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Journal of Lipid Research, Vol. 50, 2445-2454, December 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

Jing Yan^*, Yuewen Gong^*,, Yi-Min She, Guqi Wang^**, Michael S. Roberts and Frank J. Burczynski^1,*,

^* Faculty of Pharmacy, University of Manitoba, Winnipeg, Canada
Section of Hepatology, University of Manitoba, Winnipeg, Canada
Department of Internal Medicine, Department of Pharmacology and Therapeutics, and Faculty of Medicine, University of Manitoba, Winnipeg, Canada
Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
^** McColl-Lockwood Laboratory, Cannon Research Center, Charlotte, NC 28232-2861
Department of Medicine, Princess Alexandra Hospital, University of Queensland, Woolloongabba, Queensland, Australia 4102

¹ To whom correspondence should be addressed. e-mail: burczyn{at}cc.umanitoba.ca

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of 80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or -bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP's antioxidant activity is through inactivation of the free radicals by L-FABP's methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Supplementary key words hepatocyte • oxidative stress • free radical

Abbreviations: DCF, dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescin-diacetate; GST, glutathione S-transferase; L-FABP, liver fatty acid binding protein; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substance

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