STUDIES ON LOCATION OF SULFHYDRYL GROUPS IN ERYTHROCYTE MEMBRANES WITH MAGNETIC RESONANCE SPIN PROBES

Electron paramagnetic and nuclear magnetic resonance techniques which involve the spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide (MSL) and 35Cl ion, respectively, are applied to study the bovine erythrocyte membrane. Evidence from sulfhydryl blocking experiments with mercuric ion, p-mercuribenzoate, N-ethylmaleimide, and iodoacetamide indicates that the modified maleimide molecule, which serves as the spin label, is covalently bound to the membrane sulfhydryl groups. The EPR spectrum of spin label bound to ghost erythrocyte membranes consists of at least two types of sulfhydryl sites differing in the motional mobility of the spin label. The same strongly immobilized site is observed on both the intact erythrocyte and the ghost cell. The concentration of binding sites for the spin label is 9.8 × 10-18 mole per cell. Nuclear magnetic resonance measurements of the 35Cl line width in the presence of mercury give 9.1 × 10-18 mole per cell for sulfhydryl content. The NMR measurements further indicate that chloride ion exchange with the mercury labelled sulfhydryl groups is blocked, suggesting that the SH groups are located within the lipoprotein matrix. © 1969.