alpha-chymotrypsin and 1-4-dioxane

A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 degrees C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic alpha-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities (a(w)<0.5) during hydration. At a(w) about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At a(w)<0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low a(w). At a(w)>0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given a(w). This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm(-1) that was assigned to the intermolecular beta-sheet aggregation. Changes in the intensity of the 1628 cm(-1) band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of alpha-chymotrypsin depend markedly on how enzyme has been hydrated - whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity-a(w) profiles.

Topics: Animals; Cattle; Chymotrypsin; Desiccation; Dioxanes; Models, Chemical; Protein Conformation; Spectroscopy, Fourier Transform Infrared; Water

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

Topics: Chymotrypsin; Dioxanes; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Polymethacrylic Acids; Protein Denaturation; Protein Renaturation; Urea

The effects of different concentrations (20-95%) of organic solvents (ethanol, 1,4-dioxane and acetonitrile) were studied on alpha-chymotrypsin and trypsin from bovine pancreas. The changes in secondary structure were followed by CD measurements, and the apparent Michaelis constants (KMapp) and the stabilities of the enzymes were determined. Significant alterations in the CD spectra were found for both enzymes at the different organic solvent concentrations. The apparent KM values of trypsin and alpha-chymotrypsin decreased as the low solvent concentrations were elevated, but then increased in the presence of higher organic solvent concentrations. The stabilities of the enzymes changed on increase of the organic solvent concentration; trypsin exhibited a higher stability than that of alpha-chymotrypsin in all organic solvents. These results show that at an organic solvent content of 95% the manifestation of an enzyme activity similar to that measured in water can be attributed to the similar compositions of the secondary structural elements.

Topics: Acetonitriles; Animals; Cattle; Chymotrypsin; Circular Dichroism; Dioxanes; Dose-Response Relationship, Drug; Enzyme Stability; Ethanol; Hydrogen-Ion Concentration; Organic Chemicals; Protein Structure, Secondary; Solvents; Structure-Activity Relationship; Trypsin

Macroporous carriers were prepared by coating controlled pore silicas with copolymers of hydroxyalkyl methacrylate and alkaline dimethacrylate activated by epichlorhydrin (140-1000 mumol/g epoxide group content). The relationship between the preparation conditions (the monomer ratio, the nature of the solvent, the method of activation by epichlorhydrin and the reaction temperature) was studied, as well as the surface characteristics responsible for the carrier's ability to bond (the value of the surface area before and after coating with copolymers and the degree of cross-linking required for the accessibility of the reactive groups). The coupling of various amino compounds, ammonia, aminopropanol, 1,2-diaminoethane, 1,6-diaminohexane and, as a model enzyme, alpha chymotrypsin on to derivatized carriers was investigated as a function of pH, reaction time and the concentration of the compound bonded.

Topics: Amines; Ammonia; Bacillus subtilis; Chymotrypsin; Dioxanes; Drug Carriers; Endopeptidases; Epoxy Compounds; Ethylene Oxide; Glass; Kinetics; Methacrylates; Silicon Dioxide; Surface Properties

Topics: Binding Sites; Chymotrypsin; Dioxanes; Enzymes, Immobilized; Indicators and Reagents; Kinetics; Solvents

Topics: Chymotrypsin; Dioxanes; Ethanol; Heterocyclic Compounds; Methanol; Phenylalanine