Nucleotide Inhibition of Glyoxalase II Page: 52
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CHAPTER II
METHODS
Materials
Affi-gel-10 affinity chromatography material was purchased from Bio-Rad. DTA was
purchased from U.S. Biochemical. All other chemicals were purchased from Sigma. All
solvents used were of reagent grade.
Methods
Synthesis of Affi-gel 10-glutathione (Af-SG) Affinity Chromatography Matrix
The Af-SG material, in which the sulfhydryl group links the GSH to the matrix,
was synthesized by the method of Norton et al (180) with modifications. First, GSH, 350
mg, was dissolved in 50 mL of ice cold sodium acetate, 30 mM, pH 5.5. The pH was
then adjusted to 5.0 with a 10% sodium hydroxide solution. Second, a wet volume of 25
mL of Affi-gel 10 was washed with 5 volumes of ice cold sodium acetate buffer, 30 mM,
pH 5.5. Third, the washed Affigel -10 was added to the GSH solution and gently shaken
overnight at 4C. Finally, the resulting solid Af-SG material was washed with 10
volumes of 50 mM TRIS buffer, pH 7.4 to remove excess GSH. The resulting
suspension was utilized in the affinity chromatography step in the purification of Glo-II.
Abbreviations: SLG: s-D-lactoylglutathione, GSH: glutathione, DiFMOC-G: N,S-
bisflourenylmethoxycarbonyl-glutathione, DEPC: diethylpyrocarbonate, EDTA: ethylenediamine
tetraacetic acid, NEM: N-ethylmalimide, DMSO: dimethylsulfoxide, PMSF: phenylmethylsulfonyl
fluoride, DTT: dithiothreitol, GSSG: glutathione (oxidized), DTA: meso-2,5-dimercapto-N,N,N',N'-
tetramethly-adipamide, ApppA: diadenosine triphosphate, PolyA: polyadenylic acid, DTNB: dithiobis-
nitrobenzoic acid, EGTA: ethylene glycol-bis (3-aminoethyl ether)N,N,N',N'-tetraacetic acid, TRIS:
tris [hydroxymethyl] aminomethane, TES: N-trsi [hydroxymethyl] methyl-2-aminomethanesulfonic acid.52
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Gillis, Glen S. Nucleotide Inhibition of Glyoxalase II, dissertation, May 1999; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc2183/m1/61/: accessed April 18, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .