Urease Catalysis and Structure: VI. CORRELATION OF SEDIMENTATION COEFFICIENTS AND ELECTROPHORETIC MOBILITIES FOR THE POLYMERIC UREASE ISOZYMES

Abstract

Abstract Parallel experiments have demonstrated that purified α-urease, defined by its electrophoretic mobility in acrylamide gels, is the molecular species with an s20,w of 18.3 S. Gel filtration of α-urease to remove mercaptoethanol was followed by the appearance of a series of urease polymers, which were studied by ultracentrifugation and electrophoresis. The sedimentation coefficients 18.3, 27.6, 33.8, 39.8, and 44.4 S agreed with electrophoretic mobilities in indicating an integral arithmetic polymer series, with molecular weights 0.5, 1.0, 1.5, 2.0, and 2.5 million. All members were enzymatically active by catalytic gel staining; densitometry indicated a polymer distribution quite similar to that determined from relative schlieren peak areas, suggesting approximately the same specific activity for each polymer. The sedimentation constants were analyzed by means of the Kirkwood equation, manipulated so as to permit calculation of center to center distances between the monomeric units of each polymer. Close agreement was obtained for a model in which α-urease is spherical with a diameter of 125 A, the deviation of its frictional ratio from unity being due primarily to hydration, and polymerization occurs as rigid straight chain assemblies. Polymer linearity is a tentative conclusion, since concentration dependence has not been excluded except in the case of the 18.3 S polymerizing unit, where it was found to be negligible at concentrations of 6 mg per ml and below. Sedimentation ratios predicted by the Kirkwood equation for linear and compact polymers are presented, as well as formulations suitable for estimating center to center distances and geometric arrangements of any monomer-dimer-trimer series.