Turpeenniemi-Hujanen T M, Puistola U, Kivirikko K I
Biochem J. 1980 Aug 1;189(2):247-53. doi: 10.1042/bj1890247.
Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.
本文报道了两种纯化赖氨酰羟化酶的方法，两种方法均包括硫酸铵分级分离、伴刀豆球蛋白A-琼脂糖亲和层析以及用乙二醇洗脱柱子。方法A的额外步骤包括凝胶过滤和羟基磷灰石柱层析，方法B的额外步骤包括与琼脂糖偶联的胶原亲和层析和凝胶过滤。用这两种方法中的任何一种获得的最佳制剂，经十二烷基硫酸钠-聚丙烯酰胺圆盘凝胶或平板凝胶电泳检测均为纯品，但方法A获得的制剂约有一半含有少量污染物。用方法B纯化的典型制剂的比活性是鸡胚匀浆15000g上清液的134000倍，回收率约为4%。通过凝胶过滤测得纯酶的分子量约为200000，通过十二烷基硫酸钠/聚丙烯酰胺圆盘凝胶或平板凝胶电泳测得酶亚基的分子量约为85000。有人认为活性酶是由仅一种类型的单体组成的二聚体，并且先前描述的表观分子量约为550000的酶形式是这种二聚体的聚合形式。在规定条件下，用饱和浓度的合成肽底物测定的纯酶的催化中心活性约为每摩尔3-4摩尔/秒。
