A pressure-membrane technique has been developed to physically manipulate the water potential of in vitro enzyme preparations. Enzyme preparations were subjected to a range of water potentials using this technique. Indoleacetic acid oxidase and ribonuclease activities were unaffected by changes in water potential. Ribulosediphosphate carboxylase was sensitive to water potential change and its activity was reduced to 20 % of controls at a water potential of -14 bars. Loss of activity was attributed to fragmentation of the molecules to an apparent molecular weight of less than 1500. Ketose-1-phosphate aldolase lost all activity when subjected to slight water potentials (-1 bar) and fragmented in a similar fashion to ribulosediphosphate carboxylase. Fragmentation in both cases was not due to protease activity. It is concluded that water has an important structural role in maintaining the molecular integrity of ribulosediphosphate carboxylase and ketose-1-phosphate aldolase, but not in indoleacetic acid oxidase and ribonuclease.

Studies of the instability of ribulosediphosphate carboxylase at low water potentials, generated in a pressure-membrane apparatus, have been continued. The presence of phosphate, pyrophosphate, citrate, or ribulose diphosphate during treatment protects the protein against fragmentation. Tris-sulphate, sodium sulphate, tetra-alkyl ammonium salts, or polyvinylpyrrolidone do not afford protection.The results are considered in terms of water-structuring by ions, the binding of ions to the protein, and stabilizing of the protein by ions. Reported effects of ions on enzyme kinetics do not explain the difference between sulphate and phosphate with respect to protection. It is concluded that protective ions must be bound to cationic sites on the protein but must also structure adjacent water. The additional structured water presumably prevents fragmentation of the protein that can occur at low water potentials.