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Characterization of inhibitors for Cu/Zn superoxide dismutase observed by ¹?F NMR methods

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Jonathan Markley (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://www.wcu.edu/404.asp
Advisor
Jack Summers

Abstract: Superoxide dismutase (SOD) is an enzyme that disproportionates superoxide (O2-) into H2O2 and O2. The inhibition of this enzyme could be a novel method for the treatment of chronic diseases such as leukemia, cancer and malaria.2,7,8 Methods that rely on superoxide generating systems have been used previously to assay SOD activity and to search for inhibitors6, The reliability of these methods for screening inhibitors is limited due to the reactivity of superoxide as well as well as the indicators these methods used.7 Our group uses the more stable fluoride anion as an indicator of SOD activity. 19F NMR techniques that measure the rate at which fluoride relaxes (R2) were used to assay inhibitors for Cu/Zn-SOD. Inhibitors we studied included 2-methoxyestradiol (2-ME), glyoxal, flavonoids, methyl-3,4-dihydroxybenzoate (MDHB) and diethyldithiocarbamate (DDC). We have measured the kinetics of SOD inhibition as a function of DDC concentration. We have DDC inhibition of SOD to first order with an apparent rate constant of 0.0125 s-1. Our experiments indicate that quercetin does not inhibit SOD by sequestering the copper ion from enzyme active site. The effects of the flavonoids quercetin, luteolin, morin, fisitin, kaempferol, taxifolin and methoxyquercetin on SOD activities were studied as a function of flavonoid concentration. Kinetic studies of SOD inhibition indicated a two step reaction where the first step was a rapid equilibrium. The effects of flavonoid concentration on the initial SOD activity (from kinetic plots) suggested that these compounds dimerize under the conditions of the experiment, and that dimerization competes with enzyme inhibition. The effects of flavonoid concentrations on their luminescence spectra supported this hypothesis. Inhibition and dimerization equilibrium constants were estimated by fitting the curves predicted by the dimerization model to the inhibition data.

Additional Information

Publication
Thesis
Language: English
Date: 2010
Keywords
Cu/Zn-SOD, dissociation constant, flavonoid, inhibitor, kinetic, mechanism
Subjects
Superoxide dismutase -- Inhibitors