Purification and characterisation of glyoxalase II from human red blood cells

Abstract

Glyoxalase II was purified from human red blood cells. The purification factor was 83,300 and the yield was 24% or 1.7 micrograms/ml red blood cells. The purified protein was a monomer with a molecular mass of 29,200 Da and an isoelectric point of 8.3. The rate of hydrolysis of S-D-lactoylglutathione to reduced glutathione and D-lactate, catalysed by glyoxalase II, followed Michaelis-Menten kinetics where the Km and kcat values were 146 +/- 9 microM and 727 +/- 16 s-1, respectively in 50 mM Tris/HCl, pH 7.4 at 37 degrees C. Other S-2-hydroxyacylglutathione derivatives were also acceptable substrates. S-p-Nitrobenzoxycarbonylglutathione was a potent competitive inhibitor of glyoxalase II with a Ki value of 1.20 +/- 0.21 microM, and the hemithioacetal formed non-enzymically from the reaction of methylglyoxal with reduced glutathione was a weak competitive inhibitor with a Ki value of 834 +/- 98 microM.