itated proteins to avoid the rapid oxidation of SH groups

(14 ), which takes place after neutral pH is restored for the DTT reduction and mBBr conjugation. Measured basal GSSPs in human and rat RBCs are very low, and only a few previous reports found relatively close values (23 ). Conversely, most authors reported higher concentrations; one reason for these discrepancies could be that samples were frozen before the ESI-MS determination. It is possible that ferric Hb, hemicromes, and hemocromes, which can be generated by freezing (24 ), produce oxidants, increasing the Hb-SSG content. Our measures indicate that human and rat Hbs, under typical conditions, are minimally glutathionylated (9.9 nmol/g; i.e., 0.03% of chains). A negligible percentage of GSH was found bound to membrane proteins. Hb-SSG content can increase after oxidative stress produced by diamide or t-BOOH treatments. In any case, human RBC GSSP content accounts for 10% of total cellular glutathione after oxidative stress. Conversely, rat Hb was shown to produce large amounts of Hb-SSG: after treatment with diamide, all GSH was bound to Hb. This is attributable to the peculiarity of rat Hb (25 ), which is characterized by a highly reactive cysteine in position 125, in addition to cysteine 93, which is common to most mammalian Hbs. In diabetes, where oxidative stress is increased, we have measured enhanced Hb-SSG ( 648%); in addition, we have also evaluated the correlation of obtained values with other indicators of oxidative stress, i.e., protein carbonyls, isoprostanes, and malondialdehyde. Preliminary results (not shown) indicate a positive correlation with protein carbonyls and isoprostanes but not with lipid peroxidation products. In conclusion, our studies suggest, as discussed previously (9 ), that Hb-SSG could be used as a clinical marker of oxidative status and, consequently, of pathologies whose development is strongly associated with oxidative stress. The availability of a simple, sensitive, and reproducible method to detect Hb-SSG could be useful both for diagnostic and therapeutic purposes.