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Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60 degrees C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A(lim)) is proportional to protein concentration at both temperatures. The first order rate constant (k(I)) does not depend on protein concentration in the range 0.05-0.2 mg/ml at temperature 50.6 degrees C, but at temperature 60 degrees C it increases with the growth of protein concentration in the range 0.1-0.4 mg/ml. Kinetic curves, shown in coordinates {A/A(lim); t}, in experiments at 50.6 degrees C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60 degrees C, half-transformation time t(1/2) = ln2/k(I) decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60 degrees C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6 degrees C aggregation rate is limited by the stage of protein molecule denaturation, but at 60 degrees C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation.

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.

Recovery of enzymatic activity of rabbit skeletal muscle glycogen phosphorylase b preincubated with 1 mM AMP and 0.125 M K2SO4 (0.05 M glycyl-glycine buffer pH 6.8; 17 degrees C) has been studied. According to sedimentation data, preincubation conditions favor the formation of the tetrameric form of the enzyme. When registering the kinetics of the enzymatic reaction catalyzed by phosphorylase b preincubated with AMP and K2SO4, acceleration of the reaction in the course of the enzymatic process was observed. On the basis of kinetic data, the rate of constant for the dissociation of phosphorylase b tetramers into dimers has been calculated: k = (8.3 +/- 0.3) 10(-3) sec-1 (0.05 M glycyl-glycine buffer pH 6.8; 17 degrees C).

An ethanol soluble protein-trypsin inhibitor was isolated from kidney bean seeds. The inhibitor purification included gel chromatography of ethanol soluble proteins on Sephadex G-75 and affine chromatography on immobilized trypsin and chymotrypsin. The inhibitor suppressed activites of trypsin, chymotrypsin and in part trypsin-chymotrypsin activity of pronase but did not influence subtilizin. The inhibitor at a dose of 15-17.5 mug decreased by 50% the activity of 100 mug trypsin. By isoelectric focusing it was shown that the inhibitor isolated from kidney beans consisted of four isoinhibitors with pH of 4.3, 4.5, 4.7, and 4.9.

The qualitative differences in the affinity of concanavalin A (Con A), wheat-germ agglutinin (WGA) and Phaseolus vulgaris lectin to the surface of 10 microbial strains inducing various diseases in humans and agricultural animals have been demonstrated by means of the indirect immunofluorescence tests. Enterobacteria, Coxiella burnetii and Bacillus anthracis have been found to possess pronounced affinity to Con A and WGA, while Rickettsia prowazekii, Francisella tularensis and Brucella abortus, as well as Treponema pallidum, have proved to be resistant to lectins. WGA has been found to bind specifically to Brucella abortus and Treponema pallidum. Con A and WGA are seemingly suitable for use in the preliminary test for the total capacity of lectin receptors to come in contact with biological macromolecules.

Two isoinhibitors (II and III-B) have been isolated from kidney bean (Phaseolus vulgaris L.) in a highly purified state. Both were active against trypsin and chymotrypsin to the same extent. Their amino acid composition is characterized by a high content of half-cystine, aspartic acid (or asparagine) and serine, by the absence of valine, methionine and tryptophan. Glycine and serine were N-terminal in II and III-B respectively. Both isoinhibitors have C-terminal leucine.

A technique for isolation of the trypsin inhibitor from soya beans (Kunitz inhibitor) was developed with affinity chromatography as a main step, the immobilized antibodies of the inhibitor being used as a sorbent. The inhibitor obtained was homogeneous according to the data of electrophoresis in PAAG and had the specific activity equal to that of an inhibitor preparation obtained by affinity chromatography on trypsin-sepharose.