Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.     

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.     

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

Urea and methylamine effects on rabbit muscle phosphofructokinase. Catalytic stability and aggregation state as a function of pH and temperature

The effects of urea and several methylamine solutes on the catalytic stability and aggregation properties of rabbit muscle phosphofructokinase were assessed at physiologically realistic concentrations of the solutes under several pH and temperature regimes. The loss of catalytic activity observed under conditions of pH-induced cold lability was significantly reduced in the presence of trimethylamine-N-oxide, N-trimethylglycine and N-methylglycine (order of decreasing effectiveness). The concentration-dependent methylamine stabilization of the enzyme, seen with as little as 50 mM trimethylamine-N-oxide, was accompanied by increased aggregation of the enzyme to molecular weights greater than the tetramer (polytetramer) as solute concentration was raised to 400 mM. At pH 6.5-6.7 and 25 degrees C, concentrations of urea greater than 25 mM promoted a time-dependent inactivation of the enzyme which was enhanced at lower temperatures. The urea sensitivity of the enzyme exhibited with 0.8 M urea for 1 h at pH 8.0 did not result in measurable inactivation. The fluorescence emission wavelength maximum of the enzyme was shifted to longer wavelengths and the fluorescence intensity was increased as pH was lowered to 7.0, suggesting the occurrence of a protein conformation change as specific amino acid residues of the tetramer became protonated. Measurements of enzyme light scattering indicated that perturbation by urea was correlated with tetramer dissociation, which was irreversible by dialysis at 25 degrees C. The urea and methylamine influences on phosphofructokinase activity and structure were not counteracting. The synergistic interactions among pH, temperature, and solutes observed with phosphofructokinase are compared to effects on other associating-dissociating protein systems in order to evaluate possible mechanisms of action of these low molecular weight solutes.     

Temperature-induced dissociation of protein aggregates: accessing the denatured state

The thermal denaturation of lysozyme and ribonuclease A (RNase A) under reducing and nonreducing conditions at neutral pH has been monitored by Fourier transform infrared spectroscopy. In the absence of the reductant, lysozyme and RNase A undergo apparent three- and two-state denaturation, respectively, as observed from the conformation-sensitive amide I' band. For both proteins the hydrogen-deuterium exchange takes place at lower temperatures than the main denaturation temperatures, suggesting that a transient denaturation mechanism occurs. The observed transition at 51.2 degrees C during the denaturation of lysozyme is attributed to this transient effect, rather than to the loss of tertiary structure. Under reducing conditions lysozyme aggregates during the heating phase, whereas RNase A shows only a minor aggregation, which further increases during the cooling step. The reduced stability of both proteins can be correlated with the transient denaturation behavior, which is also suggested to be involved in protein aggregation at physiologically relevant temperatures. In addition, it is shown that when the temperature is further increased, the amorphous aggregates dissociate. Comparison of the dissociated states with the denatured states obtained under nonreducing conditions indicates that these states have the same conformation. By using a two-dimensional correlation analysis we were able to show that the dissociation is preceded by a conformational change. It is argued that this extends to other types of perturbation.     

Equilibrium studies on the refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH4-5tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process. At pH approximately 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 degrees C, enzyme concentration 0.4-59 mug/ml) up to 95% enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

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)     

Thermal stabilization of formaldehyde dehydrogenase by encapsulation in liposomes with nicotinamide adenine dinucleotide

The thermal stability of formaldehyde dehydrogenase (FaDH) from Pseudomonas sp. was examined and controlled by encapsulation in liposomes with β-reduced nicotinamide adenine dinucleotide (NADH). The activity of 4.8 μg/mL free FaDH at pH 8.5 in catalyzing the oxidation of 50 mM formaldehyde was highly dependent on temperature so that the activity at 60 °C was 27 times larger than that at 25 °C. Thermal stability of the FaDH activity was examined with and without liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Rapid deactivation of free FaDH was observed at 60 °C because of its dissociation into two subunits. The rate of dissociative deactivation of POPC liposome-encapsulated FaDH was smaller than that of the free enzyme. The liposomal FaDH was however progressively deactivated for the incubation period of 60 min eventually leading to complete loss of its activity. The free FaDH and NADH molecules were revealed to form the thermostable binary complex. The thermal stability of POPC liposome-encapsulated FaDH and NADH system was significantly higher than the liposomal enzyme without cofactor. The above results clearly show that NADH is a key molecule that controls the activity and stability of FaDH in liposomes at high temperatures.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.     

Denaturation-renaturation of the fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein

The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Pressure-induced structural changes of pig heart lactic dehydrogenase

Lactic dehydrogenase from pig heart can be reversibly dissociated at hydrostatic pressures above 1000 bar. The breakdown of the native quaternary structure occurs at lower pressures compared to the isoenzyme from pig, skeletal muscle. As shown by hybridization experiments of the two isoenzymes the final product of dissociation is the homogeneous monomer. Fluorescence emission spectra of the monomeric enzyme at elevated pressure are characterized by a decrease in fluorescence intensity without any red shift, indicating that no significant unfolding occurs upon high-pressure dissociation. The spectral changes are comparable to those observed after acid dissociation. The amount and rate of deactivation depend on pressure and on the conditions of the solvent. The presence of various anions (Cl^?, SO^2?₄. HPO₄^2?) has no effect on the stability of ihe enzyme towards pressure. High-pressure denaturation (as monitored by intrinsic protein fluorescence), and deactivalion (measured immediately after decompression) run parallel; the pressure dependence of their first-order rate constants is characterized by an activation volume ΔV^≠_Dc = ?140 = 10 cm³/mol. As taken from the yield of reconstitution, dissociation, denaturation and deactivation are found to be fully reversible provided the pressure does not exceed a limiting value (p = 1000 bar in Tris. pH 7.6: 24 h incubation at 20°C). After extended incubation beyond the limiting, pressure of 1000 bar. “irreversible high-pressure denaturation” occurs which is accompanied bv partial aggregation after decompression. The coenzyme, NAD⁺ stabilizes the native tetramer shifting the dissociation equilibrium to higher pressures. The overall dissociation-association reaction can be quantitatively described by a consecutive dissociation/unfolding mechanism N?4 M^'?4 M^☆ (where N is the native tetramer. and M^' and M^☆ two different conformations of the monomer). The reaction volume of the dissociation reaction N?4 M^' is found to be ΔV_Diss = ?360 = 30 cm³/mol: as indicated by the pressure dependence of the yield of reconstitution, the reaction volume of the equilibrium M^'?M^XXX is also negative.