MONOMER-DIMER EQUILIBRIUM OF UNCOMPLEMENTED M15 BETA-GALACTOSIDASE FROM ESCHERICHIA-COLI

A series of gel filtration, native polyacrylamide gel electrophoresis (PAGE) and sucrose density experiments showed that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium and that only und er some specific conditions does the equilibrium strongly favor dimeri zation. The ratio of dimer to monomer increased as a function of the p rotein concentration, and a very good fit to a theoretical plot of the effect of protein concentration on an associating system of this type was found. The K-diss (equilibrium constant for dimer dissociation) w as 2.5 x 10(-7) M. The addition of 20 mM Mg2+ lowered the K-diss to 1. 5 x 10(-7) M, and the addition of 150 mM NaCl lowered the value to 0.4 x 10(-7) M. Thiol reagents (2-mercaptoethanol and dithiothreitol) cau sed the equilibrium to shift totally to the dimeric form. The monomer- dimer equilibrium was also found to be dependent upon the pH. The diss ociation increased as the pH was raised to 8.5, but there was a revers al of the equilibrium in favor of dimer formation at pH 9.0. This sugg ests that one (or more) residues with a pK(a) value of about 8.0 is in volved. Tyr and Lys were eliminated as possible residues involved and it is, therefore, likely that one or more Cys are involved. Further ev idence that uncomplemented M15 beta-galactosidase is in a monomer-dime r equilibrium was that the gel-filtration peaks were not totally resol ved and that native PAGE bands were diffuse under all conditions excep t at high thiol concentration.