A method for the isolation and identification of pyridoxal phosphate in proteins

A method for the isolation and identification of covalently bound pyridoxal phosphate (PLP) contained in some enzymatic proteins is presented. The method involves acid hydrolysis of the protein in the presence of phenylhydrazine, separation of the adduct by elution from Sep-Pak C18 cartridges, isolation by HPLC, and either direct analysis by mass spectrometry with direct electron impact or conversion into trimethylsilyl derivatives followed by gas chromatography-mass spectrometry. Under the prescribed conditions of hydrolysis, PLP forms its phenylhydrazone and is released from the protein and hydrolyzed to the phenylhydrazone of pyridoxal, which shows a typical fragmentation in direct electron impact and in gas chromatography-mass spectrometry after silylation. The yield in phenylhydrazone of pyridoxal is on the order of 50% (+/- 5% SE, n = 15) when PLP is added to 10 mg of protein in amounts ranging from 20 to 40 nmol. Analysis of pig plasma benzylamine oxidase by this procedure confirms the presence of covalently bound pyridoxal phosphate in this enzyme.