Volanakis J E, Schrohenloher R E, Stroud R M
J Immunol. 1977 Jul;119(1):337-42.
D was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl esters (BAME) was the most sensitive substrate for D among those tested. The substrate profile of D was dinstinct when compared to that of CIs, CIr, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.
通过在Bio Rex 70、葡聚糖G - 200、Bio Rex 70和葡聚糖G - 75上连续色谱法，从过期人血浆中纯化得到D至同质。通过溶血扩散平板试验监测柱级分的D活性。按活性计算，总产率约为4%。通过沉降平衡确定分子量为22,900道尔顿。已进行氨基酸分析，并确定异亮氨酸为NH2末端。在Mg++存在下，将D与纯化的B和CoVF一起孵育，通过SDS - 聚丙烯酰胺凝胶电泳和免疫电泳判断，导致B裂解。D水解精氨酸、赖氨酸和酪氨酸的某些合成氨基酸酯。在测试的底物中，苯甲酰 - L - 精氨酸甲酯（BAME）是对D最敏感的底物。与CIs、CIr、纤溶酶、尿激酶和胰蛋白酶相比，D的底物谱不同。用10 mM二异丙基氟磷酸（DFP）处理以及还原和烷基化处理均可不可逆地抑制D的酶活性和溶血活性。
