Reactions of N-ethylmaleimide with peptides and amino acids.

N-Ethylmaleimide (NEM) has been much used for the chemical modification of proteins, and it is usually assumed that reaction occurs specifically at the thiol groups of cysteinyl residues. However, NEM has been shown to react also with the acamino group of peptides and with the imidazole group of histidine (Smyth, Nagamatsu & Fruton, 1960), and with the a-amnino group of certain amino acids (Smyth et al. 1960; Riggs, 1961). Since the reaction ofNEM with some proteins is much slower than its reaction with the thiol group of cysteine (Gregory, 1955), the possibility should be considered that, when NEM reacts with proteins, groups other than thiol may be involved. The present paper gives the rates of reaction of NEM with the ac-amino group of glycylalanine, with the N-terminal peptide of the a-chain of haemoglobin and with the N-terminal peptide of the ,B-chain of haemoglobin. The results obtained emphasize the necessity for caution in the use of NEM as a specific reagent for thiol groups of proteins. In addition, a detailed investigation is reported on the hydrolysis ofNEM addition products with cysteine, homocysteine, glutathione and reduced ribonuclease. The results obtained with these compounds are used to establish a new procedure for determining the extent of reaction of NEM with the thiol groups of a protein, and for assessing the specificity of the reaction.