Role of Protein -SH Groups in Redox Homeostasis— The Erythrocyte as a Model System

The reactivities of the sulfhydryl groups of rat, turkey, human, and calf hemoglobin were studied together with the enzyme activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutaredoxin in lysed erythrocytes to evaluate their roles in regulating redox homeostasis. The results of -SH reactivity showed rate constants spanning four orders of magnitude (k2, calf, 6.67 M−1s−1; rat -SH fast reacting, 2.8 × 104M−1s−1). Enzyme activities of glucose-6-phosphate dehydrogenase ranged from 0.402 U/ml (calf) to 0.900 U/ml (rat), glutathione reductase from 0.162 U/ml (rat) to 0.381 U/ml (human), glutaredoxin from 0.778 U/ml (rat) to 2.28 U/ml (turkey), and glutathione peroxidase from 2.07 U/ml (human) to 27.3 U/ml (rat). Blood samples of the four species were also treated with 0.5–1.5 mMtert-butyl hydroperoxide (t-BOOH) or diamide, and levels of glutathione-derived species [GSH, GSSG, and glutathione–protein mixed disulfides (GS–SP)] were determined within 120 min and related to the corresponding protein -SH group (PSH) reactivities and enzyme repertoires. In all cases t-BOOH rapidly transformed GSH into GSSG by the action of glutathione peroxidase; GSSG was in turn transformed into GS–SP, according to the reaction GSSG + PSH → GS–SP + GSH, or reduced back to GSH by glutathione reductase. The GSSG reduction was more efficient in rat and human blood, due to the contribution of the fast-reacting -SH of hemoglobin, in the rat, and to the efficiency of the enzyme repertoire of human blood. Calf blood showed a relatively low capacity to restore normal values after oxidative stress, due to its low PSH reactivity and the weak contribution of its enzymes. Diamide treatment, which is known to react nonenzymatically with thiols, gave increased GS–SP levels in rat and turkey, but not in human and calf blood, as expected from the different corresponding PSH reactivities. Species with relatively high PSH reactivity and glucose 6-phosphate dehydrogenase activity, such as the rat, therefore had a higher antioxidant capacity than species (calf) in which these parameters were relatively low.