Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability

We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

Three easy pieces

BackgroundDifferential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25 °C, may become unreliable due to difficulties in the determination of ΔC_p.MethodsThe combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.Results and conclusionsThis paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔC_p value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

The thermal stability of rhodopsin and opsin

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E_a) and entropies of activation (ΔS‡) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species —frog, squid, cattle—presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.     

Studies on the stability of 11S globulin from soybeans by differential scanning microcalorimetry

The scanning microcalorimetry method was applied to the study of thermal denaturation of 11S globulin (glycinin), dodecameric globular protein from soybeans at pH 7.6 and the range of NaCl concentrations from 0 to 0.9 m. The specific enthalpy of denaturation was shown to be the linear function of temperature. The ratio of the calorimetric enthalpy to the effective one (van't Hoff's) per protomer of glycinin was 1.23 ± 0.05. It is concluded that at the first approximation glycinin protomers denaturated independently, in conformity with the two-state model. The plotted temperature-dependent specific free energy of glycinin denaturation at different NaCl concentrations demonstrated that an increase in the salt content brought about the rise of the protein stability. The maximal glycinin stability is reached at about 273 K. The molar free energy of denaturation at 273 K in 1 m NaCl is 1320 kJ/mol.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Denaturation of cytochrome b5 by guanidine hydrochloride: evidence for independent folding of the hydrophilic and hydrophobic moieties of the cytochrome molecule.

Hepatic microsomal cytochrome b₅ is an amphipathic protein consisting of a hydrophilic (heme-containing) moiety and a Hydrophobic (membrane-binding) segment and exists in aqueous media as a micelle. Circular dichroism studies indicated that denaturation of cytochrome b₅ by guanidine hydrochloride is a two-stage process, the first transition occurring at the denaturant concentration of about 2.6 m and the second one at 5.0–5.5 m. A hydrophilic fragment of the cytochrome lacking the hydrophobic segment, on the other hand, underwent one-stage denaturation at a guanidine hydrochloride concentration of about 2.9 m. Detachment of the heme from the cytochrome and the fragment, measured by optical absorption, was effected at the denaturant concentrations of 2.6 and 2.9 m, respectively. Gel chromatography experiments showed that dissociation of the cytochrome micelle into the monomers took place concomitant with the second stage of denaturation. It is suggested that the two moieties of the cytochrome molecule exist as relatively independent domains undergoing unfolding separately and the hydrophobic domain is much more resistant to denaturation. It was further found that removal of the heme from the cytochrome by acid-acetone treatment rendered the hydrophilic domain unstable but did not affect the structure of the hydrophobic domain.     

Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg⁸⁰-Asp⁹³ as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.     

α-Chymotrypsin stability in aqueous-acetonitrile mixtures: is the native enzyme thermodynamically or kinetically stable under low water conditions?

Like many proteins, α-chymotrypsin is denatured in 50% volume aqueous-acetonitrile mixtures. However, it also shows high catalytic activity in 70% or more acetonitrile. Good activity in two different aqueous organic composition ranges has been described for several other enzymes. The stability of the native protein under low water conditions is generally believed to be a kinetic phenomenon, though there are also arguments for thermodynamic stability. We have distinguished between these possibilities by studying the effects of changing medium composition at different times. In preliminary experiments, we found catalytic activity could be recovered by adding neat acetonitrile to chymotrypsin in a 50% mixture, suggesting that the enzyme could renature under these conditions. However, in the 50% mixture, the true initial activity at 30°C is not zero, as the literature suggests. Instead, there is an initial burst of product formation over a few minutes, after which the enzyme becomes inactivated. By pre-incubating a 50% aqueous-acetonitrile mixture at 30°C prior to enzyme addition, the product burst could be eliminated. Activity could not then be recovered by slow addition of acetonitrile to the denatured enzyme. In contrast, it was possible to renature by dilution with aqueous buffer so that regeneration of catalytic activity was achieved. Thus, the good practical performance at high acetonitrile concentrations almost certainly results from a high kinetic barrier towards denaturation. The kinetics of enzyme denaturation in 50% and 70% acetonitrile were also investigated both at 30 and 20°C. Loss of catalytic activity was faster at higher temperature and at lower acetonitrile concentrations.     

Inhibition Kinetics and the Aggregation of α-Glucosidase by Different Denaturants

Kinetic changes of alpha-glucosidase from Saccharomyces cerevisiae in guanidinium chloride (GdmCl) and SDS solutions were investigated. The results showed both denaturants can lead conformational changes and loss of enzymatic activities. However, the concentrations of denaturants causing loss of activities were much lower than that of conformational changes, which suggested that the conformation of active site of α-glucosidase was more fragile than the whole molecular conformation in response to the two denaturants. According to the different kinetic process of the enzyme in the GdmCl and SDS solutions, the further investigation on the process of denaturation were made, it showed GdmCl and SDS had different types of inhibition and different types of interaction with the enzyme. Furthermore, the mechanisms of the two denaturants were discussed.     

Analysis of the Spodoptera frugiperda Nuclear Polyhedrosis Virus Genome by Restriction Endonucleases and Electron Microscopy

Restriction endonuclease analysis was used to differentiate between four strains of Spodoptera frugiperda nuclear polyhedrosis virus from different geographical areas. In addition, partial denaturation was performed, and a partial denaturation map was constructed for the Ohio strain of this virus.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Two-domain structure of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase.

NADH-cytochrome b₅ reductase is an amphiphilic protein consisting of a hydrophilic (FAD-containing) moiety and a hydrophobic (membrane-binding) segment and exists in aqueous media as an oligomeric aggregate. Circular dichroism studies have shown that denaturation of the reductase by guanidine hydrochloride in the presence of Emulgen 109P, a nonionic detergent, is a two-stage process as a function of the denaturant concentration. The first transition occurs at about 1 m guanidine hydrochloride and the second one at much higher concentrations. The guanidine hydrochloride concentration causing the second-stage unfolding depends on the concentration of Emulgen 109P. A hydrophilic fragment of the reductase lacking the hydrophobic segment undergoes one-stage denaturation at about 1 m guandine hydrochloride regardless of the presence and absence of Emulgen 109P. Both the reductase as well as the hydrophilic fragment lose their NADH-ferricyanide reductase activity and FAD also at about 1 m guanidine hydrochloride in the presence of the detergent. These findings suggest that the first-stage denaturation of the reductase represents the unfolding of the hydrophilic moiety and the second one that of the hydrophobic segment. Gel chromatography experiments have suggested that in the presence of Emulgen 109P the reductase exists as a mixed micelle with the detergent and this aggregation state persists even after the first-stage denaturation (unfolding of the hydrophilic moiety). The dissociation of the mixed micelle seems to take place concomitant with the second-stage denaturation. It is concluded that the two moieties of the reductase molecule, though linked to each other covalently, exist as independent domains undergoing unfolding separately at least in the presence of Emulgen 109P. This structural feature of the reductase is similar to that of cytochrome b₅ reported by us. The reductase is, therefore, another example of amphiphilic membrane proteins having two structurally independent domains in the molecule.     

Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli,Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase α subunit have been compared by circular dichroism measurements. The three α subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (α-2 domain) and the N-terminal domain comes from S. typhimurium (α-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 °C showed that the α-2 domain of S. typhimurium was more stable than the α-2 domain of E. coli, but the α-1 domain of S. typhimurium was less stable than the α-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 °C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Effect of acidicpH on the protein carmin from safflower seed (Carthamus tinctorius)

The effect of a decrease inpH on the structural integrity of carmin has been monitored by a variety of biophysical techniques. The protein undergoes initial dissociation up topH 3.5–4.0 without any significant denaturation. Below thispH the process of dissociation and denaturation appears to be simultaneous. Further, in thepH range of 2.5–1.6 the protein reassociates to probably a different polymer resulting from possibly, an entropically driven hydrophobic interaction. The process of dissociation appears to be reversible to a large extent. The process of denaturation appears to be governed by the kinetic path that the denatured protein molecule follows either by a sudden decrease inpH or through a gradual decrease inpH. These results are interpreted while keeping in view the oligomeric and globular structure of carmin at neutralpH. The results would help in understanding of structure-function relationship of the protein and its role in hydrogen ion bindingin vivo.     

Reversible thermal unfolding of thermostable cytochrome c-552.

Reversible thermal denaturation of cytochrome c-552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation (ΔG) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

The denaturation of orosomucoid

Highly purified human orosomucoid exhibits apparent multistate behavior on thermal denaturation, with ΔH(cal) for the transition being 119 kcal/mol at pH 7.4. Asialoorosomucoid denatured similarly. A domain structure could not be demonstrated for orosomucoid with cyanogen bromide fragments, although some reannealing of these did occur. It is suggested that the source of the apparent multistate behavior may lie in the existence of polypeptide variants, known to be present in orosomucoid. Although some polymerization occurs on heating, the unpolymerized material shows reversible thermal denaturation behavior. The presence of low concentrations of ethanol induces a significant endotherm which moves to lower temperatures with increasing ethanol concentration. This endotherm was irreversible in the continued presence of ethanol.