Two-state irreversible thermal denaturation of Euphorbia characias latex amine oxidase

Thermal denaturation of Euphorbia latex amine oxidase (ELAO) has been studied by enzymatic activity, circular dichroism and differential scanning calorimetry. Thermal denaturation of ELAO is shown to be an irreversible process. Checking the validity of two-state it really describes satisfactorily the thermal denaturation of ELAO. Based on this model we obtain the activation energy, parameter T(*) (the absolute temperature at which the rate constant of denaturation is equal to 1 min(-1)), and total enthalpy of ELAO denaturation. HPLC experiments show that the thermal denatured enzyme conserves its dimeric state. The N(2)-->kD(2) model for thermal denaturation of ELAO is proposed: where N(2) and D(2) are the native and denatured dimer, respectively.     

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.     

Kinetic study on the irreversible thermal denaturation of yeast phosphoglycerate kinase

Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins. Correspondence to: J. M. Sanchez-Ruiz     

Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins

Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed.     

Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase.

The irreversible thermal inactivation of Bacillus licheniformis alpha-amylase was studied. A two-step behaviour in the irreversible denaturation process was found. Our experimental results are consistent only with the two-step model and rule out the two-isoenzyme one. They suggest that the deactivation mechanism involves the existence of a temperature-dependent intermediate form. Therefore the enzyme could exist in a great number of active conformational states. We have shown that Ca2+ is necessary for the structural integrity of alpha-amylase. Indeed, dialysis against chelating agents leads to a reversible enzyme inactivation, though molecular sieving has no effect. Further, the key role of Ca2+ in the alpha-amylase thermostability is reported. The stabilizing effect of Ca2+ is reflected by the decrease of the denaturation constants of both the native and the intermediate forms. Below 75 degrees C, in the presence of 5 mM-CaCl2, alpha-amylase is completely thermostable. Neither other metal ions nor substrate have a positive effect on enzyme thermostability. The effect of temperature on the native enzyme and on one intermediate form was studied. Both forms exhibit the same optimum temperature. Identical activation parameters for the hydrolytic reaction catalysed by these two forms were found.     

Molecular mechanisms of the irreversible thermal denaturation of guinea-pig liver transglutaminase.

When transglutaminase is heated at temperatures above 40 degrees C, it loses its activity according to a two-step mechanism [Nury, Meunier & Mouranche (1989) Eur. J. Biochem. 180, 161-166]: N----X(TD)----D However, the nature of the molecular events responsible for the irreversible denaturation is still unknown. Investigation of the effects of dithiothreitol and 5,5'-dithiobis-2-nitrobenzoate on the kinetics of inactivation, titrations of ammonia released by deamidation and of thiol groups on the native and denatured enzymes and SDS/PAGE rule out the involvement of covalent processes during the denaturation of transglutaminase at 55 degrees C and pH 7. Of the two possible kinds of non-covalent events, i.e. unfolding of the polypeptide chain and aggregation of enzyme molecules, we show that both occur, though only the former process is responsible for the denaturation. The latter process, aggregation, follows the unfolding of the molecules.     

Molecular mechanism for osmolyte protection of creatine kinase against guanidine denaturation.

The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine-denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low concentrations of dimethysulfoxide (< 40%), glycine (< 1.5 m), proline (< 2.5 m) and sucrose (< 1.2 m) reduced the inactivation and unfolding rate constants of creatine kinase, increased the change in transition free energy of inactivation and unfolding (Delta Delta G(u)) and stabilized its active conformation relative to the partially unfolded state with no osmolytes. In the presence of various osmolytes, the inactivation and unfolding dynamics of creatine kinase were related to the protein concentrations. These osmolytes protected creatine kinase against guanidine denaturation in a concentration-dependent manner. The ability of the osmolytes to protect creatine kinase against guanidine denaturation decreased in order from sucrose to glycine to proline. Dimethysulfoxide was considered separately. This study also suggests that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also have an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations.     

Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin

A differential scanning calorimetry study of the thermal denaturation of Bacillus thermoproteolyticus rokko thermolysin was carried out. The calorimetric traces were found to be irreversible and highly scan-rate dependent. The shape of the thermograms, as well as their scan-rate dependence, can be explained by assuming that the thermal denaturation takes place according to the kinetic scheme N k----D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation, N the native state, and D the unfolded state or, more probably, a final state, irreversibly arrived at from the unfolded one. On the basis of this model, the value of the rate constant as a function of temperature and the activation energy have been calculated. It is shown that the proposed model may be considered as being one particular case of that proposed by Lumry and Eyring [Lumry, R., & Eyring, H. (1954) J. Phys. Chem. 58, 110] N in equilibrium D----I, where N is the native state, D the unfolded one, and I a final state, irreversibly arrived at from D. Lastly, some comments are made on the use of the scan-rate effect on the calorimetric traces as an equilibrium criterion in differential scanning calorimetry.     

Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.     

The role of creatine kinase and arginine kinase in muscle.

Arginine and creatine kinase activities in different muscles are compared with calculated maximum rates of ATP turnover. The magnitude of the kinase activities decreases in the following order: anaerobic muscles and vertebrate skeletal muscles greater than heart muscle greater than insect flight muscle. The maximum activity of phosphagen kinases (i.e. creatine kinase and arginine kinase), in the direction of phosphagen formation, is lower than the calculated maximum rate of ATP turnover in insect flight muscle or rat heart.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion activation of monkey muscle creatine kinase

Creatine kinase from rhesus monkey skeletal muscle is activated by acetate and other short chain fatty acids. Activation is associated with lower Km and higher Vmax values at less than saturating substrate concentrations but does not occur when both substrates are saturating. No co-operativity between subunits is evident in the activation process. It appears that acetate promotes the mutual enhancement by substrates in their binding by inducing the optimum enzyme conformation normally associated with substrate saturation. Conservation of this activation effect through the evolution of the phosphagen kinases implies that it may well be of physiological significance.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Kinetic study on the irreversible thermal denaturation of Schistosoma japonicum glutathione S-transferase

The thermal unfolding pathway of the Schistosoma japonicum glutathione S-transferase (Sj26GST) was previously interpreted by applying equilibrium thermodynamics and a reversible two-state model (Kaplan et al., (1997) Protein Science, 6, 399-406), though weak support for this interpretation was provided. In our study, thermal denaturation of Sj26GST has been re-examined by differential scanning calorimetry in the pH range of 6.5-8.5 and in the presence of the substrate and S-hexylglutathione. Calorimetric traces were found to be irreversible and highly scan-rate dependent. Thermogram shapes, as well as their scan-rate dependence, can be globally explained by assuming that thermal denaturation takes place according to one irreversible step described by a first-order kinetic constant that changes with temperature, as given by an Arrhenius equation. On the basis of this model, values for the rate constant as a function of temperature and the activation energy have been determined. Data also indicate that binding of GSH or S-hexylglutathione just exert a very little stabilising effect on the dimeric structure of the molecule.     

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.     

Folding pathway for partially folded rabbit muscle creatine kinase.

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.     

The creatine kinase system in smooth muscle

Despite the energetic flux being much lower in smooth muscle compared to striated muscles (such as the heart and skeletal muscle) creatine kinase (CK) has been found present and active in all smooth muscles studied to date. A complete CK circuit has been identified, with CK found in the mitochondria, contractile elements, membrane pumps and the cytoplasm. CK isoenzymes are coupled to many cellular energetic processes and appears to be involved in energy production and consumption by acting as an energy transducer. The CK system responds to pathological insults and development (e.g. hypertrophy and gestation respectively) by changes in sub-cellular distribution localization, isoenzymes, and specific activity. The conclusion from these observations is that creatine kinase is intimately involved in the energetic system of smooth muscle.Abbreviations CK creatine kinase - Mi-CK mitochondrial creatine kinase - Cr creatine - PCr phosphocreatiner - NMR nuclear magnetic resonance - SHR spontaneously hypertensive rat - -GPA -guanidinopropionic acid     

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate.

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate was studied. It was found that up to five of the sixteen histidine groups per enzyme subunit could be modified, and under the conditions employed, there was no evidence for formation of the disubstituted derivative of histidine. Evidence was obtained for small but significant amounts of modification of lysine and cysteine groups; tyrosine groups were not modified. Modification of the enzyme led to inactivation; this could be protected against by inclusion of substrates or, more effectively, by inclusion of the combination MgADP plus creatine plus nitrate, which is thought to produce a 'transition-stage-analogue' complex. Analysis of data on the rates of inactivation and the stoicheiometry of modification suggested that there was one essential histidine group per enzyme subunit, modification of which led to inactivation.     

DSC study of reversible and irreversible thermal denaturation of concentrated globular protein solutions

A calorimetric study of the thermal denaturation of bovine serum albumin, RNAase and catalase in concentrated solutions (crystals) has been carried out. The results obtained for RNAase studied within the pH range 2.5-8.5 show that for concentrated solutions there is an interval of pH where, on cooling of the solution which had undergone denaturation, its renaturation is observed. In the case of concentrated and dilute solutions of RNAase these intervals coincide. The study of RNAase under such conditions at various heating rates shows that there is a range of rates in which the process of denaturation of concentrated solutions can be considered as reversible. The dependences of Td and Hd on pH and concentration of solutions have been determined. The denaturation enthalpy of concentrated solutions like in dilute ones, has been found to be independent of the pH of solutions, and the experimentally registered change has been proved to be the result of its dependence on temperature. A new method of determination of protein denaturation enthalpy under the conditions of intensive molecule aggregation is suggested. The forms of irreversibility as appearing in the calorimetric experiment were determined by comparing reversible and irreversible denaturation under continuous and step-heating regimes. It is shown that the decrease in Tmax and the narrowing of the heat absorption peak in the case of decreasing heating rates of protein solutions, observed under certain environmental conditions, results from the irreversibility of the denaturation process.