alpha-chymotrypsin and 2-2-4-trimethylpentane

Differential scanning calorimetry was the method to investigate the thermostability of chymotrypsin. The transition temperature decreased by approx. 30 degrees C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbitol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the enzyme was equilibrated through the apolar solvent, isooctane, stabilization of chymotrypsin was observed at high water activities, compared to equilibration in air. The presence of isooctane resulted in a remarkable stabilization of the chymotrypsin-sorbitol complex. A sorbitol concentration of 5 mmol/g of protein was prerequisite to induce stabilization when equilibrated through isooctane at high water activities. The transition enthalpy increased with increasing amounts of sorbitol. Different hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffer salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water activity 0.97. Variations on the hydration of the preparations both offered the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalyst whose hydration was suppressed due to its environment exhibited low enzymatic activity.

Topics: Air; Buffers; Calorimetry, Differential Scanning; Catalysis; Chymotrypsin; Enzyme Stability; Freeze Drying; Octanes; Protein Denaturation; Solvents; Sorbitol; Thermodynamics; Water

The amide I region Fourier transform infrared (FTIR) spectra of alpha-chymotrypsin have been studied in deuterium oxide (D2O) solution and also in reverse micellar solution of AOT/isooctane. The Fourier second derivative was applied to all spectra, revealing that the amide I band of alpha-chymotrypsin in D2O and in reverse micellar solution consists of nine components. The band frequencies are assigned to alpha-helix, beta-sheet, random and turn structure. The second derivative spectra of alpha-chymotrypsin have been shifted in the reverse micellar solution of AOT/isooctane in comparison with its spectra in D2O. This shift has also changed the intensity of each band. Through accurate measurement of the band intensities, the relative amount of different structure of alpha-chymotrypsin can be estimated. The comparison of the calculated results obtained in D2O with those obtained in reverse micellar solution provides the possibility to analyze the effect of reverse micellar solution of AOT/isooctane on the secondary structure of alpha-chymotrypsin. The results indicate that the reverse micellar solution has decreased the amount of alpha-helix and beta-sheet structure and increased the amount of random and turn structure in alpha-chymotrypsin. The increase of the amount of random structure might loosen the structure of alpha-chymotrypsin and change the activity of the enzyme.

Topics: Amides; Chymotrypsin; Deuterium Oxide; Dioctyl Sulfosuccinic Acid; Micelles; Octanes; Protein Structure, Secondary; Proteins; Solutions; Spectroscopy, Fourier Transform Infrared

The extraction of lysozyme, alpha-chymotrypsin, and pepsin from buffered salt solutions into reverse micelles was examined at different pH values and surfactant concentrations. The reverse micelles was formed by mixing aqueous buffer supplemented with KCl and an organic phase of isooctane(2,2,4-trimethylpentane), containing the anionic surfactant, Aerosol O. T. (dioctyl ester of sodium sulfosuccinic acid). The technique of dynamic laser scattering was used to measure the size of reverse micelles which were in equilibrium with the aqueous phase. It was found that the size of the reverse micelles decreased with increasing ionic strength but increased with increasing AOT concentration. In the process of extraction, the reverse micelles might have rearranged themselves to host the protein. The sizes of protein-filled and -unfilled reverse micelles were different, and an open equilibrium could be reached between them. Under the extraction conditions, only a small number of micelles were found to contain protein.

Topics: Chymotrypsin; Dioctyl Sulfosuccinic Acid; Hydrogen-Ion Concentration; Micelles; Molecular Structure; Muramidase; Octanes; Pepsin A; Water

Structural and catalytic properties of two enzymes--alpha-chymotrypsin and horse liver alcohol dehydrogenase (LADH)--are studied in bis(2-ethylhexyl) sodium sulfosuccinate (AOT)-isooctane reverse-micelle solutions. Circular dichroism (CD) and electron paramagnetic resonance spectroscopy (EPR) studies show little change in alpha-chymotrypsin structure upon incorporation into reverse micelles. These structural properties explain, in part, the observed activity of these two enzymes in reverse micelles. alpha-Chymotrypsin retains activity in reverse micelles and, in some cases, displays enhanced activity. A sixfold increase in the turnover number was observed in w0 = 10 reverse micelles. LADH has low activity in reverse micelles compared to that in aqueous solution. At w0 = 70, the turnover number of LADH is 18% of the aqueous value. Active-site titrations show a decrease in active enzyme concentration for both enzymes upon incorporation into reverse micelles. Little change in the structure of both LADH and alpha-chymotrypsin is observed with change of water content in the reverse-micelle system.

Topics: Alcohol Dehydrogenase; Binding Sites; Chymotrypsin; Circular Dichroism; Dioctyl Sulfosuccinic Acid; Electron Spin Resonance Spectroscopy; Enzymes, Immobilized; Kinetics; Micelles; Octanes; Surface-Active Agents

On addition of cytochrome c to a AOT reverse micellar solution, the percolation process usually observed at high temperatures and surfactant concentrations, occurs at room temperature. This is observed either at relatively high water content at a given cytochrome c concentration or at low content on increasing the cytochrome c concentration. On increasing the water content a phase transition is observed with two optically transparent phases. A similar phase transition is observed on solubilizing various enzymes. The temperature of the transition appears to be strongly dependent on the location of the macromolecule in the reserve micelle.

Topics: Chymotrypsin; Cytochrome c Group; Dioctyl Sulfosuccinic Acid; Electric Conductivity; Light; Macromolecular Substances; Micelles; Molecular Structure; Octanes; Ribonucleases; Scattering, Radiation; Solubility; Spectrophotometry, Atomic; Temperature; Water