These research projects all involve the binding of the dye Coomassie Brilliant Blue (CBB) to proteins. Virtually all proteins bind the dye, and when binding occurs under acidic conditions (pH less than 1), the bound dye molecules undergo a distinct color change froµ reddish to blue. The change in absorbance occurring as a consequence of dye binding forms the basis of a sensitive method of protein assay called the Bradford method. Details of the binding mechanism on the molecular level are not particularly well understood, and the aim of the proposed studies is to provide a more complete understanding of the binding process.
- Charge State of the Protein-Binding Species
When CBB binds to proteins at low pH values, a reddish protein-binding species is thought to be converted to a blue protein-bound species. An interconversion between two differently-colored ionization states of the dye molecule is the simplest explanation of the absorbance shift which occurs during binding. The structure of the CBB molecule shows five ionizable groups and six possible ionization states for the dye. This project would involve determining the charge state of the reddish protein-binging species and of the blue protein-bound species. This determination is made easier by the fact that three of the six dye species may be isolated in solution by careful adjustment of the solution pH. Spectral evidence indicates that the reddish protein-binding species may be formed by lowering the pH below 0. There also exists a single blue species in the pH range between 3.5 and 9, and s single pinkish species also exists above pH 10. The direction and rate of migration of these individual species during zone or gel electrophoresis would allow determination of size and magnitude of the ionic charge. Similarly, passage of these species through a column containing carefullyh selected ion exchange resins should yield much the same information. The charge of the other ionic species may then be inferred.
- Determination of the Protein-Bound Dye Species
Identification of the charge states of the reddish protein-binding species and of the higher pH blue species still do not allow determination of the blue protein-bound species. We hope to accomplish this by spectrophotometric means.
The visible spectrum of the dye between pH 0 and pH 3 is a composite of the individual spectra of three differently-charged dye species in relative proportions which vary with pH. Any of these spectra in the pH range may be resolved into a set of nine Gaussian peaks, each representing the spectral transition, by means of specialized computer software (PEAKFIT). The peak analysis shows which of the nine peaks corresponds to each of the three dye species present.
It is possible to bind dye to protein in such a way as to convert all the dye present to the protein-bound form, and a spectrum of this protein-bound dye species may be measured. Peak analysis of this spectrum should reveal which, if any, of the three possible dye species conforms to the protein-bound species. A wide variety of proteins may be employed in this study.
- The Mechanism of the Binding of Dye to Protein
It has been shown that the dye binds to polyamino acids (amino acid homopolymers) such as histidine, lysine, and especially arginine. Weaker interactions with tryptophane, tyrosine, and phenylalanine have also been observed. It is also possible that dye molecules bond to already-bound dye molecules, creating a "stacking" interaction as well. In any event, the evidence probably indicated that multiple dye-binding sites exist on the surface of protein molecules. These sites may also be divided into classes on the basis of the strength of the binding interactions. We propose to carry out spectrophotometric binding studies to determine the number of binding sites of a particular class present on a particular protein molecule and also to distinguish between classes of binding sites.
These studies would be of two basic types. In constant dye studies, the amount of dye would remain constant and the spectral changes resulting from addition of increasing amounts of protein would be monitored. In constant protein studies, the amount of protein would be held constant and the changes resulting from addition of increasing amounts of dye would be followed spectrophotometrically. Results of both types of studies would be plotted according to binding equations developed for the proposed multi-site, multi-class binding mechanism. Information about the nature of the binding sites and the type of binding interaction should be provided.