Intermediate pH Values Research Articles

Tree species mediated soil chemical changes in a Siberian artificial afforestation experiment

Natural and human-induced changes in the composition of boreal forests will likely alter soil properties, but predicting these effects requires a better understanding of how individual forest species alter soils. We show that 30 years of experimental afforestation in Siberia caused species-specific changes in soil chemical properties, including pH, DOC, DON, Na+, NH4+, total C, C/N, Mn2+, and SO42-. Some of these properties –- pH, total C, C/N, DOC, DON, Na+ –- also differed by soil depth, but we found no strong evidence for species-dependent effects on vertical differentiation of soil properties (i.e., no species × depth interaction). A number of soil properties –- NO3−, N, Al3+, Ca2+, Fe3+, K+, Mg2+ and Cl− –- responded to neither species nor depth. The six studied species may be clustered into three groups based on their effects on the soil properties. Scots pine and spruce had the lowest pH, highest C/N ratio and intermediate C content in soil. The other two coniferous species, Arolla pine and larch, had the highest soil C contents, highest pH values, and intermediate C/N ratios. Finally, the two deciduous hardwood species, aspen and birch, had the lowest C/N ratio, intermediate pH values, and lowest C content. These tree-mediated soil chemical changes are important for their likely effects on soil microbiological activities, including C and N mineralization and the production and consumption of greenhouse gases.

Calculation of Weak Protein-Protein Interactions: The pH Dependence of the Second Virial Coefficient

Interactions between proteins are often sufficiently weak that their study through the use of conventional structural techniques becomes problematic. Of the few techniques capable of providing experimental measures of weak protein-protein interactions, perhaps the most useful is the second virial coefficient, B 22, which quantifies a protein solution's deviations from ideal behavior. It has long been known that B 22 can in principle be computed, but only very recently has it been demonstrated that such calculations can be performed using protein models of true atomic detail ( Biophys. J. 1998, 75:2469–2477). The work reported here extends these previous efforts in an attempt to develop a transferable energetic model capable of reproducing the experimental trends obtained for two different proteins over a range of pH and ionic strengths. We describe protein-protein interaction energies by a combination of three separate terms: (i) an electrostatic interaction term based on the use of effective charges, (ii) a term describing the electrostatic desolvation that occurs when charged groups are buried by an approaching protein partner, and (iii) a solvent-accessible surface area term that is used to describe contributions from van der Waals and hydrophobic interactions. The magnitude of the third term is governed by an adjustable, empirical parameter, γ, that is altered to optimize agreement between calculated and experimental values of B 22. The model is applied separately to the proteins lysozyme and chymotrypsinogen, yielding optimal values of γ that are almost identical. There are, however, clear difficulties in reproducing B 22 values at the extremes of pH. Explicit calculation of the protonation states of ionizable amino acids in the 200 most energetically favorable protein-protein structures suggest that these difficulties are due to a neglect of the protonation state changes that can accompany complexation. Proper reproduction of the pH dependence of B 22 will, therefore, almost certainly require that account be taken of these protonation state changes. Despite this problem, the fact that almost identical γ values are obtained from two different proteins suggests that the basic energetic formulation used here, which can be evaluated very rapidly, might find use in dynamical simulations of weak protein-protein interactions at intermediate pH values.

Removal of amino acids from water by adsorption on polystyrene resins

The adsorption of eight amino acids, L-asparagine, D,L-threonine, L-lysine, L-leucine D,L-methionine, L-tyrosine, L-phenylalanine and D,L-tryptophan, on the non-polar macroporous adsorbents Amberlite XAD-2 and XAD-4 (polystyrene–divinylbenzene copolymers) was studied. Equilibrium adsorption experiments were conducted to estimate the types of isotherm and their parameters. The effect the chemical composition and structure of the amino acids on the efficiency of adsorption was evaluated. The influence of pH and ionic strength was also studied. The data of adsorption isotherms of the examined amino acids seemed generally to approach the Freundlich isotherm model. Tryptophan isotherm adsorption data could match in some cases the Langmuir model. The majority of the adsorption isotherms were almost linear. In terms of adsorbed amino acid on both resin surfaces, the amino acids can be ranked thus: D,L-tryptophan > L-phenylalanine > D,L-methionine, L-tyrosine > L-leucine > L-lysine > D,L-threonine > L-asparagine. In low pH solution, adsorption was generally higher than that at intermediate and high pH values. Generally, as the ionic strength increases, the adsorption of the amino acids increases. © 2001 Society of Chemical Industry

Corrosion behavior of Al-Cu-Fe quasicrystals

For any application of quasicrystals or quasicrystalline coatings, e.g. for extruders, turbine blades or frying pans, corrosion resistance is of utmost importance. Therefore, corrosion of the quasicrystalline as well as related crystalline phases in the system Al-Cu-Fe was studied in particular at intermediate pH values in order to clarify the influence of the quasicrystalline structure. Salt spray tests were performed at 35°C with several single phase materials in the Al-Cu-Fe system. Highest corrosion resistance was observed in crystalline aluminides AlFe as well as Al5Fe2 where a protective oxide film had developed. The quasicrystalline alloy as well as crystalline Al7Cu2Fe exhibit severe corrosion accompanied with the formation of Cu, Cu2O and Al(OH)3. Anodic polarization of Al63Cu25Fe12 icosahedral quasicrystals reveal after a potentiodynamic scan in 0.1N NaOH the formation of an oxide layer consisting of FeO and Cu2O; there is only very little evidence for a phase transformation of the quasicrystal due to Al loss by selective corrosion. During further polarization in the passive region the oxide layer was observed to transform into probably higher oxidized states. The overall thickness of the oxide layer depends on the scan time, however, not on further polarization time at a distinct potential.

NMR study of the complexation of d-gulonic acid with tungsten(VI) and molybdenum(VI)

By using multinuclear ( 1H, 13C, 17O, 95Mo, 183W) magnetic resonance spectroscopy (1D and 2D), d-gulonic acid is found to form ten and seven complexes, respectively, with tungsten(VI) and molybdenum(VI), in aqueous solution, depending on pH and metal–ligand molar ratios. Two isomeric 1:2 (metal–ligand) complexes involving the carboxylate and the adjacent OH group are present in the pH range 2–9. At intermediate and high pH, molybdate forms a 2:1 tetradentate complex involving the four secondary hydroxyl groups, whereas tungstate forms one 2:1 terdentate species. At low and intermediate pH values, three 2:1 complexes are found for both metals, involving the carboxylate group and three secondary hydroxyl groups, as well as a 5:2 species involving the carboxylate group and all the secondary hydroxyl groups; the concentration of this species increases in time mainly at the expense of 2:1 and 1:2 complexes. Tungstate can also form two additional species, probably a 5:2 species involving the carboxylate group and all the hydroxyl groups, and a 2:1 pentadentate species involving the carboxylate group and all the secondary hydroxyl groups. In alkaline solutions, tungstate is able to form an additional 2:1 pentadentate complex involving all the hydroxyl groups.

Comparative analysis of alkali and alkaline-earth cation transfer assisted by monensin across the water∣1,2-dichloroethane interface

Abstract In the first part of this paper the electrochemical transfer of alkali cations (M+) assisted by monensin (HX) across the water ∣ 1,2-dichloroethane (DCE) interface at pH 9 the current is voltammetrically negligible. An equation for the dependence of Δowφ1/2 on pH and Na+ concentration is developed. In the second part of the paper the transfer of alkaline earth cations assisted by the same ionophore is studied. The electrochemical reactions (Me(w)2++HX(o)⇄MeHX2+(o)) and (Me(w)2++X−(o)⇄MeX+(o)) are responsible for the peaks observed at pH 9.0, respectively. As expected, in both cases ΔEp=0.030 V while at intermediate pH the electrochemical exchange reaction (Me2+(w)+HX(o)⇄MeX+(o)+H+(w)) is proposed. The net charge transfer of +1 at the interface accounts for ΔEp=0.060 V for the peak observed ΔEp in agreement with the hyper-Nernstian slope of 60 mV found for Ca2+ and Ba2+ ISE based on antibiotics with carboxylic groups at intermediate pH values. The higher selectivity for Ba2+ and the tendency in selectivity at alkaline pH found in the ISEs are also observed and thus explained according to the mechanism proposed.

Surface Forces between Hydrophilic Self-Assembled Monolayers in Aqueous Electrolytes

In this paper we present an atomic force microscopy (AFM) study on electrostatic and acid−base interactions between hydrophilic self-assembled monolayers (SAMs) with monoprotic ionizable acid functional groups (−COOH). As expected for surfaces composed of weak acid groups the surface potential and strength of interaction are sensitive to pH and ionic strength. Repulsive forces grow with the degree of ionization. At low fractional ionization, the surfaces experience attractions with a range larger than that expected for van der Waals interactions and the strength of adhesion is large. The strength of adhesion and extent of attractions diminish with increasing pH. As the surface groups ionize, at intermediate pH values, jump-in distances and adhesion grow with ionic strength and at high pH they saturate with increasing electrolyte concentration at values close to those expected for van der Waals interactions. However, the maximum charge is around 5% of the total expected for the surfaces. Thus, allowing sodium ions to bind, with a binding constant of pKNa = 3.7 and pKa = 6.3, ionic strength and pH dependencies of the surface charge can be described.

Dermal Delivery of ETH-615, a Zwitterionic Drug

ETH-615 is an amphoteric drug that forms a water-insoluble zwitterion at intermediate pH values. Increasing the aqueous solubility of ETH-615 through cyclodextrin complexation did not enhance transdermal delivery of the drug from saturated aqueous solutions. However, increasing the lipophilicity of the drug through masking of the anionic group with a pro-moiety increased the dermal and transdermal delivery of the drug. Furthermore, masking the anionic group enhanced the chemical stability of the drug, resulting in significant improvement of the shelf life of the drug in both aqueous and nonaqueous solutions.

Layer-by-Layer Growth and Condensation Reactions of Niobate and Titanoniobate Thin Films

Thin films were grown on amine-primed Si and glass substrates by sequential adsorption reactions of polyallylamine hydrochloride (PAH) and anionic colloids derived from HTiNbO5 and HCa2Nb3O10. The acid−base chemistry of polycation/polyanion adsorption was studied in detail for PAH/HTiNbO5. The pKa of PAH, defined as the pH at which it is half protonated, is 8.7. Titanoniobate colloids, prepared by reaction of HTiNbO5 with tetra(n-butylammonium) hydroxide, TBA+OH-, are unilamellar at pH ≥ 8.5 and restack below pH 7.0. Efficient tiling of a PAH-terminated surface by a layer of unilamellar titanoniobate sheets occurs only at intermediate pH values (8.5−9.0). At lower pH, the colloid restacks on the surface, and at higher pH, only partial coverage by single sheets is observed by atomic force microscopy (AFM). At pH 8.5, high-quality multilayer films can be grown by sequentially adsorbing PAH with either the titanoniobate or niobate colloid. TGA/DTA studies of bulk PAH/titanoniobate intercalation compounds sho...

Lifetime-Based pH Sensors: Indicators for Acidic Environments

We characterized the pH-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-domain fluorometry, with the objective of identifying lifetime-based sensors for low pH values. These three probes were originally designed as dual excitation wavelength–ratiometric probes, with high photostability and high quantum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays analysis reveals that these probes have double exponential intensity decays at intermediate pH values and that the decay time amplitudes are greatly dependent on pH. The longer lifetime components originated from the unprotonated forms and the shorter components from the protonated forms. Both forms can emit fluorescence at intermediate pH values. The apparent pKavalues were also determined from the titration curves of phase angles and modulations versus pH for the purpose of pH sensing. The apparent pKavalues range from pH 3 to 5, a range where lifetime-based sensors are not presently reported. Since these probes show low pKavalues and display substantial phase and modulation changes with pH, they are suitable as lifetime-based pH sensors to monitor the pH changes in acidic environments. One potential application of these probes is to trace the pH in different cellular compartments.

Molecular Arrangement in Two-Dimensional Streptavidin Crystals

We studied the molecular arrangement of two-dimensional streptavidin crystals at the air−water interface over a range of pH values. We quantified the varying amounts of coexisting P1, P2, and C222 crystals in the different morphologies observed at pH 4.5−6.5. Chiral, needlelike crystals at pH 4.5 consist of P1 crystals with frequent line defects. Larger chiral domains near pH 5 are essentially all P1 coexisting with a small amount of P2, whereas at slightly higher pH values (near pH 5.5), H-shaped domains contain 4 times as much P1 coexisting with a P2/C222 mixture. Morphologies intermediate to these shapes exhibit intermediate compositions. Between pH ∼6−7, crystals all display a characteristic dendritic-X morphology, but arrangement at the molecular level is quite different compared with lower pH values. Crystals are mostly P2 in symmetry near pH 6, but at pH 7 and above, crystals have C222 symmetry. Coexistence of P2 and C222 crystals occurs at intermediate pH values. We determined the orientation and arrangement of streptavidin molecules in P1, P2, and C222 crystals relative to the directions exhibiting faster growth. The direction of faster growth in P1 crystals includes both interactions between biotin-free subunits and interactions between biotin-bound subunits. In the P2 arrangement, growth in the direction of intermolecular biotin-free subunits is preferred, whereas growth is faster along the biotin-bound direction of C222 crystals. We developed a model of the molecular arrangement for the observed solid-phase coexistence in these crystals.

Process Effects of By-Product Carbon Dioxide Production from Transketolase-Catalysed Condensations

The production of carbon dioxide from transketolase-catalysed condensations has been studied. The effects of carbamate formation, carbon dioxide dissociation and gas/liquid interfacial area were evaluated as potential causes of activity loss on account of by-product carbon dioxide production. At low pH values physical effects (gas/liquid interfacial area) were dominant, while at higher pH values carbamate effects dominated. However, neither of these effects were found to lead to a significant additional activity loss under the conditions chosen. Residual activity loss was evaluated at intermediate pH values and attributable to non-specific protein oxidation, indicating the need to operate the process under reducing conditions.

Interaction of Et 2SnCl 2 with 5′-IMP and 5′-GMP

The interactions of Et 2SnCl 2 with 5′-IMP and 5′-GMP have been studied in aqueous solutions by 1H-and 31P-NMR spectroscopy as a function of pH. At low pH values (<4.0) Sn(IV) interacts with the pyrophosphate oxygens of these nucleotides. At intermediate pH values (4–9.5) no interaction of the metal with the nucleotides take place, while at pH > 9.5 the sugar O 2′ and O 3′ atoms are the preferred coordination sites. In addition, the solid adducts obtained from aqueous solutions at pH=3–4 of the above interactions correspond to formulae; (Et 2Sn) 2(5′-IMP) 2(H 2O) and (Et 2Sn) 3(5′-GMP) 2(OH) 2(H 2O) 2 as their elemental analysis show. IR spectra and solid state 13C, 31P-NMR spectra 119Sn Mössbauer and solution 119Sn-NMR spectra once more confirm the pyrophosphate involvement in bonding with Sn(IV) in oligomeric or polymeric structures and trigonal bipyramidal or octahedral geometries.

Procain interaction with DPPC multilayers: an ESR spin label investigation

A spin label electron spin resonance (ESR) study has been carried out on the interaction between the local anesthetic procaine (PRC) and multilamellar dispersions of di-palmitoylphosphatidylcholine (DPPC). The investigation has been performed either in the gel or in the liquid crystal phase of DPPC varying the anesthetic/DPPC molar ratio as well as the pH of the dispersion medium and using spin labeled DPPC molecules (5- and 16-PCSL) and the di-tert-butyl nitroxide (DTBN) spin probe. On increasing the anesthetic concentration in the dispersion medium up to PRC/DPPC molar ratio of 33 mol.%, a lyotropic transition of the bilayer from the lamellar gel phase Lβ, to the interdigitated Lβi one is observed either at pH 5.0 or at pH 10.0. At intermediate pH values, about pH 7.0, such a PRC concentration dependent transition is absent. A decrease of the partition of the DTBN molecules between the dispersion medium and the fluid hydrophobic phase of DPPC multilayers is observed at pH 5.0 with anesthetic concentration, while an opposite behaviour is evidenced at pH 10.0. The pre- and main-phase transition temperatures of the DPPC multilayers are also affected although to different extent. These results are interpreted in terms of adsorption of the PRC molecules at the surface of the model membrane at the extreme pH values investigated, while at about neutral pH values they intercalate among the DPPC molecules. The protonation state of procaine plays an important role as it determines the way of surface adsorption of the PRC molecules in the neutral form at the high pH value and in the charged cationic form at pH 5.0.

Effects of solution pH, temperature, nitrate/ammonium ratios, and inhibitors on ammonium and nitrate uptake by Arabica coffee in short‐term solution culture

Solution pH, temperature, nitrate (NO3 ‐)/yammonium (NH4 +) ratios, and inhibitors effects on the NO3 ‐ and NH4 + uptake rates of coffee (Coffea arabica L.) roots were investigated in short‐term solution culture. At intermediate pH values (4.25 to 5.75) typical of coffee soils, NH4 + and NO3 ‐ uptake rates were similar and nearly independent of pH. Nitrate uptake varied more with temperature than did ammonium. Nitrate uptake increased from 0.05 to 1.01 μmol g‐1 FWh‐1 between 4 and 16°C, and increased three‐fold between 16 to 22°C. Between 4 to 22°C, NH4 + uptake rate increased more gradually from 1.00 to 3.25 μmol g‐1 FW h‐1. In the 22–40°C temperature range, NH4 + and NO3 ‐ uptake rates were similar (averaging 3.65 and 3.56 umol g‐1 FW h‐1, respectively). At concentrations ranging from 0.5 to 3 mM, NO3 ‐ did not influence NH4 + uptake rate. However, NO3 ‐ uptake was significantly reduced when NH4 + was present at 3 mM concentration. Most importantly, total uptake (NO3 ‐+NH4 +) at any NO3 ‐/NH4 + ratio was higher than that of plants fed solely with either NH4 + or NO4 ‐. Anaerobic conditions reduced NO3 ‐ and NH4 + uptake rate by 50 and 30%, respectively, whereas dinitrophenol almost completely inhibited both NH4 + and NO3 ‐ uptake. These results suggest that Arabica coffee is well adapted to acidic soil conditions and can utilize the seasonally prevalent forms of inorganic N. These observations can help optimizing coffee N nutrition by recommending cultural practices maintaining roots in the temperature range optimum for both NH4 + and NO3 ‐ uptake, and by advising N fertilization resulting in a balanced soil inorganic N availability.

BRAZIL NUT SEEDLING ESTABLISHMENT AND AUTECOLOGY IN EXTRACTIVE RESERVES OF ACRE, BRAZIL

In the western Amazonian state of Acre, Brazil, extractive reserve residents are examining strategies to intensify their extraction-based system without compromising biological conservation. One such strategy is to increase production from their major source of income, Brazil nut (Bertholletia excelsa), by augmenting the species’ density within the reserves. We evaluated the success of Brazil nut seedling establishment at three types of planting sites on extractivist landholdings: forest gaps, shifting-cultivation plots, and pastures. Nursery-grown seedlings were planted according to a randomized complete block design at five extractivist landholdings with planting sites as the main treatment. Average photosynthetic photon-flux density was approximately four times higher in shifting-cultivation plots and pastures than in forest gaps. Soil pH and extractable Ca, K, Mg, and P were the highest in shifting-cultivation plots. Pasture sites had the most compact soils and an intermediate pH value; forest-gap soils were the most acidic. There were no differences in total soil N, total soil C, and percent organic matter across the three sites. After two years, mean seedling height and diameter in the shifting-cultivation plots were almost double those of seedlings established in pastures and forest gaps, while seedling survival at all three sites was comparable, at approximately 40%. Seedlings in shifting-cultivation plots and pastures had higher predawn water potential values, foliar nutrient levels, and root biomass than seedlings in forest gaps. Although light, nutrients, and water resources were available in pastures, enrichment plantings in this site proved to be the least compatible with the overall extractivist system because of high labor inputs necessary for establishment, particularly if grazing animals were part of the system. Although forest gaps were appropriate enrichment sites from a socio-economic perspective, light availability was low, and both belowground resources, nutrients and water, appeared to be very limiting so that gap seedlings demonstrated slow growth. Shifting-cultivation plots were the best sites for Brazil nut enrichment plantings in extractive reserves. Light, nutrients, and water resources proved to be readily available, seedlings performed the best, and overall compatibility with the extractivist system was good.

Brazil Nut Seedling Establishment and Autecology in Extractive Reserves of Acre, Brazil

In the western Amazonian state of Acre, Brazil, extractive reserve residents are examining strategies to intensify their extraction-based system without compromising biological conservation. One such strategy is to increase production from their major source of income, Brazil nut (Bertholletia excelsa), by augmenting the species’ density within the reserves. We evaluated the success of Brazil nut seedling establishment at three types of planting sites on extractivist landholdings: forest gaps, shifting-cultivation plots, and pastures. Nursery-grown seedlings were planted according to a randomized complete block design at five extractivist landholdings with planting sites as the main treatment. Average photosynthetic photon-flux density was approximately four times higher in shifting-cultivation plots and pastures than in forest gaps. Soil pH and extractable Ca, K, Mg, and P were the highest in shifting-cultivation plots. Pasture sites had the most compact soils and an intermediate pH value; forest-gap soils were the most acidic. There were no differences in total soil N, total soil C, and percent organic matter across the three sites. After two years, mean seedling height and diameter in the shifting-cultivation plots were almost double those of seedlings established in pastures and forest gaps, while seedling survival at all three sites was comparable, at approximately 40%. Seedlings in shifting-cultivation plots and pastures had higher predawn water potential values, foliar nutrient levels, and root biomass than seedlings in forest gaps. Although light, nutrients, and water resources were available in pastures, enrichment plantings in this site proved to be the least compatible with the overall extractivist system because of high labor inputs necessary for establishment, particularly if grazing animals were part of the system. Although forest gaps were appropriate enrichment sites from a socio-economic perspective, light availability was low, and both belowground resources, nutrients and water, appeared to be very limiting so that gap seedlings demonstrated slow growth. Shifting-cultivation plots were the best sites for Brazil nut enrichment plantings in extractive reserves. Light, nutrients, and water resources proved to be readily available, seedlings performed the best, and overall compatibility with the extractivist system was good.

Adhesive and Friction Forces between Chemically Modified Silicon and Silicon Nitride Surfaces

We report the results of probing adhesion and friction forces between surfaces with functional terminal groups with chemically modified scanning probe microscopy (SPM) tips. Surfaces with terminal groups of CH3, NH2, and SO3H were obtained by direct chemisorption of silane-based compounds on silicon/silicon nitride surfaces. We studied surface properties of the resulting self-assembled monolayers (SAMs) in air and aqueous solutions with different pHs. Work of adhesion, “residual forces”, and friction coefficients was obtained for four different types of modified tips and surfaces. Absolute values of the work of adhesion between various surfaces, Wad, were in the range 0.5−8 mJ/m2. The work of adhesion for different modified surfaces correlated with changes of solid−liquid surface energy estimated from macroscopic contact-angle measurements. Friction properties varied with pH in a register with adhesive forces showing a broad maximum at intermediate pH values for a silicon nitride/silicon nitride mating pair. Similar broad maxima were observed in the acid range for a NH2-terminated SAM and in the basic range for a SO3H-terminated SAM. This behavior can be understood considering the changes of the surface charge state determined by the zwitterionic nature of silicon nitride surfaces with multiple isoelectric points.

Electrochemical and Ion Exchange Properties of Polyaniline‐Poly(vinylsulfonate) Composites in Aqueous and Nonaqueous Media

A composite of polyaniline and poly(vinylsulfonate) was obtained by electrochemical polymerization of aniline in a solution containing poly(vinylsulfonate) ions. The irreversible retention of the polyelectrolyte was confirmed using Fourier transform infrared reflection‐absorption spectroscopy. The symmetric stretching of the group is observed at ca. , in addition to the bands due to polyaniline. The polyelectrolyte is retained even after extensive cycling in acidic aqueous or neutral nonaqueous solutions not containing polyelectrolyte. The composite film is electroactive in aqueous (pH < 7) and nonaqueous media. The specific charge in aqueous media is ca. 84 (±2) Ah/kg, as measured using an electrochemical quartz crystal microbalance (EQCM) (both redox processes). The ion exchange of the modified electrode was studied using probe beam deflection and EQCM. The charge compensation in aqueous solution involves a mixed proton and anion exchange. Three different regimes are observed at different pH values. At pH below −1 and pH above 2, the dominant process of charge compensation is proton exchange. At intermediate pH values (between −1 and 2), there is an increased contribution of anion insertion to the charge‐compensation process. In nonaqueous media, it seems that cations are retained inside the film and the charge is compensated only by anion exchange.

The native and the heat-induced denatured states of α-chymotrypsinogen A: thermodynamic and spectroscopic studies

We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of α-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly “softens” at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less “tight”. This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, k M, of the protein “native” state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.