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J. Biol. Chem., Vol. 282, Issue 42, 31085-31093, October 19, 2007

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Nitro-fatty Acid Reaction with Glutathione and Cysteine

KINETIC ANALYSIS OF THIOL ALKYLATION BY A MICHAEL ADDITION REACTION^*

Laura M. S. Baker, Paul R. S. Baker¹, Franca Golin-Bisello, Francisco J. Schopfer², Mitchell Fink, Steven R. Woodcock^¶, Bruce P. Branchaud^¶, Rafael Radi^||3, and Bruce A. Freeman⁴

From the Departments of Pharmacology and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, the ^¶Department of Chemistry, University of Oregon, Eugene, Oregon 97403, and the ^||Departmento de Bioquímica, Facultad de Medicina, Universidad de la Republica, 11800 Montevideo, Uruguay

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO₂) and linoleic acid (LNO₂) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO₂ and LNO₂ reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M^-1s^-1 and 355 M^-1s^-1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A₂ and 15-deoxy-^12,14-prostaglandin J₂. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO₂ and LNO₂, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pK_a of target thiols increased. Increasing concentrations of the detergent octyl--D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

Received for publication, May 17, 2007 , and in revised form, August 22, 2007.

^* This work was supported in part by National Institutes of Health Grants HL58115 and HL64937 (to B. A. F.) and AHA Grant 0450118Z (to B. P. B.), and a Department of Education GAANN (Graduate Assistance in Areas of National Need) award (to S. R. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the American Diabetes Association.

² Supported by a Beginning Grant In-Aid from the AHA.

³ Supported by the Howard Hughes Medical Institute (U. S. A.) and International Centre of Genetic Engineering and Biotechnology (Italy).

⁴ To whom correspondence should be addressed: Dept. of Pharmacology, Thomas E. Starzl Biomedical Science Tower, 200 Lothrop St, University of Pittsburgh, Pittsburgh, PA 15213. Tel.: 412-648-9319; Fax: 412-648-2229; E-mail: freerad{at}pitt.edu.

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