[26] Purification and assay of 15-ketoprostaglandin Δ13-reductase from bovine lung

摘要: Publisher Summary This chapter presents procedure for purification and assay of 15-ketoprostaglandin Δ 13 -reductase from bovine lung. The described in the consists two column steps—carboxymethyl-Sephadex 2',5'-ADP-Sepharose—that takes about 3 days. yield was 10–20%, enzyme purified to homogeneity. 5-Keto-PGE 1 forms a labile red chromophore alkaline solution. property is basis routine detection activity during purification. reaction mixture contains 0.1 M sodium phosphate, pH 7.4, mM EDTA, mercaptoethanol, 14.2 μM 15-keto-PGE , NADH. initiated by addition give final total volume 2.0 ml. After incubation at 44 °, usually 45 rain, concentration remaining substrate quantified after 0.5 ml 2 N NaOH solution measuring maximum absorption 500 nm, reached ca. rain alkali addition. Under these conditions maximal molar extinction coefficient 37.4 cm –1 . value varies according conditions.