Speaker: Arynn Yaeger
Candidate for Bachelor of Science in Biochemistry
Time: 4:30 PM
Location: Trustee Hall 118
Supervisor: Dr. Eamonn Healy
Title: Development of Quantitative Assay for Glutathione Reductase

Abstract: Glutathione Reductase has been implicated as the critical enzyme for maintenance of GSH during oxidative stress. We report here steps towards development of a quantitative assay for Glutathione Reductase using biotin. Biotinylation has achieved widespread and general utility in a variety of bioanalytical applications due to the ready formation of biotin-avidin complexes (1015 M-1). We have succeeded in coupling a biotin to GSSG (reduced glutathione), with detection by HPLC at 260nm. The GSSG-biotin is then complexed with Glutathione Reductase, and subsequently complexed in a Glutathione Reductase : GSSG-biotin : avidin triplex. By purifying the resulting complex and spectroscopically determining the concentration, Glutathione Reductase can be accurately assayed. The avidin-biotin system utilized involves two ligands with different affinities, biotin and 4-hydroxyazobenzene-2-carboxilic acid (HABA). Avidin is affinity labeled with the dye HABA, so that upon exposure to the high-affinity ligand biotin with Glutathione attached, HABA is expelled from avidin in stoichiometric proportions easily detected and quantified by spectroscopy.