title: Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation.
abstract: The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) 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 target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.
authors.affiliation: Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
authors.forename: Krishna
authors.initials: K
authors.lastname: Harohalli
authors.forename: Charles E
authors.initials: CE
authors.lastname: Petersen
authors.forename: Chung-Eun
authors.initials: CE
authors.lastname: Ha
authors.forename: Jimmy B
authors.initials: JB
authors.lastname: Feix
authors.forename: Nadhipuram V
authors.initials: NV
authors.lastname: Bhagavan
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism*; D015538:Nitrosation; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D009573:Nitrites; D011994:Recombinant Proteins; D012709:Serum Albumin; D009569:Nitric Oxide; D014364:Tryptophan