342 3. A-ETHYLMALEIMIDE 



Reaction with Proteins 



The SH groups of native proteins are frequently quite unreactive to 

 iV-ethylmaleimide and this reagent is not reliable for the titration of total 

 protein SH groups. However, certain accessible SH groups react readily 

 and this provides iV-ethylmaleimide with a certain specificity of attack 

 one does not have with the mercurials and many other SH reagents. In- 

 deed, the advantages of iV-ethylmaleimide as an SH reagent may be sum- 

 marized as (1) probable high selectivity for SH groups, (2) reaction with 

 only certain accessible SH groups on enzymes, making possible specific 

 metabolic inhibitions, and (3) reasonably good penetration into cells due 

 to the uncharged nature of the compound. Alexander (1958) did not 

 apply his method for SH group estimation extensively to proteins, but 

 noted that A^-ethylmaleimide does not react with all the SH groups and 

 felt that its use would be limited in this field. The reaction with bovine 

 seralbumin is quite rapid (Fig. 3-2), half-reaction time being around 

 3 min (Roberts and Rouser, 1958), but essentially no reaction occurs with 

 /?-lactoglobulin unless it is denatured with urea or guanidine, in which 

 case reaction is rapid and complete (Habeeb, 1960; Stark et al., 1960). 

 The half-reaction time for denatured /5-lactoglobulin is less than 2 min 

 (Leslie et al., 1962 a). The same holds for ovalbumin, the rate being in- 

 significant with the native protein but complete within 5 min if denatured 

 (Leslie et al., 1962 b). Thus proteins, and presumably enzymes, differ 

 markedly in the reactivity of their SH groups. 



Human globin reacts with 4.8-5.0 equivalents of A^-ethylmaleimide in 

 10 min, and an additional 0.7-0.8 equivalent is taken up over the next 

 5 hr, while globin denatured with lauryl sulfate takes up 5.0 equivalents 

 (Cole et al., 1958). Thus globin is a protein which readily reacts with 

 iV-ethylmaleimide in both native and denatured states. That the reaction 

 is with SH groups is established by the fact that previous treatment with 

 Hg++ prevents binding of iV-ethylmaleimide (Riggs and Wells, 1960). 

 Reaction of hemoglobin with SH reagents alters the oxygen dissociation 

 curves (inhibition of Bohr effect); iV-ethylmaleimide reacts much faster 

 with HbOa than with Hb, and treatment with iV-ethylmaleimide increases 

 the affinity of Hb for oxygen (Riggs, 1961). By using iV-ethylmaleimide- 

 C^^ it was possible to show that at least 97% of the radioactivity is associ- 

 ated with the /5-chain of hemoglobin and thus the reactive SH groups are 

 in this part of the protein, and consequently it is the /?-chain which is in- 

 volved in the Bohr effect. The effect of A-ethylmaleimide on the oxygenation 

 of hemoglobin may be interpreted in terms of changes in the configurations 

 of the protein chains (R. F. Benesch and R. Benesch, 1962). The initial 

 attachment of iV-ethylmaleimide to the reactive hemoglobin SH groups 

 does not alter the Bohr effect or modify the solubility, but a secondary 

 reaction occurs, whereby the bound iV-ethylmaleimide interacts with the 



