Studies on Protein Multimers

It has been shown that the action of 90% 1,2-propanediol on urease results in a dissociation that is symmetrical and results in 240,000 dalton units. Ultraviolet absorption and optical rotatory dispersion studies indicate no conformational change, and no loss of enzymatic activity was observed. The 240,000-dalton form of urease reassociated to a value twice this when the pH was reduced from 9.2 to 7.0. The product of reassociation was identical with the starting material by the criteria of electrophoresis, ultracentrifugation and measurements of ultraviolet absorption, optical rotatory dispersion, and enzymatic activity. The results indicated a single binding area which does not interact with the active site of the enzyme. The interaction between the 240,000 dalton units in the 480,000-dalton form during reassociation does not involve disulfide bonds. Dissociation at pH values lower than 9.2, initially described, has been accomplished, indicating that pH change is not a determining factor for the process. It is proposed that the action of glycols or glycerol in this dissociation can be attributed to interference with the water structure of the solvent around the binding areas of the urease subunits. This results in a favorable entropy change predominantly contributing to the free energy of dissociation. Ultracentrifuge studies in the presence of 1,2-propanediol under the dissociating conditions supported the above conclusions based on studies in buffers. Comparative studies with ethylene glycol, 1,2-propanediol, and glycerol indicate that the same type of dissociation occurs in all three. Studies of the stability of the dissociated urease show that, upon long exposure in ethylene glycol or 1,2-propanediol, changes occur that lead to loss of enzymatic activity. These changes are accompanied by an apparent reassociation followed by polymerization or denaturation. The apparent reassociation is distinguishable from normal association-dissociation and conditions are described for its elimination. A suggested cyclic mechanism of dissociation and reassociation is presented in a scheme that correlates the phenomena observed.