66] Formaldehyde dehydrogenase

Abstract

This chapter discusses the determination of formaldehyde dehydrogenase. The assay of the enzyme in crude extracts is based on the formation of acid. Alcohol dehydrogenase present in the extracts reacts with formaldehyde and reduced diphosphopyridine nucleotide (DPNH) to yield methanol and DPN. The overall reaction catalyzed by formaldehyde dehydrogenase and alcohol dehydrogenase is a dismutation of formaldehyde to methanol and formic acid. Catalytic amounts of DPN (nicotinamide adenine dinucleotide, NAD) and GSH are required. The reaction is followed by measuring manometrically the rate at which the acid produced releases carbon dioxide from bicarbonate buffer. After acetone treatment, the enzyme can be assayed spectrophotometrically by observing the initial rate of increase of absorbancy at 340 mμ because of the reduction of DPN (NAD). Residual alcohol dehydrogenase is inhibited by hydroxylamine. A unit of enzyme is the amount necessary to oxidize 1 micromole of formaldehyde per minute under the conditions of the assay. Specific activity is expressed as units per milligram of protein. Protein was determined by the biuret test or the method of Warburg and Christian.