The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates

The aim of this work was to study the effect of the formation of more heat-stable conformers of chicken egg ovalbumin during incubation at basic pH (9.9) and elevated temperature (55 degrees C) on the protein aggregation properties at neutral pH. Native ovalbumin (N-OVA) is converted on the hours time-scale into more heat-stable forms denoted I- (intermediate) and S-OVA, that have denaturation temperatures 4.8 and 8.4 degrees C, respectively, higher than that of N-OVA. The conversions most likely proceed via I-OVA, but direct conversion of N-OVA into S-OVA with slower kinetics can not be excluded. It is demonstrated that both I- and S-OVA have similar denaturation characteristics to N-OVA, except that higher temperatures are required for denaturation. The presence of even small contributions of I-OVA does, however, reduce the Stokes radius of the aggregates formed upon heat treatment of the material at 90 degrees C about 2-fold. This affects the gel network formation considerably. Since many (commercial) preparations of ovalbumin contain varying contributions of the more heat-stable forms mentioned, proper characterization or standardization of the isolation procedure of the material is essential to control or predict the industrial application of this protein.     

Aspects of morphine chemistry important to persons working with cold-blooded animals, especially fish

The relative amounts of the different forms of morphine, and many other pharmacologic agents, depend on temperature and pH. Some forms are more efficacious because they are uncharged and can penetrate lipid membranes more easily than the charged forms. Persons who administer pharmacologic agents to ectotherms (that is, cold-blooded animals) should consider the effect of temperature on the relative amounts of the different forms of drugs. For example, the fraction of morphine present in the uncharged form is twice as high in a fish or frog at 5 degrees C as in a mammal at 37 degrees C. Moreover, because the pH of blood, plasma, and tissues of ectotherms is higher when they are held at lower temperatures, the combined effect of temperature and pH on the speciation of pharmacologic agents also should be considered. In addition, the total solubility of morphine and other pharmacologic agents depends on temperature and pH. The purpose of this overview is to describe how temperature and pH influence the solubility and speciation of morphine.     

Maximum-entropy calculation of free energy distributions for two forms of myoglobin

The temperature dependence of the heat capacity of myoglobin depends dramatically on pH. At low pH (near 4.5), there are two weak maxima in the heat capacity at low and intermediate temperatures, respectively, whereas at high pH (near 10.7), there is one strong maximum at high temperature. Using literature data for the low-pH form (Hallerbach and Hinz, 1999) and for the high-pH form (Makhatadze and Privalov, 1995), we applied a recently developed technique (Poland, 2001d) to calculate the free energy distributions for the two forms of the protein. In this method, the temperature dependence of the heat capacity is used to calculate moments of the protein enthalpy distribution function, which in turn, using the maximum-entropy method, are used to construct the actual distribution function. The enthalpy distribution function for a protein gives the fraction of protein molecules in solution having a given value of the enthalpy, which can be interpreted as the probability that a molecule picked at random has a given enthalpy value. Given the enthalpy distribution functions at several temperatures, one can then construct a master free energy function from which the probability distributions at all temperatures can be calculated. For the high-pH form of myoglobin, the enthalpy distribution function that is obtained exhibits bimodal behavior at the temperature corresponding to the maximum in the heat capacity (Poland, 2001a), reflecting the presence of two populations of molecules (native and unfolded). For this form of myoglobin, the temperature evolution of the relative probabilities of the two populations can be obtained in detail from the master free energy function. In contrast, the enthalpy distribution function for the low-pH form of myoglobin does not show any special structure at any temperature. In this form of myoglobin the enthalpy distribution function simply exhibits a single maximum at all temperatures, with the position of the maximum increasing to higher enthalpy values as the temperature is increased, indicating that in this case there is a continuous evolution of species rather than a shift between two distinct population of molecules.     

Renaturation of ovotransferrin under two-step conditions allowing primary folding of the fully reduced form and the subsequent regeneration of the intramolecular disulfides

A two-step procedure was found to be useful for the efficient refolding of a complex protein, ovotransferrin. In the first step, the reduced and denatured form of the protein was incubated at a low temperature in a nondenaturing buffer containing reduced glutathione; in the second step, the reduced form was reoxidized at a higher temperature in the presence of oxidized glutathione. Under these conditions, the fully reduced forms of ovotransferrin and its half-molecules were almost quantitatively reoxidized to regain iron-binding abilities and conformations, very similar to the native form. The circular dichroism spectra revealed that at low temperatures the fully reduced forms have partially folded conformations, which are fluctuating like "molten globule" states. The reoxidization kinetics compared between whole ovotransferrin and the two half-molecules supported independent refolding of the N- and C-terminal domains.     

Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.     

Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH

Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.     

Long-time molecular dynamics simulations of botulinum biotoxin type-A at different pH values and temperatures

Botulinum neurotoxins type A (BoNT/A) are highly potent toxins, but are also useful in the treatment of illnesses. We studied the properties of BoNT/A at various temperatures and pH values in order to understand its toxicity and structure variations. The pH values of the environment of BoNT/A are obtained by changing the protonation states of certain titratable residue groups. Our results show that certain parts of the protein are active at acidic pH environments or at high temperatures. The protein is more stable in neutral environments at normal human body temperature, whereas, at high temperature, the protein is more stable in acidic environments. Also, the three domains of the protein tend to have relative motion rather than within individual domains.     

Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).     

Structural conversion from non-native to native form of recombinant human epidermal growth factor by Brevibacillus choshinensis

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40 degrees C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.     

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.     

Circular dichroism study of polyriboxanthylic acid.

We report in the present paper the circular dichroism spectra of poly(X) at different pH and temperature values. The spectra are characteristic of three stable forms of poly(x) in the pH range of protonation of xanthosine. An electrostatic barrier is proposed to account for the hysteresis and metastability observed in a certain pH range. Some results on oligo(X) at basic pH are also presented. Poly(X) at basic pH is investigated also by hydrodynamic techniques.     

Structural Conversion from Non-native to Native form of Recombinant Human Epidermal Growth Factor by Brevibacillus choshinensis

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40°C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.     

The effects of temperature and pH on the reaction between 1-chloro-2,4-dinitrobenzene and glutathione, catalyzed by the major glutathione S-transferase from Galleria mellonella

The nucleophilic substitution reaction between glutathione and 1-chloro-2,4-dinitrobenzene has been studied at temperatures between 4 and 42°C and pH values between 6.99 and 10.80. The apparent enthalpy, entropy and free energy of ionization of the thiol group have been estimated as have the apparent enthalpy, entropy and free energy of activation of the reaction between the glutathione thiolate anion and the aromatic electrophile. The results obtained permit the calculation of values of the second order rate constant governing the reaction at a range of temperatures and pHs. These values are in accord with those reported in the literature from experimental work by others. The major glutathione S-transferase from Galleria mellonella has been studied with respect to its kinetic responses to changes in pH and temperature. There appear to be two kinetically critical ionizations governing the reaction at high pH. These ionization events are characterized by apparent pK_a values of 8.61 ± 0.15 and 9.16 ± 0.22. A thermodynamic model of the kinetic behavior of the enzyme permits the prediction of its activity over a range of pH and temperature values. The apparent free energy of activation for the enzyme catalyzed reaction is only 7% lower than that for the non-catalyzed reaction between 1-chloro-2,4-dinitrobenzene and glutathione thiolate anion. This observation is compatible with the suggestion that promotion of the ionization of the glutathione thiol group is the major mechanism of catalysis.     

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.     

Human erythrocyte glutathione reductase: pH dependence of kinetic parameters

Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH, 2',3'-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2',3'-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's for ATP-ribose and 2',5'-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2'-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467'-Glu-472' ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.     

Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH

The pH of conventional Tris-glycine SDS-PAGE gels during a run is determined to be 9.5, in contrast to Bis-Tris-Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10mM acrylamide and pH 7.2 is more than 4h at room temperature. Rates are significantly lower in Bis-Tris-Mes gels than in Tris-glycine gels, reducing the risk of adventitious protein modification. Commercial Bis-Tris-Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris-glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis-Tris gels.     

Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations

The conformation of heat-denatured ovalbumin aggregates has been examined at several concentrations and pH values, using measurements of optical rotation dispersion (ORD), circular dichroism (CD) and viscosity. The protein was subjected to heating at relatively low temperatures, ranging from 48.5 to 76 degrees; the particular temperature chosen depended on pH. The heat treatment was sufficient to remove the ability of the molecules to absorb heat on re-heating. The denatured molecules were shown to be rather compact, i.e. not much larger than the native molecule, and to retain a significant amount of secondary structure; this was also the case for molecules present in small aggregates. It is suggested that this type of ovalbumin monomer builds three-dimensional networks in denatured solutions at higher concentrations, and that gelation should be looked upon as arising from surface contacts between hydrated globules. The present results also imply that such globules have gelation properties which depend on whether pH is acidic or basic compared to the isoelectric point of the protein.     

Novel injectable pH and temperature sensitive block copolymer hydrogel

A novel pH and temperature sensitive block copolymer was prepared by adding pH sensitive moiety to temperature sensitive block copolymer. This block copolymer solution showed a reversible sol-gel transition by a small pH change in the range of pH 7.4-8.0 and also by the temperature change in the region of body temperature. The very precise molecular weight control of block copolymer and the prudential tuning of hydrophilic-hydrophobic balance were needed to control the phase diagram. This block copolymer solution forms a gel at 37 degrees C, pH 7.4 (human body). When the block copolymer solution is at room temperature and pH 8.0 as a sol state, both the temperature and pH change are needed for the gelation. This material can be employed as injectable carriers for hydrophobic drugs and proteins, etc. Gelation inside the needle can be prevented by an increase in the temperature during injection, because it does not change into the gel form with only increasing temperature. This material can be used for even a long guide catheter into the body. The block copolymer hydrogel which shows the sol-gel transition by the small pH change from pH 8.0 to pH 7.4 has merits in the delivery system for protein and cells which show cytotoxicity in acidic (below pH 6.5) or basic (above pH 8.5) conditions. This block copolymer system could be used as a template technology for injectable delivery systems.     

The human serpin proteinase inhibitor-9 self-associates at physiological temperatures

The metastable serpin architecture is perturbed by extremes of temperature, pH, or changes in primary sequence resulting in the formation of inactive, polymeric conformations. Polymerization of a number of human serpins in vivo leads to diseases such as emphysema, thrombosis, and dementia, and in these cases mutations are present within the gene encoding the aggregating protein. Here we show that aggregation of the human serpin, proteinase inhibitor-9 (PI-9), occurs under physiological conditions, and forms aggregates that are morphologically distinct from previously characterized serpin polymers. Incubation of monomeric PI-9 at 37 degrees C leads to the rapid formation of aggregated PI-9. Using a variety of spectroscopic methods we analyzed the nature of the structures formed after incubation at 37 degrees C. Electron microscopy showed that PI-9 forms ordered circular and elongated-type aggregates, which also bind the fluorescent dye Thioflavin T. Our data show that in vitro wild-type PI-9 forms aggregates at physiological temperatures. The biological implications of PI-9 aggregates at physiological temperatures are discussed.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.