Purification of human platelet monoamine oxidase b by high performance liquid chromatography

Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chrom-atography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0-1%) of octyl-B-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently-labeled with [³H]-pargyline [³H(G)] or catalytic activity using [¹⁴C-methylene]-benzylamine as substrate. [³H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit M (approximately 59,000) of MAO B were determined by sodium-dodecyl^r sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.