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 a ctivity was also remarkably increased by Ca2+ or a cationic detergent (cety ltrimethylammonium chloride). Carboxyl group modification of HSA resulted i n about 40-fold increase of the antioxidant activity. The chemical modifica tion study indicated that in addition to functional cysteine(s), cationic a mino acid residues such as histidine, arginine and lysine appeared to invol ve in the antioxidant reaction. MSA also exhibited alkaline-pH dependent pe roxidase activity to remove fatty acid hydroperoxide, nt neutral pH, only t wo thiols of Cys-289 and free Cys-34 of NSA were modified by a thiol-specif ic modification reagent, 5-((((2- iodoacetyl)amino)ethy)amino)naphthalene-1 -sulfonic acid (I14), regardless of the presence or absence of dithiothreit ol (DTT), and the resultant antioxidant activity was not decreased, suggest ing that Cys-289 and Cys-34 did not participate in the antioxidant reaction , At alkaline pH, I14 modified several additional HSA thiols in the presenc e, but did not in the absence of DTT. The antioxidant activity of the modif ied HSA was remarkably decreased to as much as 30% of the antioxidant activ ity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-trea ted 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 fu nctional cysteine(s)-mediated catalysis. (C) 2000 Academic Press.