The kinetics of the inactivation of the soybean trypsin inhibitor by heat processing]

The investigations of kinetics of the thermal degradation of the antinutritious substances in the soya beans were carried out. The mathematical analysis of the obtained experimental data helped to determine the kinetic constants D and Z necessary to calculate the time of heat treatment to obtain the minimal value of the inhibitor at any temperature level.     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

The inactivation of dilute solutions of crystalline trypsin by x-radiation. I. Kinetics and characteristics

The activity of dilute solutions of crystalline trypsin is destroyed by x-rays. The inactivation is an exponential function of the radiation dose. The reaction yield of inactivation is independent of the intensity at which the radiation is delivered or the quality of the x-rays. The reaction yield increases with increasing concentration of trypsin, varying from 0.06 to 0.7 micromoles per liter per 1000 r for trypsin solutions ranging from 1 x 10(-7) to 2 x 10(-4)M.     

Mechanism of inactivation of trypsin by antithrombin

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.     

Intermolecular Overhauser effects in fluoroalcohol solutions of cyclo-alanylglycine

Interactions between the diketopiperazine cyclo-alanylglycine and four fluorinated alcohols in water-fluoroalcohol mixtures were examined by (1)H[(19)F] intermolecular nuclear Overhauser effects (NOE) experiments. The alcohols studied were trifluoroethanol, hexafluoroacetone trihydrate, 1,1,1,3,3,3-hexafluoroisopropanol and perfluoro-t-butanol. The experimental methods used permit detection of solvent-solute NOEs of 0.1% or less. Solute and solvent diffusion coefficients were determined and apparent molecular radii of the fluoroalcohols estimated. Using these data, it was shown that observed (1)H[(19)F] intermolecular NOEs are consistent with expectations based on theory. A method for extending conventional theory to take into account the shape of a solute and the exposure of its hydrogens to solvent is described. This approach gives reasonable agreement with experimental results, particularly if it is assumed that solute-solvent interactions take place in such a way that the fluorines of a fluoroalcohol are preferentially oriented toward the solute during solute-solvent encounters. The results support the suggestion that intermolecular (1)H[(19)F] NOEs may become a useful tool for studies of peptide and protein conformations in fluoroalcohol-water solvent mixtures.     

Influence of inactivation of trypsin on immunoreactivity and serum immunoreactive trypsin concentration measured by radioimmunoassay

The influences of various active site-specific reagents of trypsin and protease inhibitors on the immunoreactivity of trypsin and serum trypsin concentration have been studied by radioimmunoassay (RIA). The RIA using inactivated 125I-trypsin as tracer showed lower Bo/T than the RIA using active 125I-trypsin, but the coefficient of variance of the former was smaller than that of the latter. Normal serum trypsin concentrations were 26.12-36.38 ng/ml with the RIA using inactivated 125I-trypsin as antigen tracer, and 201.15 ng/ml with the RIA using active 125I-trypsin as tracer. The recovery experiment showed that the difference was due to the interaction of serum protease inhibitors and labeled active trypsin.     

Study of ultrafiltration of trypsin solutions]

The dependence of the rate of trypsin ultrafiltration on the concentration, pressure and structure of membranes was studied. During ultrafiltration of diluted trypsin (0.3 mg/ml), water and sodium chloride the flow rate increased linearly with a pressure increase in the range of 0.4-4.2 kg/cm2. During ultrafiltration of trypsin solutions of a concentration of 1 mg/ml and over at a pressure of 2-3 kg/cm2 deviations from linear proportionality occurred which enhanced with an increase in the protein concentration and a decrease in membrane permeability.