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 NO₂^–. The pattern of NO release from BSA nitrosated with acidified NO₂^– 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 NO₂^– 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 NO₂^–, 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 NO₂^– 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 N₂O₃, the reactive nitrosating species formed in aqueous acidified NO₂^– solutions.

Detection of N‐nitrosomelatonin and other N‐nitrosotryptophan derivatives by transnitrosation of APF and DAF‐2

“…In order to quantitatively determine N ‐nitrosoindole compounds, the lifetime of the N ‐nitrosotryptophan derivative must be long in regard to the period of the detection procedure. Unfortunately, this prerequisite is not evident for N ‐nitrosotryptophan derivatives at pH 7.4 because they slowly decay ( t 1/2 ∼ 100–140 min) [8, 17, 22, 23] via an acid‐catalyzed hydrolysis reaction: …”

Biomedical Vignette

“…Harohallli et al [8] compared the patterns of NO release from nitrosated bovine serum albumin, human serum albumin and a number of recombinant human serum albumin mutants. They found that Cys-34 is the primary target for nitrosation in bovine serum albumin.…”