Affinity-chromatography purification of alkaline phosphatase from calf intestine.
A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.
D000327:Adsorption; D000469:Alkaline Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001151:Arsenic; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004589:Electrophoresis, Disc; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D011506:Proteins / Q000032:analysis; D012685:Sepharose / Q000031:analogs & derivatives
D011506:Proteins; D012685:Sepharose; D000469:Alkaline Phosphatase; D001151:Arsenic