Reactivity of sulfenic acid in human serum albumin.
Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.
Turell
Lucía
L
Laboratorios Enzimología, Facultad de Ciencias, Universidad de la República, Uruguay.
Botti
Horacio
H
Carballal
Sebastián
S
Ferrer-Sueta
Gerardo
G
Souza
José M
JM
Durán
Rosario
R
Freeman
Bruce A
BA
Radi
Rafael
R
Alvarez
Beatriz
B
D018053:Arsenites / Q000737:chemistry; D003512:Cyclohexanones / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D015394:Molecular Structure; D009579:Nitrobenzoates / Q000737:chemistry; D010084:Oxidation-Reduction; D012709:Serum Albumin / Q000737:chemistry*; D017670:Sodium Compounds / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013434:Sulfenic Acids / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D018053:Arsenites; D003512:Cyclohexanones; D009579:Nitrobenzoates; D012709:Serum Albumin; D017670:Sodium Compounds; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; C011136:thionitrobenzoic acid; C017947:sodium arsenite; C004774:dimedone; D006861:Hydrogen Peroxide