FAST-REACTING THIOLS IN RAT HEMOGLOBINS CAN INTERCEPT DAMAGING SPECIES IN ERYTHROCYTES MORE EFFICIENTLY THAN GLUTATHIONE

The S-conjugation rates of the free-reacting thiols present on each co mponent of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) ( DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mn), three classes of thiols have bee n defined as follows: fast reacting (fHbSH), with t(1/2) <100 ms; slow reacting (sHbSH), with t(1/2) 30-50 s; and very slow reacting (vsHbSH ), with t(1/2) 180-270 s, Under paraphysiological conditions, fHbSH (i dentified with Cys-125 beta(H3)) conjugates with DTNB 100 times faster than glutathione and similar to 4000 times more rapidly than (v)sHbSH (Cys-13 alpha(All) and Cys-93 beta(F9)). Such characteristics of fHbS H reactivity that are independent of the quaternary state of hemoglobi n are mainly due to the following: (i) its low pK (similar to 6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123 beta (H1)) and (ii) the large exposure to the solvent (as measured by analy sis of a model of the molecular surface) and make these thiols the kin etically preferred groups in rat erythrocytes for S-conjugation. In ad dition, because of the high cellular concentration (8 mM, i.e. four ti mes that of glutathione), fHbSHs are expected to intercept damaging sp ecies in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategie s of antioxidant defense) to cysteinyl residues in proteins.