Kinetic and optical spectroscopic studies on the purple acid phosphatase from beef spleen

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Abstract
A new purification scheme was developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only 5 steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate and [p-(acetylamino)benzyl]phosphonate, a nonhydrolyzable substrate analog; the last 2 of these show simple competitive inhibition. Cyanide, azide, tartrate and p-nitrophenol show no inhibition at concentrations up to 10 mM. MW estimations by gel electrophoresis and gel permeation chromatography give a value of 40,000 for the native enzyme, which consists of 2 subunits of apparent MW 24,000 and 15,000. Careful metal analyses indicate the presence of 2.1 .+-. 0.1 Fe atoms per enzyme molecule, and < 0.1 Cu, Zn, Ni or Mn atom per enzyme. The purple enzyme (.lambda.max 550 nm) is reversibly converted to a pink, active form (.lambda.max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. EPR spectroscopy at several temperatures showed weak g = 4.3 signals (< 0.1 spin/molecule) for the native, reduced and inhibited forms of the enzyme.