Dye Colour
The suffix "R" in the name of Coomassie Brilliant Blue R-250 is an abbreviation for Red as the blue colour of the dye has a slight reddish tint. For the "G" variant the blue colour has a more greenish tint. The "250" originally denoted the purity of the dye.
The colour of the two dyes depends on the acidity of the solution. The "G" form of the dye has been studied in detail. At a pH of less than 0 the dye has a red colour with an absorption maximum at a wavelength of 470 nm. At a pH of around 1 the dye is green with an absorption maximum at 620 nm while above pH 2 the dye is bright blue with a maximum at 595 nm. At pH 7 the dye has an extinction coefficient of 43,000 M−1cm−1.
The different colours are a result of the different charged states of the dye molecule. In the red form, all three nitrogen atoms carry a positive charge. The two sulfonic acid groups have extremely low pKa's and will normally be negatively charged, thus at a pH of around zero the dye will be a cation with an overall charge of +1. The green colour corresponds to a form of the dye with no net overall charge. At neutral pH (pH 7), only the nitrogen atom of the diphenylamine moiety carries a positive charge and the blue dye molecule is an anion with an overall charge of -1. The pKa's for the loss of the two protons are 1.15 and 1.82. The final proton is lost under alkaline conditions and the dye becomes pink in colour (pKa 12.4).
The dye molecules bind to proteins including wool (keratin) to form a protein-dye complex. The formation of the complex stabilises the negatively charged anionic form of the dye producing the blue colour, even under acid conditions when most of the molecules in solution are in the cationic form. This is the basis of the Bradford assay which is used to quantify the concentration of protein in a solution.
The dye also forms a complex with the anionic detergent sodium dodecylsulfate. The formation of this complex stabilises the neutral green form of the dye. This effect can interfere with the estimation of protein concentration using the Bradford assay. It is also likely that the anionic detergent competes with the dye for binding to the protein.
Read more about this topic: Coomassie Brilliant Blue
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