Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation |
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Authors: | Krishna Harohalli Charles E. Petersen Chung-Eun Ha Jimmy B. Feix Nadhipuram V. Bhagavan |
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Affiliation: | Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA. |
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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. |
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Keywords: | Nitric oxide Human serum albumin Site-directed mutagenesis Cardiovascular disease |
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