Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/1997
Publication Date: N/A
Citation: Interpretive Summary: Damaged proteins that collect in the body are an important component of the biology of human aging. The biochemical events that produce these damaged proteins are not well understood. We studied the effect of hypochlorite, the active ingredient in bleach, on the protein hormone insulin. Hypochlorite is important because it is produced in the body as part of microbial defenses against infection. One form of protein damage is the accumulation of carbonyl groups on proteins. We found that after treatment with hypochlorite these groups formed at the end of the insulin molecule, but not at sensitive sites within the molecule. Our findings will help clarify the nature of the processes that produce protein damage during aging or nutrient deficiency in humans.
Technical Abstract: Bovine insulin was reacted at pH 4.0 with submolar amounts of hypochlorite. At least 1 molecule of insulin was modified per 2 molecules of hypochlorite added, as estimated by HPLC of native and modified insulin. About 5% of the hypochlorite-modified insulin reacted with dinitrophenylhydrazine (DNPH), a reagent which specifically labels carbonyl groups. The major DNPH-labeled product was isolated from the native insulin on reverse-phase HPLC, using TFA/water/acetonitrile gradients. The UV-spectrum of the major peak on the HPLC-diode array detector was representative of DNPH-adducts, with lambda-max = 365 nm. Several methods, including total amino acid analysis, tryptic digestion, and collision-induced disassociation electrospray MS (CID-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 indicated that tyrosine was also degraded by hypochlorite, but we could not detect a carbonyl group formed at tyrosine. These findings suggest that the terminal amino groups of proteins are highly vulnerable to carbonyl 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-specific reagent, can be a useful approach to the study of reactive sites on proteins.