RESUMO
Enzymes entrapped in systems formed with water, phospholipids, toluene, and Triton X-100 show a catalytic activity that is much lower and a thermostability that is much higher than that observed in totally aqueous systems or in other types of reverse micelles. By phase boundary titrations and dynamic light scattering, this work characterizes reverse micelle systems formed in either toluene or propylbenzene with Triton X-100 and water. Four regions with distinct structural features were encountered. Up to one molecule of water per one Triton X-100 molecule, the system was transparent; light scattering measurements of this region indicated that water hydrated Triton X-100 monomers. A turbid region was formed as water content was increased to water:Triton X-100 ratios of 7.6 in toluene and 4.2 in propylbenzene. This thermodynamically unstable region was formed by large polydisperse structures. Transparent systems containing small size (27-150 A) thermodynamically stable reverse micelles were formed when the ratio of water to Triton X-100 molecules in the reverse micelle was in the range of 7.6 to 26.8 in toluene and 4.2 to 15.1 in propylbenzene. In this region, micellar size increased with water content. Water concentrations higher than the latter values resulted in phase separation. A similar titration of the aforementioned systems in the presence of phospholipids revealed that in the first region of transparency up to 10 molecules of water hydrated a phospholipid molecule. The inclusion of phospholipids to the Triton X-100 systems caused a displacement of the boundaries of the second region of transparency toward higher water contents. Copyright 1998 Academic Press. Copyright 1998Academic Press
RESUMO
Catalysis, stability, and thermostability of yeast hexokinase were determined in the microenvironments of two organic solvent/Triton X-100/phospholipids systems. In the abscence of enzyme, phase diagrams showed two transparent/turbid transitions, and reverse micelles were only observed in the second region of transparency (T2), where particle size as a function of water content shows a minima (see previous paper in this issue). In the present work, enzyme activity was detected throughout the four regions of the phase diagrams of these systems. Catalysis increased with water content; nevertheless, the maximum activities that were reached in the toluene and propylbenzene systems were 30 and 1.6%, respectively, of the activity in all aqueous media. Because in the T2 region in the propylbenzene system, micelles are much smaller than in toluene (see preceding paper), it would appear that expression of catalysis depends on the size of the micelles. However, a comparison of the dimensions of hexokinase and those of reverse micelles in the T2 region, suggests that in this region, hexokinase entrapment increases the inner volume of the micelle. High enzyme thermostability was only observed in the first transparent region (T1) of the system that contained phospholipids. In this region, hexokinase induced the formation of reverse micelles from dispersed surfactant monomers. There is a striking similarity in the dimensions of hexokinase entrapped in reverse micelles as determined by dynamic light scattering measurements in the T1 region with those of hexokinase as obtained from X ray diffraction studies of the enzyme in a crystalline environment. This suggest that high thermostability, and low catalytic rates result from restrictions in mobility imposed by a low water environment. Copyright 1998 Academic Press. Copyright 1998Academic Press
