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Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John WileySons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure.

Examples of application of high hydrostatic pressure for modulating important properties of enzymes, namely catalytic activity, substrate specificity and thermal stability are presented in this paper. Enzymatic reactions characterised by negative values of activation volumes (ΔV≠) are accelerated at high pressures as is the case with hydrolysis of anilides by α-chymotrypsin. Simultaneous action of elevated temperature and high pressure raises the rate of enzymatic hydrolysis by more than 50 times as compared to the reaction studied at 20 °C and atmospheric pressure. Due to different signs in ΔV≠ for hydrolysis of anilides and esters, a 100-fold increase in anilide-hydrolysing over ester-hydrolysing activity of α-chymotrypsin in the sysytem of reversed micelles was observed at 2000 atm, in comparison with the rates measured at 1 atm. Another positive effect of hydrostatic pressure is in its ability to significantly decelerate heat inactivation of α-chymotrypsin : a 5–10-fold decrease in the rate of thermal inactivation of the enzyme at 50 °C was observed by applying pressure of 1.500–2.000 atm. This protection becomes even more important in the presence of glycerol. Possible explanations of activation and stabilisation effects of high hydrostatic pressure are discussed.

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

The local cerebral blood flow, oxygen tension, local impedance and intragastric pressure were examined in 13 patients with meningiomas of the middle third of the apical sagittal sinus at various stages of surgery including those after ablation of the intrasinus node and its reconstruction. In all the patients the decompressive cranial trepanation showed a blood stream enhancement but on the meningioma side. It is concluded that the degree of disturbances of oxygenative metabolism and blood circulation after the removal and reconstruction of the intrasinus node was dependent on the variant of the sinus injury.

The formation of non-covalent complexes with polyelectrolytes has been suggested to enhance the resistance of enzymes towards inactivation by organic solvents in their homogeneous mixtures with water. Existence of such complexes in water-cosolvent media was proved by experiments with a fluorescence dye, eosin. In the case of catalysis by α-chymotrypsin, formation of the complex with polyelectrolytes produced two major eflects: i) considerable increase in enzyme activity at concentrations of ethanol and N,N-dimethylformamide of 10–30 % v/v; ii) conservation of the enzymatic activity at cosolvent concentrations of more than 40% v/v, where the native enzyme is completely inactive. General character of the observed activation and stabilization phenomena was shown by example of several experimental systems.

The combined effects of high pressure and reversed micelles have been studied to modulate the catalytic behaviour of butyrylcholinesterase. The purpose of this study was to determine whether the conformational plasticity of the enzyme is altered by entrapment in reversed micelles. The presence of soman, an irreversible inhibitor of cholinesterase was used to bring to the fore a possible modification of the enzyme behaviour in this system under pressure. Results show differences between enzyme in conventional medium and in reversed micelles regarding the mechanism of BuChE catalyzed hydrolysis of acetylthiocholine. In both systems, the enzyme displays a non-Michaelian behaviour with this substrate. In conventional medium the kinetics is multiphasic with an activation phase followed by an inhibition phase at high concentration. In reversed micelles there is inhibition by excess substrate but the activation phase is missing. This behaviour may be the result of a change of the enzyme conformational plasticity when is entrapped in reversed micelles.

Biocatalytic transformations in reversed micelles formed by anionic surfactant Aerosol OT in octane have been studied at high pressures by an example of alpha-chymotrypsin-catalyzed hydrolysis of N-carbobenzoxy-L-tyrosine p-nitrophenyl ester and N-succinyl-L-phenylalanine p-nitroanilide. For the first time it has been found that the enzyme retains high activity in these water-in-oil microemulsions up to a pressure of 2 kbar. The value of the activation volume (delta V*) for the enzyme reactions shows a dependence on the water content in the system. When the size of the micellar aqueous inner cavity (as evaluated at 1 atm) approaches the molecular size of alpha-chymotrypsin, delta V* becomes significantly different from the value in aqueous solution and in the micelles with a larger size. Possibilities of regulating the enzyme activity by pressure in systems with a low content of water are discussed.

The authors analyze the results of surgical management of 242 patients with upper sagittal sinus meningiomas who have been treated in the past 20 years. All the patients were divided into 3 groups in accordance with the surgical policy and techniques of interventions and anesthesiological support. The type of sinus wall tumor lesion and its associated cerebral blood outflow disorders detected by electrophysiological studies in pre- and intraoperative periods determined surgical policy which along with microsurgical technique of recovery of the intact sinus and superficial veins of the brain enhanced the efficiency of operations up to 92.2% and reduced fatal outcomes to zero.

The paper deals with improvement of procedures for autografting a greater omental fragment to the vascular layer of the brain in its experimental ischemic lesions. The findings suggest that a functional vasculature has formed between the omentum and the meninx vasculosa just at hour 24 postautografting. This provides evidence to recommend the method for clinical application to compensate cerebral circulatory disorders in tumor-induced and vascular abnormalities in the brain.

Catalytic activity of covalently modified alpha-chymotrypsin in water/cosolvent solutions was investigated. The stability of chymotrypsin increases upon modification with hydrophilic reagents, such as glyceraldehyde, pyrometallic and succinic anhydrides, and glucosamine. Correlation was observed between the protein's stability in organic solvents and the degree of hydrophilization of the protein's surface. The protein is the more stable, the higher are the modification degree and the hydrophilicity of the modifying residue. At a certain critical hydrophilization degree of chymotrypsin a limit of stability is achieved. The stabilization effect can be accounted for by the fact that the interaction between water molecules on the surface and protein's functional groups become stronger in the hydrophilized protein.

A correlation between the stability of alpha-chymotrypsin against irreversible thermal inactivation at high temperatures (long-term stability) and the coefficient of Setchenov equation as a measure of salting-in/out efficiency of solutes in the Hofmeister series has been found. An increase in the concentration of salting-in solutes (KSCN, urea, guanidinium chloride, formamide) leads to a many-fold decrease of the inactivation rate of the enzyme. In contrast, addition of salting-out solutes has a small effect on the long-term stability of alpha-chymotrypsin at high temperatures. The effects of solutes are additive with respect to their salting-in/out capacities; the stabilizing action of the solutes is determined by the calculated Setchenov coefficient of solution. The correlation is explained by a solute-driven shift of the conformational equilibrium between the 'low-temperature' native and the 'high-temperature' denatured forms of the enzyme within the range of the kinetic scheme put forward in the preceding paper in this journal: irreversible inactivation of the high-temperature form proceeds much more slowly compared with the low-temperature form.

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

It is suggested that chemical methods of improvement of protein stability can be a real challenge to the genetic methods. Multi-point immobilization is efficient against inactivation proceeding through protein unfolding, whereas single-point covalent modification and “medium engineering” (by adding low-molecular weight compounds) are good in inhibiting “incorrect refolding” of proteins. Hundred- and thousand- fold effects of deceleration of irreversible inactivation by chemical methods have not been yet achieved by using genetic procedures. The urgent task of today biotechnology is to work out the strategies for increasing protein stability [1]. Along with well developed genetic methods, chemical procedures such as covalent immobilization, chemical modification and medium engineering are beneficial to this end. Modern protein design by chemical means is methodically simple and gives a possibility to change protein structure without a loss of functional activity.

A correlation was found between the thermal stability of alpha-chymotrypsin and the coefficient Ks of the Sechenov equation as a quantitative measure of the "salting-in" or "salting-out" capacity of solutes. At high temperatures, an increase in the concentration of "salting-in" agents (KSNC, GuHCl, urea, formamide) resulted in thermal stabilization of alpha-chymotrypsin. The maximal (about 100-fold) stabilizing effect in concentrated solutions of salting-in agents was comparable with those induced by covalent modification with hydrophilic reagents or immobilization. Conversely, an increase in the concentration of "salting-out" agents stabilized the enzyme only marginally at high temperatures. An additivity of solutes' action on the thermal stability of the protein has been demonstrated. The observed correlation was explained in terms of the solutes' action on the reversible conformational transition of the enzyme native form into a much more stable form existing at high temperatures.

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The stability of α-chymotrypsin covalently modified with a strongly hydrophilic modifier, pyromellitic dianhydride, against solvent-induced denaturation in water—organic solvent binary mixtures has been studied. It was found that the hydrophilization results in a strong stabilization of the enzyme against denaturation by organic solvents. The stabilizing effect is explained in terms of the enhanced ability of the hydrophilized enzyme to keep its hydration shell, which is indispensable for supporting the native protein conformation, from denaturing stripping by organic solvents

Stabilizing effect of denaturing salts on irreversible thermoinactivation of native and modified alpha-chymotrypsin at elevated temperatures is observed. The effect is caused by a shift of conformational equilibrium, at the primary step of reversible unfolding in the course of thermoinactivation, to a more unfolded form which is not able to refold "incorrectly". The stability of alpha-chymotrypsin is regulated within a wide range by medium alteration: the stabilizing effects are similar to those achieved by multipoint attachment of the enzyme to a support or by hydrophilization of protein by covalent modification.

An unusual ("zig-zag") temperature dependence of the rate of irreversible thermoinactivation of enzymes was observed for native and covalently modified alpha-chymotrypsin and trypsin. This dependence was characterized by alternation of plots with positive and negative apparent values of activation energy for the thermoinactivation process. A kinetic scheme which reflects the observed regularities in thermoinactivation for which the temperature-dependent conformational transition is an essential feature, is proposed. Fluorescence spectroscopy data suggest that the conformational transition predicted by the scheme is of the unfolding type. Substantial differences in thermostabilities of "high temperature" and "low temperature" conformations of enzymes may be due to different mechanisms of their irreversible thermoinactivation.

Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.

Basing on the hypothesis that contact of hydrophobic surface clusters of proteins with water is thermodynamically disadvantageous, it is suggested to carry out the hydrophilization of protein surface by covalent modification in order to increase its thermostability. Hydrophilic fragments were introduced into the surface of alpha-chymotrypsin using acylation by anhydrides of aromatic carboxylic acids and reductive alkylation by aliphatic aldehydes. As a result of the hydrophilization the stability of the enzyme against irreversible thermoinactivation increased thousand-fold. The correlation is observed between the degree of hydrophilization of the protein surface and the increase in thermostability of modified alpha-chymotrypsin. The level of thermostability achieved by covalent modification of alpha-chymotrypsin is practically equal to thermostability of proteinases from extreme thermophiles, the most stable proteolytic enzymes currently known.

Covalent immobilization increases thermostability of thermitase, a stable thermophilic protease from Thermoactinomyces vulgaris. At 68°C, the catalytic activity of the immobilized enayme is more than 10 times greater than that of the native thermitase.

One method of describing wave motion in a fluid with continuous stratification is to use normal waves (modes). The propagation of internal gravity waves in closed rectangular regions whose boundaries coincide with planes through which there is no normal motion is essentially different from wave motion in an unbounded medium [1, 2]. This paper describes a theoretical and experimental investigation of the propagation of internal waves in an exponentially stratified fluid in a horizontal channel of finite height.

The catalytic activity, thermostability (resistance to monomolecular thermo-inactivation) and molecular mobility of chymotrypsin and trypsin mechanically entrapped into polymethacrylate and polyacrylamide gels have been studied. It has been established that the thermostability of the enzymes does not depend on the concentration of electroneutral polyacrylamide gel over the range of 0–50 w/w%. However, in polymethacrylate gel of concentration higher than 30 w/w%, when a high catalytic activity is retained, the thermostability of chymotrypsin dramatically increases: in 50 w/w% gel the first-order rate constant for thermoinactivation of the enzyme at 60°C is 10−5 that in water. Based on these data and also on experimentally obtained results on transitional and rotational diffusion of both native and modified enzymes, the following mechanism of enzyme stabilization is formulated and proved. In principle, the protein molecule of an enzyme may form with the three-dimensional lattice of polyelectrolyte gel multiple noncovalent linkages (via electrostatic or hydrogen bonds); as a result, the structure of the enzyme becomes more rigid and its thermostability should increase. However, since these bonds are relatively weak, in diluted gels they can hardly be realized, as the “quenching” of the transitional movement of the enzyme molecules, accompanying complex formation would have required a heavy entropy loss. At the same time, in concentrated gels, this unfavourable entropy contribution is absent as the polymer's lattice provides significant steric hindrances for the transitional diffusion, so that the molecules almost stop moving. That is why weak linkages between the protein globule and the support can be realized here. That the complex formation does take place is indicated by the fact the rotational diffusion of the protein molecules is almost completely frozen. When there is no specific protein-support interaction (in polyacrylamide gel), no deceleration of the rotational movement of the protein molecules occurs and no noticeable increase in the thermostability of the enzymes is observed. It is possible that the mechanism discovered by us functions in vivo and is responsible for the stability (and, which is important, for stability regulation) of the proteins incorporated in biomembranes. On the other hand, the results obtained by us may enrich enzyme engineering, as they allow the general strategy of production of stabilized enzymes to be outlined.

The question concerning the effect of low temperature on the fatigue characteristics of titanium alloys is addressed in the literature in connection with the latters' use in cryogenic engineering. This applies, among other things, to titanium alloy VT5-1kt (similar to alloy Ti-SAI-2~ in the United States). A number of studies devoted to the cyclic strength and crack resistance of this alloy and other titanium alloys in the 293-294 K interval are known [1-6]; as yet, however, there is no well-defined notion concerning this effect. Tobler and Reed [2] indicate that the cooling of specimens down to the helium temperature does not change the growth rate of fatigue cracks in both the single-phase alloy Ti-5AI-2.5Sn, and the two-phase alloy Ti-6AI-4V. In addition to this, researchers have noted an increase in the rate of crack growth with high stress intensity factors in a singlephase alloy of similar composition when loaded at 20 K [3], and even at 77 K [6].

A possible mechanism for so-called irreversible thermoinactivation of enzymes is suggested. We postulated that in many cases “irreversibly thermoinactivated enzyme” is an incorrectly folded polypeptide chain which, during a reasonable experimental interval, cannot transform into a correctly folded, thermodynamically stable and catalytically active conformation because of high kinetic barriers (intrinsic steric hindrances). Proceeding from this model, reactivation of “irreversibly” thermoinactivated trypsin (covalently attached to CNBr-activated Sepharose to prevent intermolecular processes such as aggregation or autolysis) was carried out. Thermoinactivated trypsin was first transformed to random coil by reduction of its disulphide bonds in 8M urea solution and then renaturated into a catalytically active form by reoxidation of disulphide bonds in the presence of dithiothreitol and in the absence of the denaturant.

The dependence of the catalytic activities of alpha-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.

1. For steels of the transition (07Kh16N6) and martensitic classes (03Kh12N10MT) the fracture toughness is fairly high under plane strain conditions down to 4.2°K. 2. The fracture toughness of aluminum alloys 1201 and 01381 increases with decreasing temperatures down to 4.2°K, although the presence of defects in weld seams cuts the structural strength in half. 3. The fracture toughness of titanium alloy VT5-1kt is approximately the same as that of the steels at 4.2°K. The value of KIc decreases in liquid hydrogen, which is due to the interaction of the metal with gaseous hydrogen.

The results of a theoretical and experimental investigation of the phase structure of three-dimensional internal waves in a horizontal channel occupied by an exponentially stratified fluid are presented. The stationary internal waves excited by a source moving uniformly along the bottom of a channel with rigid covers are considered. A dispersion relation, on the basis of which the phase structure of the internal waves in the far zone is investigated, is obtained within the context of the linear theory. The dependence of the boundary of the region of wave disturbances on the internal Froude number F is found. The equation of the lines of constant phase is obtained in the first approximation for large F. By means of an IAB-451 shadowgraph instrument, using the dark field method with vertical illumination, the phase patterns of the internal waves are obtained. The angles of the wave disturbance zone are measured in the region of relatively large F. Similarity of the phase patterns for the first three-dimensional mode is obtained experimentally for large F.

A comparison of the structure of (a) proteins from thermophilic and mesophilic microorganisms, (b) closely related proteins with different thermostability from various mesophilic sources, and (c) mutationally altered enzymes with those from wild strains has been carried out. The main molecular mechanisms existing in nature for the creation of thermostable proteins have been elucidated. The most important mechanism is the strengthening of hydrophobic interactions in the interior of the protein globule. This mechanism has been employed to advance a novel approach to enzyme stabilization which consists of the following steps. A protein is first made to unfold into a random coil-like state and then the folding of the protein is performed in one of three ways: (1) in ‘non-native’ conditions, (2) in the presence of substances which can interact with the protein in a noncovalent fashion, (3) after covalent modification of the unfolded protein.

The behavior of internal waves in a vertically bounded channel differs considerably from the wave motion in an infinite stratified fluid. In [1] the phase structure of the internal waves in an exponentially stratified layer of fluid between rigid horizontal planes was experimentally and theoretically investigated. A characteristic feature of such a channel is the boundedness of the phase and group velocities of each mode. Below, the case of an exponentially stratified channel between layers of homogeneous unbounded fluid is considered.

Reductive alkylation of amino groups in α-chymotrypsin by glyoxylic acid in the presence of sodium cyanoborohydride results in essential stabilization of the enzyme against irreversible thermoinactivation. At 60°C, the stabilizing effect is higher than one thousand, and it increases even more with rising the temperature.

The problem of the inter-relation between the structure and stability of proteins includes two main questions: firstly, how can we discover the molecular characteristics of the structure of proteins which are responsible for their stability? Secondly, how, knowing the general molecular causes of stability, can we alter the structure of a specific protein in order to increase its stability, i.e. to stabilise it? In the present review an attempt is made to present the current state of both the first (fundamental) and second (applied) aspects of this problem. The bibliography includes 250 references.

The experimental results of studying the effect of homogeneous stratification of the fluid on the conditions of generation of a Karman vortex street [1] developing in the wake of a cylinder in steady horizontal motion are described. In a homogeneous medium at Reynolds numbers Re >5 two symmetrical regions of vorticity of opposite sign are formed behind the cylinder and move together with the latter. As the speed of the cylinder increases, the link between the vortices and the cylinder grows weaker, the vortices are stretched out along the flow and at Re > 40 begin to separate alternately, forming a vortex street in the wake. At first, the frequency of vortex separation increases sharply with increase in Re, but then levels off. It is found that in a uniformly stratified fluid the onset of vortex separation from the moving cylinder is delayed. The dependence of the critical Reynolds number (onset of vortex separation) on the internal Froude number is obtained. The effect of stratification of the fluid on the frequency of separation of the vortices in the Karman street is investigated. The effect of the Froude number on the dependence of the Strouhal number on the Reynolds number is established.

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.2) covalently immobilized on Sepharose or in polyacrylamide gel has been irreversibly denatured at 70–90°C and then reactivated in an almost 100% yield. Thermoinactivated enzyme in first made to unfold under the action of urea with SS bonds being simultaneously reduced and then made to refold (under the optimal conditions for the thiol-disulfide exchange) into its native conformation. It is demonstrated that the ‘irreversible monomolecular thermoinactivation-reactivation’ cycle can be repeated many times. The contribution of various mechanisms to thermoinactivation of the enzymes is discussed. Based on the data obtained, the irreversible thermoinactivation of enzymes under investigation should be ascribed only to changes in their secondary and tertiary structures; the primary structure is not likely to be affected.

The molecular mechanisms of protein inactivation, i.e. aggregation, thiol-disulphide exchange, alteration of the primary structure, dissociation of cofactor molecules from the active centre, dissociation of the oligomeric proteins into subunits and conformational changes have been analysed. All these mechanisms are closely interrelated during inactivation of proteins. However, in many cases, the conformational changes accompany and trigger other inactivation processes. Reactivation of irreversibly inactivated proteins is·discussed. Reactivation can be successful when inactivation has been caused by aggregation, modification of SH-groups (or S-S bonds) or as a consequence of irreversible conformational changes.

alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.

Prevention of the denaturation of enzymes is frequently indispensable to their technological application. This is exemplified by the following five operational factors of importance in the practical use of enzymes: 1. Enzymes isolated from their in Vivo environment usually become labile, and their lifetime sometimes does not exceed minutes (1–4). For example, the stability of immobilized aspartase proved insufficient for technological use of this enzyme (5). 2. For many processes, elevated temperature is desirable. The rates of chemical reactions, including enzymatic, increase with temperature; so that at higher temperatures, the required conversion of the substrate will be achieved in a shorter time or with a smaller amount of immobilized enzyme. This is very important for technological processes where the cost of the enzyme is a limiting factor, e. g., glucoamylase (6). Also, higher temperatures allow germ-free conditions to be maintained (7), which are indispensable for, say, the food industry (8,9). On the other hand, denaturation of biocatalysts intensifies (1–3) at elevated temperatures. 3. Reaction equilibrium may be such that the required products are obtained only if the reaction is carried out in an aqueous-organic mixture with a high proportion of the organic component. Under such conditions enzymes as a rule lose their catalytic activity or specificity (2,10; see also references in 11). 4. Sometimes the pH optimum of an enzymatic reaction and the pH range within which the enzyme is stable do not coincide. A good example is synthesis of penicillin antibiotics under the action of penicillin amidase; equilibrium in the synthesis is shifted toward the product in an acidic medium, where the enzyme is rather unstable (12). 5. It may not be convenient to use an enzyme immediately upon harvesting or isolation. Hence there is a need for stabilization against inactivation during prolonged storage (13).

Numerous examples of protein inactivation are considered. Several principal inactivation mechanisms are distinguished: aggregation, thiol-disulfide exchange, alteration in the protein primary structure, dissociation of cofactor molecules from the enzyme active site, dissociation of proteins with quaternary structure into subunits, and conformational changes. All the mechanisms interweave during protein inactivation. In many cases, however, conformational transformations in protein play an important role; they accompany and often trigger other mechanisms of inactivation which would be impossible without preliminary alterations in protein structure. The published data on reactivation of inactivated proteins are considered. Reactivation was a success when inactivation was caused by aggregation, chemical modification of SH-groups of S-S bonds, or resulted from "irreversible" conformational changes.