Effect of an exposure to chloramine-T on the immunoreactivity of glucagon

Various preparations of glucagon treated with chloramine-T under different conditions have been studied with respect to their immunoreactivity toward two different glucagon antisera; one specific for pancreatic glucagon and the other capable of reacting with enteroglucagon as well. The glucagon preparations exposed to chloramine-T for different periods reacted almost identically with the nonspecific antibody whether they were used as tracer or standard. On the contrary, treatment with chloramine-T under severe conditions led to reduced immunoreactivity toward the specific antibody. Inclusion of dimethyl sulfoxide (DMSO) in the chloramine-T reaction resulted in preservation of the immunoreactivity of the treated preparations. The cyanogen bromide cleaved-glucagon, (1-26) homoserine lactone, showed little cross-reactivity with the specific antibody whereas it reacted to a similar extent with the nonspecific antibody as natural glucagon did. Amino acid analysis of the hormone exposed to chloramine-T demonstrated that the methionine residue at position 27 in the glucagon molecule had been oxidized to methionine sulfoxide. In addition, tryptophan had also been affected. DMSO protected methionine and tryptophan from the oxidative action of chloramine-T. We postulate from these results that the change in the immunoreactivity toward the specific antibody of glucagon exposed to chloramine-T is mainly due to oxidation of the methionine residue at position 27 in the molecule. The usefulness of DMSO in the iodination process is also discussed.