Unusual (saw-like) temperature dependence of the rate of irreversible thermoinactivation of enzymes

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.     

Correlation of enzyme thermostability and its surface hydrophobicity (using a modified alpha-chymotrypsin as an example)

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.     

High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: alpha-Chymotrypsin

Based on the idea that proteins can be stabilized by a decrease in the thermodynamically unfavorable contact of the hydrophobic surface clusters with water, alpha-chymotrypsin (CT) was acylated with carboxylic acid anhydrides or re-ductively alkylated with aliphatic aldehydes. Modification of CT with hydrophilic reagents leads to 100-1000-fold increase in stability against the irreversible thermoinactivation. The correlation holds: the greater the hydrophilization increment brought about by the modification, the higher is the protein thermostability. After some limiting value, however, a further increase in hydrophilicity does not change thermostability.It follows from the dependence of the thermoinactivation rate constants on temperature that for hydrophilized CT there is the conformational transition at 55-65 degrees C into an unfolded state in which inactivation is much slower than that of the low-temperature conformation. The thermodynamic analysis and fluorescent spectral data confirm that the slow inactivation of hydrophilized CT at high temperatures proceeds via a chemical mechanism rather than Incorrect refolding operative for both the native and low-temperature form of the modified enzyme. Hence, the hydrophilization stabilizes the unfolded high-temperature conformation by eliminating the incorrect refolding. (c) 1992 John Wiley & Sons, Inc.     

Operational stability of copolymerized enzymes at elevated temperatures

The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.     

Study of the kinetics of thermoinactivation of lysyl-tRNA-synthetase from the liver of x-irradiated rats

A study was made of the kinetics of thermoinactivation of lysyl-tRNA-synthetase isolated from rat liver at early times of radiation damage development after the effect of a minimum absolutely lethal X-radiation dose (0.21 C/kg). The thermostability of a dimer form of the enzyme was shown to be higher than that of a monomer. It was established that substrates had a stabilizing effect on the enzyme during thermoinactivation. On the basis of the data obtained from the studies in the kinetic properties of the enzyme and the thermoinactivation a conclusion is made that lysyl-tRNA-synthetase is stabilized during subunit aggregation. The thermostability of the enzyme was decreased by irradiation.     

Thermostabilization of enteroviruses by estuarine sediment

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Thermostabilization of enteroviruses by estuarine sediment.

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.     

Interaction between 5-lipoxygenase and allosteric effector--sodium dodecyl sulfate

The role of allosteric effector--sodium dodecyl sulfate (SDS) in the lipoxygenase catalysis in micelle system has been studied. The effect of the stable hydrophobic bis-nitroxides, blocking the free radical transformation, on the oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase from potato tuber has been investigated. The inhibiting effect of nitroxide compounds on oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase depends on SDS concentration. The inhibition percentage is determined by the substrate nature and presence of allosteric effector. The presence of SDS did not lead to an appreciable change in the pKa values of ionogenic enzyme groups. The effect of SDS and micellar system on thermodynamic parameters for thermoinactivation of 5-lipoxygenase was studied. It was found that thermoinactivation rate constants and activation energy of enzyme thermoinactivation were increased in the presence of SDS. It is suggested that interaction of 5-lipoxygenase and allosteric effector--SDS intensifies the dissociation of radical intermediates from the active site of the enzyme. These findings are of physiological significance in the light of the lipoxygenase involvement in the membrane lipid peroxidation.     

Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.     

Sweetness of sweet protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions

Thermostability of thaumatin and mechanisms of thermoinactivation were examined at 80 degrees C in the pH range from 2 to 10. The sweetness of thaumatin disappeared on heating at pH above 7 for 15 min, but the sweetness remained even after heating at 80 degrees C for 4 h at pH 2. This indicated that the sweet protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions. Prolonged heating of thaumatin under acid conditions slowly reduced sweetness, and produced a heterogeneous population of molecules, all of which was soluble and monomeric. The resultant molecules were clearly distinct from those generated by heating at pH above 7. Hydrolysis of peptide bonds and other irreversible chemical reactions slowly took place in the molecule heated under acid conditions, and it would be, in part, a cause of thermoinactivation of thaumatin under acid conditions. The thermostability of thaumatin and the mechanism of thermoinactivation were largely dependent on pH.     

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes in supercritical carbon dioxide

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes was performed in supercritical CO(2) and liquid CO(2) at 308.2 and 333.2 K at pressures of 6.1 and 10.1 MPa. The enzymatic activity of coated alpha-chymotrypsin complexes for dipeptides synthesis at 10.1 MPa in supercritical CO(2) (SC-CO(2)) was higher than that in a liquid CO(2) and ethyl acetate solution at 6.1 MPa. The behavior of alpha-chymotrypsin in SC-CO(2) was similar to that in liquid ethyl acetate. And increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in SC-CO(2). Furthermore, the control of the water content in the reaction media had a dominant effect on the enzymatic activity. The maximum conversion for the dipeptide synthesis by the surfactant-coated alpha-chymotrypsin was obtained at 4% water content. The alpha-chymotrypsin complexes exhibited a higher enzymatic activity than native alpha-chymotrypsin in SC-CO(2). The nonionic surfactants l-glutamic acid dialkyl ester ribitol amide and sorbitan monostearate were more favored than the anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate.     

Bicarbonate protects the water-oxidizing complex of photosystem II against thermoinactivation in intact <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Currently available data about bicarbonate (BC) action on the Mn-containing water-oxidizing complex (WOC) of the photosystem II (PSII) were obtained almost solely in vitro, e.g. on subchloroplast membrane fragments enriched with PSII. To investigate the in vivo BC effect on the PSII donor side, we used the method of dark thermoinactivation of intact Chlamydomonas reinhardtii cells. Photosynthetic activity of PSII was measured as photoinduced changes in the PSII chlorophyll fluorescence yield and as the rate of photosynthetic oxygen evolution. To exclude a “direct” effect of the absence of BC on the PSII activity, before measurements of the photosynthetic activity, the concentration of BC in all samples was equalized by addition of NaHCO₃ to each of them (except for those that contained 5 mM of NaHCO₃ during thermoinactivation) to reach the final concentration of 5 mM. This allowed registering only so-called “irreversible” (i.e., not reversible by subsequent addition of BC) effect of the absence of BC during thermoinactivation. It was shown that, if 5 mM NaHCO₃ was added to the medium before thermoinactivation, the rate of inactivation of the PSII donor side was lower than in BC-depleted medium 1.5-to 2-fold. The obtained results are interpreted as an indication that BC protects the donor side of PSII against thermoinactivation in vivo, in intact C. reinhardtii cells. This proves the correctness of the earlier proposition that BC is an integral constituent of the Mn-containing water-oxidizing complex of PSII. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 342–349. The article was translated by the authors.     

On the role of reversible denaturation (unfolding) in the irreversible thermal inactivation of enzymes

The contribution of the reversible thermal unfolding of an enzyme toward the overall irreversible thermoinactivation process has been examined both theoretically and experimentally. Using bovine pancreatic ribonuclease as a model, we have studied the effect of such variables as pH and salts both on the equilibrium constant of reversible denaturation and on the rate constant of the overall irreversible process. It has been demonstrated that at temperatures where a significant fraction of the enzyme molecules are in the native conformation, there is a correlation between the enzyme thermostabilities with respect to the reversible and irreversible inactivations: greater stability against the former is accompanied by greater stability against the latter. On the other hand, at very high temperatures (where essentially all of the enzyme molecules are unfolded), such a correlation does not exist. These findings are considered in terms of a kinetic model for irreversible enzyme thermoinactivation, and the implications of the derived relationship are discussed.     

Properties of alpha-chymotrypsin enclosed into polycarbonate microcapsules. Estimation of the diffusion effect

The conditions for the microencapsulation of alpha-chymotrypsin into semi-permeable polycarbonate membranes are selected. The diameter of the microcapsules is 100-150 microns. The microencapsulation can be carried out at any value of pH. The effect of the diffusion in the hydrolysis of some esters by microcapsulated alpha-chymotrypsin was estimated. The thermostability of the microencapsulated enzyme was studied.     

Why do solution additives suppress the heat‐induced inactivation of proteins? Inhibition of chemical modifications

Thermoinactivation of proteins is prevented by several kinds of solution additives such as chaotropes, amino acids, amino acid derivatives, and polyamines. Here, we investigated the molecular mechanisms of action of the various additives that prevent thermoinactivation of bovine pancreatic ribonuclease A and hen egg white lysozyme. The thermoinactivation of both proteins in the presence of additives showed clear correlations with deamidation and β-elimination of the proteins. Thus, experimental evidences indicated that the effects of additives on thermoinactivation of proteins are highly due to the suppression of chemical modifications. To our surprise, not only the suppressive effect of the additives on heat-induced inactivation but also that on the chemical modification of proteins is remarkably similar by comparison of two unrelated proteins. This finding indicates the generality of the effects of additives on heat-induced chemical modification of proteins.     

Kinetics of adsorption,desorption, and exchange of alpha-chymotrypsin and lysozyme on poly(ethyleneterephthalate) tracked film and track-etched membrane

Adsorption kinetics of (125)I-radiolabeled alpha-chymotrypsin at pH 8.6 was studied in a laminar regime between two walls of poly(ethyleneterephthalate) tracked films and membranes. Adsorption kinetics in the presence of solution (10 microg/mL), desorption by rinsing with buffer, and the following exchange of proteins by flowing unlabeled solution were measured. At pH 8.6, alpha-chymotrypsin is almost neutral and can be mostly removed from the film surface, contrary to positive lysozyme adsorbed at pH 7.4. Results suggest that alpha-chymotrypsin is irreversibly adsorbed in pores, while desorption and exchange occur on membrane flat faces. A method is proposed to determine adsorption kinetics in the pores. Kinetics of desorption and exchange of alpha-chymotrypsin from the film surface can be described by stretched exponential functions in the examined time domain with the same exponent, beta approximately 0.62, which does not depend also on the former adsorption duration. However, the mean residence time at the interface is about 2.5 times greater in the presence of only the buffer than that in the presence of solution. This effect could be explained by a fast exchange at the arrival of unlabeled solution for a part of the adsorbed population.     

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.