Purification and properties of glyoxalase I from sheep liver

1 Glyoxalase I has been obtained in electrophoretically pure form from sheep liver by a procedure which includes ammonium sulfate and poly(ethylene glycol) fractionations and column chromatographies on hydroxyapatite, Cibacron Blue–Sephadex G-100 and DEAE-cellulose. The specific activity of the homogeneous preparations is about 4000 units/mg of protein (25° C). Three separate peaks of activity were obtained in the last column on DEAE-cellulose (DE-32). No signs of heterogeneity were seen in the previous steps. Purified but not crude preparations gave two activity peaks on disc gel electrophoresis. 2 The isoelectric point of metal-free apoglyoxalase I is 5.0 by isoelectric focusing. The apparent molecular weight of glyoxalase I is 45 900 from gel chromatography. From dodecylsulfate gel electrophoresis the enzyme has a subunit molecular weight of 21000. 3 In addition to methylglyoxal, phenylglyoxal and kethoxal are good substrates of sheep liver glyoxalase I. Hydroxypyruvaldehyde and glyoxal react more slowly. 4 Catalytically inactive apoenzyme of sheep liver glyoxalase I has been prepared by dialysis against EDTA and Chelex-100. All of the activity is restored by Mg2+ ions; Zn2+, Mn2+, Co2+, Ni2+ and Ca2+ ions, in decreasing order of maximum velocity, give partial reactivation. Of these metals, Mg2+ has the highest (2.8 mM) and Zn2+ the lowest (0.007 mM) apparent half-saturation concentration when assayed in 80 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (Hepes) buffer pH 6.8. 5 Several chelating agents are inhibitors of sheep liver glyoxalase I. In all cases Mg2+ reverses the inhibition after a short incubation time.