unprotonated species absorb. This dye protein complex absorbance, however, varies with the type of dye used. For example dye- protein
complex is formed only when blue (deprotonated) form of dye binds to protein
molecules, or may occur when green (protonated) form of free dye interacts with
protein molecules (Atherton et al. 1996). The assay is monitored using spectrophotometer at 595 nm and can be performed in 10 min or less. The greatest advantage
of this assay is its ease and high sensitivity, perceived linearity and speed of determination of protein concentration in wide variety of protein samples (Sapan et al.
1999).Moreover, this assay is comparatively resistant to interference from polyphenols, carbohydrates such as sucrose, cations such as sodium potassium present in
protein sample except detergents such as sodium dodecyl sulphate.
The two measure formats of this assay are micro-assay and macro-assay that can
measure protein concentration between 1 and 20 μg protein cm
−3
and 20 and 100 μg
protein cm
−3
respectively. The assay can be modified by using perchloric acid
(3.15%) or hydrochloric acid in place of perchloric acid as solvent for dye that varies maximum absorbance of dye-protein complex from 595 to 620 nm (Sedmark
and Grossberg 1977).
Recent Advances in Analysis of Food Proteins
1. Biuret method, in which a copper ion (Cu + 2) complexes with peptide bonds of
protein and peptide and under appropriate alkaline conditions is reduced to Cu+,
which reacts with Folin reagent, and
2. Folin-Ciocalteau reaction, which involves the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper-catalysed oxidation of
aromatic amino acids tyrosine and tryptophan (Kolakowski 2005)
This blue color can be quantified by its absorbance at 660 nm. The intensity of blue
color is partly dependent on amount of tyrosine and tryptophan amino acids present
in protein sample. This technique is more sensitive to low concentrations of proteins
than the Biuret method detecting down to 10 μg cm
−3
of protein. However, interference by an exhaustive number of compounds such as Tris, zwitterionic buffers such
as Pipes and Hepes, and EDTA affects the effectiveness of this method. These interferences are eliminated by number of modifications including heating sample before
and after treating it with Folin reagent, addition of perhydrate, SDS, chloramines-T
and lipid removal by extraction with organic solvents (Kolakowski 2001).
Bradford Method (CB Dye-Binding Assay)
Bradford assay is a colorimetric method that relies on electrostatic interactions
between basic amino acid molecules such as arginine, lysine and histidine with
Coomassie brilliant blue G-250 (CBB) in an acidic matrix which results in a dyeprotein complex with srectral shift from reddish to bluish form of dye (RedmileGordon et al. 2013). At low PH free dye is protonated with an absorption spectrum
maximum at 465 nm. On binding to protein, a metachromatic response is observed
due to formation of bluish dye-protein complex with maximum absorbance at
595 nm. At this wavelength the unprotonated species absorb. This dye protein complex absorbance, however, varies with the type of dye used. For example dye- protein
complex is formed only when blue (deprotonated) form of dye binds to protein
molecules, or may occur when green (protonated) form of free dye interacts with
protein molecules (Atherton et al. 1996). The assay is monitored using spectrophotometer at 595 nm and can be performed in 10 min or less. The greatest advantage
of this assay is its ease and high sensitivity, perceived linearity and speed of determination of protein concentration in wide variety of protein samples (Sapan et al.
1999).Moreover, this assay is comparatively resistant to interference from polyphenols, carbohydrates such as sucrose, cations such as sodium potassium present in
protein sample except detergents such as sodium dodecyl sulphate.
The two measure formats of this assay are micro-assay and macro-assay that can
measure protein concentration between 1 and 20 μg protein cm
−3
and 20 and 100 μg
protein cm
−3
respectively. The assay can be modified by using perchloric acid
(3.15%) or hydrochloric acid in place of perchloric acid as solvent for dye that varies maximum absorbance of dye-protein complex from 595 to 620 nm (Sedmark
and Grossberg 1977).
Recent Advances in Analysis of Food Proteins