Abstract

An oxygen-dependent, covalent interaction of radioactive 6-hydroxydopamine, 5,6-dihydroxtryptamine, and related compounds with several model proteins in vitro is described. 6-Hydroxydopamine and 5,6-dihydroxytryptamine react with bovine serum albumin in the presence of oxygen to yield a total of about 12 moles of amine bound per mole of protein. The nature of the oxidation product which reacts rapidly has not been defined, but in the case of 6-hydroxydopamine the amine moiety of the side chain, and hence cyclization to a dihydroxyindole, is not required. 5,7-Dihydroxytryptamine, norepinephrine, and dopamine react much more slowly, and tyramine, serotonin, and nonphenolic amines, not at all. The reaction of 6-hydroxydopamine and 5,6-dihydroxytryptamine appears to occur almost exclusively with sulfhydryl groups of proteins, but both amines appear capable of generating additional free sulfhydryl groups by reduction of disulfide bonds in proteins. Denaturation of proteins such as albumins or hemoglobins with 6 M urea results in a marked increase in the amount of radioactive amine bound per mole of protein. This binding approaches the theoretical number of potential free sulfhydryl groups, suggesting that in the native proteins certain sulfhydryl and/or disulfide bonds are inaccessible to the amine. In proteins which do not contain disulfide bonds, blockade of free sulfhydryl groups with N-ethylmaleimide renders the protein resistant to interaction with 6-hydroxydopamine. Polylysine reacts only slowly with 6-hydroxydopamine. The reaction product of radioactive 6-hydroxydopamine and bovine serum albumin consists primarily not of monomeric protein but of a series of polymeric proteins as determined by gel filtration and polyacrylamide gel electrophoresis. The cross-linking of bovine serum albumin to form polymers which occurred with both 6-hydroxydopamine and 5,6-dihydroxytryptamine was irreversible. Incorporation of more than 2 moles of amine per mole of protein appeared to be required for cross-linking. Acid hydrolysis of 6-hydroxydopamine-cross-linked bovine serum albumin yielded two radioactive, ninhydrin-positive products, which are suggested to be cysteine derivatives. The covalent binding and induction of cross-linking of proteins in vitro by 6-hydroxydopamine and 5,6-dihydroxytryptamine is discussed from a mechanistic standpoint and as a possible model for the mechanism of the cytotoxic actions of these compounds in vivo.