A simple rapid purification scheme for hydroxymethylbilane synthase from human erythrocytes.

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.