Site-directed mutagenesis defines tryptophan as the principal nitrosation site in human serum albumin

Human serum albumin (HSA) is plasma a protein involved in transporting ligands. This unique protein has three domains and two subdomain. i.e. A and B. HSA binds different ligands at various sites and thus plays a key role in modulating the effects of drugs and endogenous ligands on their biological targets. In the present study patterns of NO release from nitrosated bovine serum albumin, human serum albumin and a number of recombinant human serum albumin mutants were compared. All albumin samples were nitrosated by incubation with acidified NO₂– and by incubation with the NO donor S-NO 3-mercaptopropionic acid. Definitive conclusions about the roles of Cys-34 and Trp-214 in the nitrosation of human serum albumin are presented. Specifically, while the pattern of NO release from nitrosated bovine serum albumin agreed with previous reports, which indicate that Cys-34 is the primary target for nitrosation in bovine serum albumin. The pattern of NO release from nitrosated human serum albumin, which also contains a Cys-34 residue, indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential nitrosation target. in BSA, mutants lacking Trp-214 the only tryptophan residue in human serum albumin were synthesized. A mutant lacking Cys-34, the only free thiol in HSA was also synthesized. Studies with these mutants showed that in contrast to bovine serum albumin the primary nitrosation target in human serum albumin is tryptophan. Mutation of Trp-214 lead to an increase in Cys-34 nitrosation indicating a competition between the two residues for reaction with NO+ in wild type human serum albumin. Experiments with W214L/Y411W, a mutant in which tryptophan had been moved to a new location suggested that the reactivity of tryptophan with NO + varies with its local environment. The implication of these studies for the interpretation of previous work on NO physiology using nitrosated bovine serum albumin as a model compound are discussed. The following recombinant human serum albumin species were produced in the yeast species Pichia pastoris were studied: Wild type, C34S, W214L, W214E and W214L/Y411W.