A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.
Coomassie brilliant blue G (CBB) is the basis of a popular method of protein assay. Normally, the assay is carried out at low pH where the addition of protein to a CBB reagent results in an increase in absorbance at 595 nm due to formation of a protein-dye complex. The absorbance change is proportional to the amount of protein present. It has been found that it is also possible to detect protein at elevated pH, and binding studies have been carried out for a group of seven standard proteins at pH 7.0. The sensitivity of protein detection, linearity of the assay plots of absorbance versus mass of protein, variability of color development among proteins, nature of the dye-protein complex, and rapidity of the binding process were all compared at low and neutral pH. At low pH, it was found that sensitivity is greater, the assay plots are more linear, and the assay is less subject to color variability than at neutral pH. The formation of the dye-protein complex is slower at neutral pH, and the complex is similar at low and neutral pH based on molar absorptivity and lambda max measurements. It was also possible to calculate values for v, the number of dye molecules bound per molecule of protein, from the assay data. At low pH, the maximum value of v correlates well with the arginine and lysine content of the protein. This study also showed conclusively that the blue ionic form of the dye is that which binds to proteins, since at neutral pH only the blue form is present.
Chial
H J
HJ
Chemistry Department, Gustavus Adolphus College, St. Peter, Minnesota 56082.
Splittgerber
A G
AG
D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012394:Rosaniline Dyes / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D011506:Proteins; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue