Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change.
Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.
Lee
H
H
National Creative Research Initiative Center for Antioxidant Proteins, Department of Biochemistry, Paichai University, 439-6 Doma-2-Dong Seo-Gu, Taejon, 302-735, Republic of Korea.
Cha
M K
MK
Kim
I H
IH
D000975:Antioxidants / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000975:Antioxidants; D008054:Lipid Peroxides; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D010544:Peroxidases; D003545:Cysteine; D004229:Dithiothreitol