ISOLATION AND IDENTIFICATION BY GAS-CHROMATOGRAPHIC MASS-SPECTROMETRYOF THE CARBONYL-ACTIVE SITE OF PIG-KIDNEY DIAMINE OXIDASE

An adduct with phenylhydrazine was formed with the purified pig kidney diamine oxidase and in parallel with the I-tyrosine decarboxylase fro m Streptococcus faecalis. The labeled enzymes were hydrolyzed by chemi cal hydroIysis and the adducts released by hydrolysis were isolated an d identified first in HPLC and successively in GC-MS. Both enzymes gav e the same adduct which was identified as the phenylhydrazone of pyrid oxal. The isolated adduct had the same retention time as the phenylhyd razone of pyridoxal in HPLC and in gas chromatography and showed the s ame molecular weight in mass spectrometry when chemical ionization was used and the same fragmentation in mass spectrometry when electronic impact was used. The reported results show that pig kidney diamine oxi dase contains pyridoxal in the form of covalently linked pyridoxal pho sphate which can be released from the enzyme only by chemical hydrolys is. Pig kidney diamine oxidase is therefore a pyridoxal enzyme such as I-tyrosine decarboxylase as hypothesized in the past but never clearl y demonstrated. (c) 1994 Academic Press, Inc.