A spectral study of the charge forms of Coomassie blue G

The Coomassie brilliant blue protein assay is commonly used because of its superior sensitivity, but it is not well understood on the molecular level. This paper attempts to gain better understanding of the assay by studying the three charge forms of the free dye present at the usual pH of the assay. A linear least squares method is outlined which allows calculation of the spectra of the red, green, and blue charge forms of the dye and also calculates the two related pKa's with values of 1.15 and 1.82. The pure component dye spectra were found to differ substantially from the spectrum of the dye-protein complex. The presence of a fourth, pink, ionic state of the free dye at high pH (pKa = 12.4) is also shown. The signs and magnitudes of the ionic charges for the free dye forms are deduced and discussed. The results of this investigation are also discussed in terms of the potential for improvement of the CBB protein assay, and the conclusion is drawn that the assay conditions have been well optimized by earlier workers.