Formaldehyde Dehydrogenase from Human Liver: PURIFICATION, PROPERTIES, AND EVIDENCE FOR THE FORMATION OF GLUTATHIONE THIOL ESTERS BY THE ENZYME

Abstract

We have purified formaldehyde dehydrogenase (EC 1.2.1.1) 1390-fold from human liver. The final preparation, which has a specific activity of 3.20 i.u. per mg of protein (25°) is homogeneous according to electrophoretic criteria. S-Formylglutathione rather than formate is formed from formaldehyde and reduced glutathione in the reaction catalyzed by purified formaldehyde dehydrogenase. The enzyme is not strictly NAD-specific; NADP can also be used although NADP has a much higher Km value than NAD, especially at high pH values. S-Formylglutathione is reduced by the enzyme to formaldehyde with either NADH or NADPH as cofactors. Methylglyoxal and some other ketoaldehydes also can be used as the substrates of formaldehyde dehydrogenase. The product obtained from methylglyoxal is probably S-pyruvylglutathione. S-Formylglutathione is hydrolyzed very actively in crude human liver preparations. All of this activity is removed during the purification of formaldehyde dehydrogenase. Formaldehyde dehydrogenase has an apparent molecular weight of 81,400 according to gel filtration and a subunit molecular weight of 39,500 according to dodecyl sulfate gel electrophoresis. Isoelectric focusing experiments gave an isoelectric point, pI of 6.35. The enzyme is very sensitive to mercaptide-forming reagents. NAD and NADH protect the enzyme. NAD and especially NADH stabilize the enzyme during storage and against denaturation at high temperatures.