In a previous communication the reaction of N-ethylmaleimide (NEM) with the thiols of intact human erythrocytes was described.1 The principal sites of reaction are the p-chains of hemoglobin2 and intracellular glutathione. Average values of 2.35 and 0.45 equivalents of HbSH and GSH respectively per mole of hemoglobin have been found for normal red cells. The conditions used were NEM/Hb = 8,-1 hr, 250C, pH 6.8. NEM is relatively unstable above pH 7 and reacts readily with thiols at pH 6.8.3 As shown in Figure 1, the uptake of NEM by both erythro-3.0 e-~~~~GSH 2.5 _ 2.0J 0 Uj 1.0 , 0.5 , 10 20 30 40 50 60 70 M N U T E S FIG. 1.-Rate of NEM-reaction with intact erythrocytes and dialyzed stroma-free hemolysates. NEM 0.002 M, pH 6.8, 23 1 10C, molar ratio NEM/Hb = 8. See Table 1 for procedure and calculation of equiv-SH/mole Hb, uncorrected for GSH.cytes and dialyzed hemolysates, measured by decrease in absorption at 300 m/, is rapid during the first 30 minutes and very slow after one hour. The reagent exhibits a relatively high degree of specificity in reacting rapidly with -SH groups.3-8 No significant amounts of NEM are bound when the -SH groups of hemoglobin are alkylated8 and the measurement of -SH by amperometric titration has been correlated with OD3oo.2 Reactions with other sites on the protein, as well as the imidazole-catalyzed polymerization of NEM,9 are apparently secondary. Correlations of OD)oo with S-cysteinosuccinic acid formed on hydrolysis of NEM-treated proteins also indicate that the rapid reaction of NEM with proteins is limited to condensation with -SH groups.7' 10The purpose of the present communication is to report evidence that the reactive -SH groups of hemoglobin undergo reversible changes on oxygenation-deoxygena-, tion. As measured by reaction of NEM with erythrocytes for one hr at pH 6.8, the number of -SH groups decreased on deoxygenation from about 2 (2.3 ± 0.2) to 1057

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