REVERSIBLE AND IRREVERSIBLE INHIBITION OF SHEEP LIVER SORBITOL DEHYDROGENASE WITH CIBACRON-BLUE-3GA AND ERIOCHROME-BLACK-T

Due to the central role of sorbitol dehydrogenase in diabetic cataract , it is important to examine this enzyme's interaction with different inhibitory compounds such as dyes. The aim of the study was to investi gate the binding of Cibacron Blue and Eriochrome Black T to the active site in sorbitol dehydrogenase. These dyes' effect on the enzyme was studied by steady state and affinity labelling kinetics, Both dyes wer e coenzyme competitive inhibitors with K-El values around 0.5 mu M. Es sentially the same K-El values were obtained using the dyes as protect ing ligands against the affinity label D,L-alpha-Bromo-beta-(5-imidazo lyl)-propionic acid. Both dyes were also able to inhibit the enzyme ir reversibly through an affinity labelling mechanism, with K-El' values for Cibacron Blue and Eriochrome Black T of 2.2 and 3.1 mM, respective ly, Dithiothreitol and NADH were competitive protecting ligands agains t both dyes, The rate of inactivation was fastest for Cibacron Blue at acid pH values, while the opposite was the case with EBT. Both Cibacr on Blue and Eriochrome Black T bind to sorbitol dehydrogenase in two d ifferent ways, In both cases the complex formed prior to irreversible inhibition is the weakest, The tighter reversible complexes are sugges ted to share a common epitope in the coenzyme binding region. Both irr eversible complexes involve binding close to the zinc ion at the activ e site and the sugar binding site, Due to different pH dependences it can be concluded that the affinity labelling mechanism is different fo r the two dyes and in neither case is the inactivation due to removal of the active site zinc ion.