McCarthy R C, Jakubowski H V, Markowitz H
Clin Chim Acta. 1983 Aug 31;132(3):287-99. doi: 10.1016/0009-8981(83)90007-4.
Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48 000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with alpha-naphthyl phosphate at 30 degrees C and para-nitrophenyl phosphate at 37 degrees C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 micrograms of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9-7.7% with serum at 1.3 to 5.6 micrograms PAP/l.
通过硫酸铵沉淀和亲和层析法从良性前列腺增生组织中分离出前列腺酸性磷酸酶。通过二维凝胶电泳对纯化后的酶进行表征，结果显示在9摩尔/升尿素中，等电点为5.9至6.3时，该酶有一组表观分子量为48000的蛋白质斑点。纯化后酶的比活性在30℃时以α-萘基磷酸为底物为723 U/mg蛋白，在37℃时以对硝基苯磷酸为底物为659 U/mg蛋白。用纯化后的酶对兔子进行免疫，制备抗体并用于放射免疫分析（RIA）。与使用pH 7.0或7.3的磷酸盐缓冲液或通过氯胺-T法碘化前列腺酸性磷酸酶（PAP）的RIA相比，使用pH 6.6的磷酸盐缓冲液和通过博尔顿-亨特法对PAP进行碘化提高了分析的精密度。前一种RIA在每升血清中含有2微克酶时可取代50%的示踪剂。对于每升血清中PAP含量为1.3至5.6微克的血清样本，11次分析的批间变异系数范围为3.9 - 7.7%。
