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.

Nonlinearity in protein assays by the Coomassie blue dye-binding method.

The Coomassie brilliant blue (CBB) method for protein determination takes advantage of the fact that a low-pH red form of CBB reverts to a blue form when CBB binds to protein. The increase in absorbance at 595 nm of a protein-dye mixture compared to a blank containing only the dye reagent has been used to estimate the total amount of protein present in the sample mixture. A disadvantage of this method of protein determination is that the assay plot of absorbance at 595 nm versus total protein is not linear.

Steady state and pre-steady state kinetic properties of rat liver selenium-glutathione peroxidase.

The kinetic properties of partially purified rat liver selenium-glutathione peroxidase were studied under various conditions. Steady state kinetic measurements show sigmoidal saturation curves, parabolic double reciprocal plots, and Hill coefficients greater than unity. Although these kinetic results appear to show cooperative interactions between subunits, they more reflect the presence of several oxidation-reduction forms of the catalytic site. A substrate-induced transition between enzyme forms was evidence by the occurrence of a lag in the attainment of the final steady state velocity under certain preincubation conditions. This hysteretic behavior was evident only when the enzyme was incubated in the absence of reduced glutathione, the donor substrate. Thus, reduced glutathione induces the transition to the fully active form of the enzyme, a slow process requiring about 0.5 min after addition of glutathione, depending on conditions. The length, tau, of the lag period is dependent on the concentrations of enzyme and glutathione, but to a first approximation, this lag period is independent of the concentration of the hydroperoxide acceptor substrate. The lag period is also relatively independent of the nature of the hydroperoxide species. A model for the transition process that is compatible with these observations and with the possible oxidation-reduction properties of the selenium moiety of the enzyme is suggested.

The binding interaction of Coomassie blue with proteins.

The Coomassie brilliant blue protein assay is commonly used because of its sensitivity and convenience, but it is not well understood on a molecular level. This study attempts to gain better understanding of the assay system through spectrophotometric binding studies carried out on selected proteins under solvent conditions characteristic of the normal protein assay. The studies were generally conducted at high protein/dye concentration ratios, where only the high-affinity dye binding sites would be occupied. Modified Scatchard and Hill analyses show that these high-affinity sites are few in number compared to the total number of dye-binding sites on a given protein. The magnitudes of the high-affinity binding constants are typical of noncovalent binding interactions.

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.

A mathematical model for the description of the Coomassie brilliant blue protein assay.

The Coomassie brilliant blue dye-binding method for protein assay has become important relatively recently. The basis of the assay method is the binding of dye to protein, with production of a dye-protein complex which absorbs light intensely at 620 nm, but the mechanism of the binding process is not well understood. In this paper, two mathematical models for the binding process are developed, one involving the binding of both protonated (green) and deprotonated (blue) forms of the dye. The second model allows only binding of the blue species to proteins. These models are tested for their ability to estimate number of dye-binding sites (n) and binding constants (Kd) from protein assay data. The models are also tested for their ability to reproduce the experimental assay curve using either known values or reasonable estimates of the equation parameters. The models are shown to be approximately equal in ability to reproduce experimental data related to the protein assay, which somewhat favors the simpler of the two models. In this paper, a method for estimating n and Kd from standard curve-fitting procedures is established. Hitherto, binding constants were only available from assay data taken under conditions of very large molar protein/dye ratios. The possibility of protonated forms of the dye binding to proteins was not ruled out by this study, but for many purposes the use of the simple dye-binding model, in which only the deprotonated dye species binds, is sufficient.