The behavior of horse liver alcohol dehydrogenase in guanidine hydrochloride solutions

Abstract

The Z average molecular weight of horse liver alcohol dehydrogenase (LADH) in dilute neutral buffer was found to be 78,200 by sedimentation equilibrium. One molar guanidine hydrochloride (GuHCl) reversibly inhibited LADH activity. The inhibition was competitive with limiting nicotinamide adenine dinucleotide and mixed with limiting ethanol. When EDTA was added to LADH in 1 M GuHCl, zinc was removed followed by aggregation. Sedimentation velocity experiments indicated that no subunit was formed under these conditions. Higher concentrations of GuHCl resulted in dissociation of LADH. In 3 M GuHCl, the sedimentation equilibrium data indicated the presence of a reversible association-dissociation system involving subunit, dimer, and trimer. EDTA removed zinc but had no effect on the molecular weight of LADH in 3 M GuHCl. Reduction and alkylation of LADH in 3 M GuHCl resulted in almost complete dissociation into two subunits. Apparently, sulfhydryl residues are involved in subunit association, while zinc plays little or no role. The Z average molecular weight of LADH in 5 M GuHCl containing mercaptoethanol was found to be 45,700, a value higher than expected. The discrepancy may be attributed to incomplete dissociation, a small error in apparent partial specific volume, or solvent binding. Measurements of molecular weight, sedimentation coefficient, and intrinsic viscosity of LADH in 5 M GuHCl indicate that the structure of LADH is composed of two subunits.