Reversible Changes in the Nem-Reactive —Sh Groups of Hemoglobin on Oxygenation-Deoxygenation

Abstract

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

Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

“…As previously observed (Taketa and Morell, 1969) and confirmed here for native hemoglobin, conversion of the cross-linked hemoglobins to the carbonyl derivative slightly decreases the reactivity with DTDP relative to that of the corresponding oxyhemoglobin derivatives. Furthermore, deoxygenation of the cross-linked hemoglobins decreased reactivity of the reactive thiol as previously observed for the native protein (Riggs, 1961;Morell et al, 1962;Benesch and Benesch, 1962). At alkaline pH (8.4) the rate constants for reaction of these proteins with DTDP is increased to the same degree (three-to fivefold relative to those at pH 7.0) as observed for native HbA 0 .…”

Antioxidant functions for the hemoglobin β93 cysteine residue in erythrocytes and in the vascular compartment in vivo

“…The high degree of B93C conservation in vertebrates [9], combined with its allosterically-controlled reactivity [13, 17] and high intracellular concentration suggest a prominent functional role in vivo. In the present work, we tested whether the β93Cys residue plays a role within the RBC antioxidant network.…”

“…This residue is conserved amongst vertebrates but its function remains unclear. Previous studies have shown that the reactivity of the β93Cys residues towards thiol-reactive agents is allosterically controlled by oxygen-dependent changes in hemoglobin conformation with the β93Cys in the R-state being more reactive towards nitrosating and alkylating agents [11–14] and to mercurials [15, 16] than T-state hemoglobin [17]. Moreover, the β93Cys can affect electron transfer reactions and limit ferrous heme-derived superoxide production and reactivity with other RBC components [18–21].…”

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation

“…The quaternary conformation changes the environment of the βCys-93 residue on the proximal side of the haem. In the R state it is exposed to the solvent and reacts with sulphhydryl reagents [3,[42][43][44]. In the T state, solvent access to the cysteine is impeded by the salt bridges formed in deoxyhaemoglobin [3] and exogenous sulphhydryl reagents react much more slowly.…”