A radioassay for nonoxidized methionine in peptides. A method for identifying (after isoelectric focusing) and for estimating biologically active forms of corticotropin and other hormones.

A radioassay for nonoxidized methionine in peptides is described; it has advantages over other methods currently used because of its simplicity, sensitivity, accuracy, and applicability to individual peptide components in mixtures and to many samples at a time. Methionyl residues were S-carboxymethylated with iodo[2-14C]acetic acid; iodo[2-3H]acetic acid did not provide a stable radioactive tracer. The labeled peptide was isolated by carboxymethylcellulose chromatography or by isoelectric focusing (IEF) or electrophoresis in polyacrylamide gel, and its radioactivity measured. The assay was applied to corticotropins, alpha-melanotropin, bombesin, glucagon, substance P, parathormone, and calcitonin. Twenty-four to thirty samples were conveniently analyzed at a time with a lower detection limit of less than 1 nmol of methionine per sample. The accuracy of the assay, assessed also by reverse-phase high-performance liquid chromatography, is a consequence of its precision, the specificity of the reaction with iodoacetic acid, and the use of an appropriate standard of the peptide being assayed. Methionine was identified, and could be estimated, in individual peptide components of a mixture by using IEF to separate simultaneously the labeled peptide from iodo[2-14C]acetic acid and from other peptide and protein components. This was facilitated by a convenient method for detecting and quantifying these peptides after IEF. The assay is particularly useful for several peptide hormones whose biological activity depends on their sole methionine residue being in a nonoxidized state. It can be used for monitoring their isolation or synthesis and their stability during processing and storage, as well as for evaluating differences in biological potency between preparations and analogues.