Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

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Abstract
A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 .times. 103 over the initial aqueous butan-1-o1 extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalyzing the hydrolysis of 1 .mu.mol of p-nitrophenyl phosphate/min at 35.degree. C in 0.1 M-2-amino-2-methylpropan-1-o1/HCl buffer, pH 10.5, containing 10 mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a MW of 156,000 and 160,000, respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising 2 subunits of about 80,000 MW. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.