Smythe E, Williams D C
Department of Biochemistry, Trinity College, Dublin, Ireland.
Biochem J. 1988 Apr 1;251(1):237-41. doi: 10.1042/bj2510237.
Hydroxymethylbilane synthase from human erythrocytes was purified 47,000-fold to greater than 95% homogeneity and 7.5% yield by a simple and rapid procedure using heat treatment (80 degrees C, in the presence of proteinase inhibitors, to convert one of two chromatographically separable forms into the other), DEAE-cellulose and Cibacron Blue F3G-A-Sepharose chromatographies and Sephadex G-75 gel filtration. The purified enzyme was similar to the enzyme purified from other species in showing hyperbolic dependence of velocity on substrate concentration, a non-linear progress curve for uroporphyrinogen appearance, and was monomeric, having an Mr of 44,000 by gel filtration on Sephadex G-100 and h.p.l.c. and an Mr of 45,000 on SDS/polyacrylamide-gel electrophoresis. The enzyme showed a sharp pH profile for Vmax, and various folates were shown to accelerate neither the enzymic formation of hydroxymethylbilane nor ring-closure of hydroxymethylbilane.
通过一种简单快速的方法，利用热处理（80℃，在蛋白酶抑制剂存在下，将两种可通过色谱分离的形式之一转化为另一种）、DEAE-纤维素和Cibacron Blue F3G-A-琼脂糖凝胶色谱以及Sephadex G-75凝胶过滤，将人红细胞中的羟甲基胆色素原合酶纯化了47000倍，纯度大于95%，产率为7.5%。纯化后的酶与从其他物种纯化得到的酶相似，表现出速度对底物浓度的双曲线依赖性、尿卟啉原生成的非线性进程曲线，并且是单体，通过在Sephadex G-100上的凝胶过滤和高效液相色谱法测得其Mr为44000，在SDS/聚丙烯酰胺凝胶电泳上测得其Mr为45000。该酶的Vmax显示出明显的pH曲线，并且各种叶酸均未显示出能加速羟甲基胆色素原的酶促形成或羟甲基胆色素原的环化反应。
