Selective purification of tartrate-inhibitable acid phosphatases: rapid and efficient purification (to homogeneity) of human and canine prostatic acid phosphatases.

Abstract

We describe the synthesis of a long-chain monoamide derivative of L(+)-tartaric acid and its attachment to Sepharose 4B. Procedures are then described for use of this material in purifying human prostatic and (for the first time) canine prostatic acid phosphatases to constant specific activity and electrophoretic homogeneity. Depending on sample size, such purification is possible in one step, and is clearly faster and more efficient than are previously described methods. These materials and procedures have significant potential in studies of the comparative biochemistry and clinical chemistry of tartrateinhibitable acid phosphatases.

Isolation, characterization, and spontaneous interconversion of two forms of human prostatic acid phosphatase

“…Procedures for the purification of HPAP have been reviewed by Bodansky [6] and recent methods have utilized a combination of different techniques including exclusion and ion-exchange chromatography [7,11,18], concanavalin A affinity chromatography [9,28,29], and tartrate affinity methods [13,30,31]. Only Choe et a1 [9,29] purified the two enzyme forms to homogeneity in order to determine their antigenic properties, whereas others have concentrated on isolating only the major enzyme form.…”

“…The specific activity of the tissue extract was increased by the initial purification steps, which were adaptations of the method of Ostrowski et a1 [18]. Although HPAP can be purified to a high degree and in excellent yield by the simple affinity column procedure [13] outlined in Table IA, the binding capacity of the tartramic acid affinity column for HPAP is limited to 3 or 4 mg when a preparation of low (enzymatic) specific activity is employed. By increasing the specific activity of the enzyme solution which is applied, the binding capacity of the column for HPAP is significantly increased, but the overall yield is only slightly decreased.…”

“…As the major step in the purification, HPAP solution of various stages of purity were applied to the tartramic acid affinity column [ 131. AS shown in Figure 1 Chromatography was performed using a 2.5 X 3.5-cm column of Sepharose 4B-bound aminohexyl-L-tartramic acid [13]. A crude enzyme preparation (280 mg) obtained after the buffer dialysis step (Table I) was applied to the column and eluted with 600 ml of 50 mM sodium acetate (pH 4.8) containing 50 mM NaCI.…”

“…DEAExellulose chromatographic separation of two forms of HPAP and their subsequent interconversion. Chromatography was performed on a 1.5 X 25-cm column of DEAE-cellulose starting with enzyme previously purified by L-tartramic acid affinity chromatography [13]. A linear gradient made up from 350 ml of 10 mM sodium phosphate (pH 7.0) and 350 ml of 80 mM sodium phosphate (pH 6.0) was applied and 8ml fractions were collected at a rate of 12 ml/hr.…”

“…We present a method for the purification and separation of two forms of HPAP and we compare their physical, biochemical, and kinetic properties. The purification procedure utilizes a further simplified version of the N-(6-aminohexyl)tartramic acid affinity column method of Van Etten and Saini [13].…”