Purification and some properties of diacetyl reductase from beef liver.

1.1. Diacetyl reductase (acetoin:NAD+ oxidoreductase, EC 1.1.1.5) from beef liver has been partially purified by a series of steps involving acetone precipitation of water extracts of acetone cakes, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose. By following this procedure we have reached a specific activity 300-fold that of beef liver water extracts.2.2. Enzyme purified in this manner is highly specific for one of the substrates, diacetyl (butane-2,3-dione), which can not be replaced by pentane-3-one, pentane-2,4-dione, hexane-2,5-dione, pyruvate, oxaloacetate, α-ketoglutarate or acetoin (acetyl methyl carbinol), but not for the other; NADPH can be substituted for NADH. Since beef liver crude extracts catalyze acetoin reduction by NADH to butyleneglycol, it is deduced that butyleneglycol dehydrogenase (2,3-butyleneglycol:NAD+ oxidoreductase, EC 1.1.1.4) and diacetyl reductase are two different enzymes in beef liver.3.3. Diacetyl reductase shows maximal activity at pH 6.1 and does not require metallic ions. At pH 6.1 the reaction in the diacetyl-acetoin direction follows Arrhenius's law, at least from 3.5 °C to 28–30 °C; activation energy has been estimated to be 14 400 cal/mole. It has not been possible to detect any activity in the backward direction (acetoin-diacetyl).4.4. Sephadex-gel filtration resolved the enzyme preparations into two peaks of activity; the molecular weights of the enzymatic species were estimated as 26 000 and 76 000. The ratio of the peaks sizes varied with the eluant (phosphate buffer pH 6.3) molarity. This is taken as indicative that diacetyl reductase can be in two different states of association (as a monomer and a trimer), the equilibrium being somehow regulated by the ionic strength of the buffer.