INTERACTION OF CIBACRON BLUE 3G-A AND RELATED DYES WITH NUCLEOTIDE-REQUIRING ENZYMES
BEISSNER, ROBERT STEVEN
Doctor of Philosophy
Cibacron Blue 3G-A substituted on an agarose or dextran matrix has become widely used as an absorbant for enzyme purification. Its mode of interaction with the proteins which bind to it is not entirely clear. One popular theory for proteins' attraction for the dye is that it is a specific structural probe for the supersecondary protein structure called the dinucleotide fold (Stellwagen E. (1977) Acct. Chem. Res. 10, 92-98). The purpose of this investigation is to more clearly define this dye's mode of affinity for proteins. It has been determined through both competitive inhibition kinetic studies and the use of dyed agarose matrixes that the major portion of Cibacron Blue 3G-A that is involved in its binding to the selected dehydrogenases and kinases studied is 1-amino-4(4'-aminophenylamino)anthraquinone-2,3'-disulfonic acid. Effective interaction of a structural isomer and smaller moieties of Cibacron Blue 3G-A with these selected kinases and dehydrogenases indicate a largely nonspecific interaction with respect to the exact structure and conformation of the dye. There is much evidence available that a combination of hydrophobic and electrostatic interactions are taking place at a hydrophobic patch or pocket on the enzymes. This interaction is not sufficiently specific to be used as a probe for a defined protein supersecondary structure. These studies have shown the potential of dyes for determining an enzyme's kinetic mechanism through competitive inhibition. The unusual inhibition patterns exhibited by Cibacron Blue 3G-A with phosphoglycerate kinase allowed a clear demonstration of the random mechanism of that enzyme. Two new columns were developed for use in the purification of mainly nucleotide requiring enzymes using dyes covalently bound to an agarose matrix. These columns represent the feasibility of developing a large number of new chromatographic materials that are inexpensive, easily prepared, and offer a wide variability.