FORMATION OF CARBONYLS DURING ATTACK ON INSULIN BY SUBMOLAR AMOUNTS OF HYPOCHLORITE

Bovine insulin was reacted at pH 4.0 with submolar amounts of hypochlo rite. At least one molecule of insulin was modified per two molecules of hypochlorite added, as estimated by HPLC of native and modified ins ulin. About 5% of the hypochlorite-modified insulin reacted with dinit rophenylhydrazine (DNPH), a reagent which specifically labels carbonyl groups. The major DNPH-labeled product was isolated from the native i nsulin on reverse-phase HPLC, using trifluoroacetic acid/water/acetoni trile gradients. The UV spectrum of the major peak on the HPLC diode-a rray detector was representative of DNPH adducts, with lambda(max) = 3 65 nm. Several methods, including total amino acid analysis, tryptic d igestion, and collision-induced dissociation-electrospray MS, indicate that the major carbonyl in the DNPH-labeled product was on the amino- terminal phenylalanine of the insulin B-chain. Amino acid analysis ind icated that tyrosine was also degraded by hypochlorite, but we could n ot detect a carbonyl group formed at tyrosine. These findings suggest that the terminal amino groups of proteins are highly vulnerable to ca rbonyl formation during hypochlorite attack. The use of relatively low amounts of active oxygen species (such as hypochlorite), followed by chromatographic isolation of the protein labeled with a carbonyl-speci fic reagent, can be a useful approach to the study of reactive sites o n proteins. (C) 1998 Academic Press.