alpha-chymotrypsin and sodium-sulfate

Topics: Acetyl Coenzyme A; Acetylation; Aminosalicylic Acid; Animals; Arylamine N-Acetyltransferase; Chymotrypsin; Gastric Juice; Humans; Intestinal Absorption; Intestine, Large; Intestine, Small; Isoniazid; Phenobarbital; Rats; Sodium Chloride; Sulfanilamides; Sulfates; Sulfathiazole; Sulfathiazoles

alpha-Chymotrypsin (alpha CT) was used as a model protein to study the effects of salt-induced precipitation on protein conformation. Process parameters investigated included the type and amount of salt used to induce precipitation. The salts studied included Na2SO4, NaCl, NaBr, KBr and KSCN. Precipitate secondary structure content was examined via laser Raman spectroscopy. Conventional and saturation transfer electron paramagnetic resonance spectroscopy were employed to probe the tertiary structure of the active site in spin-labelled alpha CT precipitates. As the molal surface tension increment of the inducing salt increased, the beta-sheet content increased and the alpha-helix content decreased. There was no significant variation in secondary structure with the amount of salt used. The fraction of precipitate that recovered activity on redissolution was correlated with the change in secondary structure content. Spin-labelled precipitate spectra indicated that the active site remains unaltered during precipitation. Molecular modelling was employed to investigate how physical property of alpha CT were affected by these types of conformational change. Estimated physical property changes could not account entirely for observed deviations from current equilibrium theory for salt-induced precipitation. The spectroscopic observations were also combined with activity/solubility results to propose a mechanism for the salt-induced precipitation of globular proteins.

Topics: Animals; Bromides; Chemical Precipitation; Chymotrypsin; Crystallography; Electron Spin Resonance Spectroscopy; Models, Molecular; Potassium; Potassium Compounds; Protein Conformation; Salts; Sodium; Sodium Chloride; Sodium Compounds; Spectrum Analysis, Raman; Structure-Activity Relationship; Sulfates; Thiocyanates

The influence of inorganic salts on the trans-cinnamoyl-chymotrypsin deacylation kinetics at 25 degrees C and various pH values has been studied: KCl (pH 6.0-13.4), CsCl (pH 9.8 and 13.1), NaCl and Na2SO4 (pH 9.8). Each salt was found to accelerate the reaction with different effectiveness which depended upon pH. The data show that the acyl-enzyme occurs in two forms, which are seen kinetically by different salting effects in deacylation; the equilibrium between the forms is controlled by an ionizing group in the enzyme with pKa about 12.

Topics: Acylation; Cesium; Chlorides; Chymotrypsin; Hydrogen-Ion Concentration; Kinetics; Osmolar Concentration; Potassium Chloride; Salts; Sodium Chloride; Sulfates