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ORIGINAL RESEARCH ARTICLE

Target Size Analysis by Radiation Inactivation: The Use of Free Radical Scavengers

Gene C. Ness^*,1, Laura C. Pendleton^* and Michael J. McCreery

^* Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612; and Medical Research Institute of Chemical Defense, U.S. Army Edgewater Area, Aberdeen Proving Ground, Maryland 21010

To whom requests for reprints should be addressed at ¹ Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. E-mail: gness{at}hsc.usf.edu

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.

Key Words: radiation inactivation analysis • free radical scavengers • glucose-6-phosphate dehydrogenase • glutamate dehydrogenase • target size determinations • functional units of proteins