BINDING OF VANADATE TO HUMAN ERYTHROCYTE-GHOSTS AND SUBSEQUENT EVENTS

Spectroscopic techniques were used to investigate the interaction betw een vanadate and human erythrocyte ghosts, Direct evidence from V-51 n uclear magnetic resonance (NMR) studies suggested that the monomeric a nd polymeric vanadate species map bind to the anion binding sites of b and 3 protein of the erythrocyte membrane. The results of V-51 NMR stu dies and the quenching effect of vanadate on the intrinsic fluorescenc e of the membrane proteins indicated that in the low concentration ran ge of vanadate (<0.6 mM), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mM. The experiments of sulfhydryl content titration by the method of E llman and residue sulfhydryl-labeled fluorescence spectroscopies clear ly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (C. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of i ntact ghost membrane were reduced by the reaction with vanadate, as sh own by the ESR studies employing the protein-and lipid-specific spin l abels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate map oxidize the residue sulfhydryl g roups of membrane proteins, and thus decrease the fluidity of erythroc yte membrane.