STUDIES IN THE PHYSICAL CHEMISTRY OF AMINO ACIDS,PEPTIDES, AND RELATED SUBSTANCES : XI. THE SOLUBILITY OF CYSTINE IN THE PRESENCE OF IONS AND ANOTHER DIPOLAR ION

1. As an introduction to the relations that obtain in biochemical systems containing several components, some ionic, some dipolar ionic, the solubility of cystine has been investigated in the presence of glycine and neutral salts. 2. Both glycine and sodium chloride increase cystine solubility at all concentrations. The interaction between cystine and ions is, however, diminished with increase in glycine concentration, and the interaction between cystine and glycine with increase in ionic strength. 3. Sodium sulfate also increases the solubility of cystine, but at concentrations greater than one molal its solvent action is smaller than its salting-out effect, which is greater at all concentrations than that of sodium chloride, and greater the higher the glycine concentration. 4. These interactions are defined by an equation giving the solubility ratio of cystine in terms of salting-out constants, constants related to the electric moments of cystine, and to the ionic strength and dielectric constant of the solution. 5. The higher the concentration of glycine and therefore the dielectric constant of the solution, the smaller that part of the interaction between ions and dipolar ions which depends upon Coulomb forces and the greater appears the salting-out effect. 6. Conversely, the greater the ionic strength and the salting-out effect the smaller the interaction between dipolar ions in solution.     

Hydrophobic binding of ribosomes and polysomes to agarose gels

Rat liver ribosomes and polyribosomes could be immobilized in agarose gels at 4°C and pH 7.6, using KCl or NaCl molarities of 0.25 or higher. The binding could be effected in the presence of excess protein and/or detergents. Polysomes attached to endoplasmic membrane fragments did not bind to agarose even at 0.5m KCl; tRNAs were also not bound. The larger (60 S) subunit of liver ribosomes was also completely immobilized at 0.3m KCl, while the immobilization of the smaller (40 S) subunit was poor even at 1m KCl. The ribosomal subunits could be essentially quantitatively desorbed at 4°C by a low ionic strength elution, while the recovery of gel-bound polysomes was of the order of 80 to 85% under these conditions. The polysomes that recovered from agarose at low ionic strength were active inin vitro incorporation of amino acids into polypeptides.     

Hydrolysis of starchy materials by repeated use of an amylase immobilized on a novel thermo-responsive polymer

A copolymer of methacrylic acid (MAA) and N-isopropyl acrylamide (NIPAM) was used as a novel, reversibly soluble-insoluble support whose solubility changes depending on the temperature of the solution. Amylase (Dabiase K-27) immobilized covalently on the thermo-responsive polymer showed good solubility response: the immobilized enzyme (D-MN) was in a soluble state below 32°C, but insoluble above 42°C. D-MN in a soluble state has a high specific activity for the hydrolysis of soluble or uncooked starch. The solubility response of D-NM to changes in the temperature of the solution was more sensitive when 0.5% NaCl was added to a buffer solution (pH 4.5) with D-MN than in the buffer solution without NaCl. D-MN was used successively for repeated hydrolysis reactions of soluble and uncooked starches, in which D-MN was insolubilized either by changing the temperature of the reaction mixture from 30°C to 36°C with 0.5% NaCl or by adjusting the NaCl concentration of the reaction mixture from 0% to 1% at 30°C. In the repeated hydrolysis, glucose was produced successively from the soluble and uncooked starches, and D-MN could be repeatedly used after being recovered from the reaction product by centrifugation at the end of each batchwise hydrolysis.     

Initial protein concentration effects on precipitation by salt

The influence of initial protein concentration on the performance of salting-out precipitation is examined. In the precipitation of bovine albumin by ammonium sulfate, a peak occurs in the plot of protein solubility versus initial protein concentration; that is, the solubility first increases and then falls with increasing initial protein concentration. In addition, the dependence of solubility on the initial protein concentration is less significant if using higher salt concentrations. The solubility behavior of bovine albumin may be representative, because it covers all possible alternatives; namely, the solubility is independent of, increases with, decreases with, or first increases and then decreases with the initial protein concentration. The appearance of a solubility peak can be explained based on the occurrence of a primary particle during the precipitation process. However, inclusion of the influence of initial protein concentration into the Cohn equation is not feasible with the use of a logarithmic scale, which does not sensitively reflect the change in protein solubility. Increasing initial protein concentration favors protein recovery because it reduces the resultant volume of the supernatant phase. (c) l997 John Wiley & Sons, Inc.     

Correlations for the partition behavior of proteins in aqueous two-phase systems: effect of overall protein concentration

The effect of protein concentration in partitioning in PEG/salt aqueous two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000/phosphate system with 3% w/w NaCl was used for alpha-amylase. The concentration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on their physicochemical properties. In the top phase, maximum protein concentration was determined mainly by a steric exclusion effect of PEG, and hydrophobic interaction between PEG and proteins. In the bottom phase, maximum concentration was determined mainly by a salting-out effect of the salts present. As the ionic strength was increased in the systems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting-out effect. Amyloglucosidase had a very low maximum concentration in the PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the salt-rich bottom phase due to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and, hence, a stronger salting-out effect. The protein concentration in each of the two phases was correlated with a "saturation"-type equation. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between the "saturation" equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. (c) 1996 John Wiley & Sons, Inc.     

Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B²²), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B²² was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B²² is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B²² decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B²² parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B²² correlates with protein solubility as well as physical protein stability.     

Solubility and phase transitions in the water-protein-salt system

The previously derived formulas for the curves corresponding to sol-gel, liquid-liquid, and liquid-solid phase transitions, which correlate the critical molar composition of the water-protein-salt system with individual characteristic features of its component (protein charge z, the number of ions adsorbed v, the function of electrolyte activity A) are presented as curves in ordinary coordinates of protein solubility logS against salt concentration m3. Tendencies in changes in phase transition lines versus the v, z, and v/z ratio have been determined. Correlations of the salting-out curve and the salting-out coefficient with phase transitions are discussed.     

7S Globulins from Phaseolus vulgaris L.: Impact of Structural Aspects on the Nutritional Quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS–PAGE showed major bands corresponding to the phaseolin subunits (43–53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 °C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of β-sheet and β-turn structures, together with a contribution at 1687 cm^?1 that was assigned to intramolecular β-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Interactions of macromolecules with salt ions: an electrostatic theory for the Hofmeister effect

Salting-out of proteins was discovered in the nineteenth century and is widely used for protein separation and crystallization. It is generally believed that salting-out occurs because at high concentrations salts and the protein compete for solvation water. Debye and Kirkwood suggested ideas for explaining salting-out (Debeye and MacAulay, Physik Z; 1925;131:22-29; Kirkwood, In: Proteins, amino acids and peptides as ions and dipolar ions. New York: Reinhold; 1943. p 586-622). However, a quantitative theory has not been developed, and such a theory is presented here. It is built on Kirkwood's idea that a salt ion has a repulsive interaction with an image charge inside a low dielectric cavity. Explicit treatment is given for the effect of other salt ions on the interaction between a salt ion and its image charge. When combined with the Debye-Hückel effect of salts on the solvation energy of protein charges (i.e., salting-in), the characteristic curve of protein solubility versus salt concentration is obtained. The theory yields a direct link between the salting-out effect and surface tension and is able to provide rationalizations for the effects of salt on the folding stability of several proteins.     

Effects of different movement directions on electromyography recorded from the shoulder muscles while passing the target positions

PurposeWe compared electromyography (EMG) recorded from the shoulder joint muscles in the same position for different movement directions.MethodsFifteen healthy subjects participated. They performed shoulder elevation from 0° to 120°, shoulder depression from 120° to 0°, shoulder horizontal adduction from ?15° to 105°, and shoulder horizontal abduction from 105° to ?15°. The target positions were 90° shoulder elevation in the 0°, 30°, 60°, and 90° planes (0°, 30°, 60°, and 90° positions). EMG signals were recorded from the supraspinatus (SSP) muscle by fine-wire electrodes. EMG signals from the infraspinatus (ISP), anterior deltoid, middle deltoid, and posterior deltoid muscles were recorded using active surface electrodes.ResultsDuring elevation and horizontal abduction, the SSP showed significantly higher activity than that shown during depression and during horizontal adduction in the 0°, 30°, and 60° positions. During elevation, the ISP showed significantly higher activity than during depression and during horizontal adduction in the 90° position. During horizontal abduction, the ISP showed significantly higher activity than during depression in the 90° position.ConclusionsWhen the movement tasks were performed in different movement directions at the same speed, each muscle showed characteristic activity.     

The ionic effects of NaCl on physiology and gene expression in rice genotypes differing in salt tolerance

The effects of ionic stress on the physiology and gene expression of two rice genotypes (IR4630 and IR15324) that differ in salt tolerance, were investigated by evaluating changes in the biomass, Na⁺ and K⁺ concentrations and applying the cDNA-AFLP technique to highlight changes in gene expression. Over 8 days of salinisation, the effect of NaCl on the reduction of biomass (dry weight) was apparent from 24 h after salinisation (the first time point), indicating that the consequences of the build up of Na⁺ (and Cl^-) in the leaves of both lines was rapid. Furthermore, root growth of IR15324 was much more sensitive to salt than that of IR4630 (the reduction in root dry weight compared to non-salinised plants was three times greater in IR15324 than IR4630). The two rice lines also differed in their Na⁺ accumulation in saline conditions, a difference that was more marked in the shoots, particularly at the final harvest, than in the roots. Under salt stress, the K⁺ content (µmol/shoot) increased over four successive harvests (24, 48, 96, 192 h) in both lines, but was always greater in IR4630 than in IR15324: differences in Na⁺/K⁺ ratio appear to be an important determinant of salt tolerance in rice. To separate osmotic from ionic effects of salt, mannitol was applied as a non-ionic osmoticum at an osmotic potential estimated to be equivalent to 50 mM NaCl. Messenger RNA was sampled at 0.5, 6, 24, 48 and 192 hours after salinisation. Several products (AFLP-bands) were detected, which were upregulated in the response to ionic effects of salt in the tolerant line (IR4630) and not expressed in the sensitive line (IR15324). Bioinformatic analysis indicated three of these AFLP-bands have a high-degree of sequence similarity with the genes encoding a proline rich protein, senescence associated protein and heat-shock protein. The data are novel in that they differentially highlight changes induced by the ionic rather than osmotic effects of salt and in a tolerant rather than a sensitive genotype. The possible roles of the products of these genes are discussed.     

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.     

Influence of temperature and salinity on the germination of Lotus creticus (L.) from the arid land of Tunisia

Effects of salinity, temperature and their interactions on the rate and final percentage of germination were evaluated for two populations (Msarref, Oued dkouk) of the invasive glycophyte Lotus creticus Linné, grown under arid environmental conditions of the Tunisia. Seeds that were not treated with NaCl germinated well in a wide range of temperatures. For both populations, maximum germination occurred in distilled water at 25°C and lowest germination for all salinities was at 35°C. Germination was substantially delayed and significantly reduced with an increase in NaCl to levels above 300 mm . Compared to the Oued dkouk population, final germination and germination rate of the Msarref population was completely inhibited at 300 mm NaCl. The interactive effect of temperature and NaCl concentration on final germination and germination rate was significant (P < 0.01), indicating that the germination response to salinity depended on temperature. The inhibition of Oued dkouk population seed germination at high salt concentration was mostly due to osmotic effects while ionic effects were noted at Msarref population. The germination behaviour of the Oued dkouk population would therefore imply adaptive mechanisms to saline environments, while in the Msarref population such mechanisms seem to be absent. Since seed germination is more sensitive to salinity stress than the growth of established plants, the greater tolerance to salinity of Oued dkouk population would be an adaptive feature of this population to saline environment.     

Role of some growth regulators on cytogenetic activity of barley under salt stress

The effects of gibberellic acid (GA₃), kinetin (KIN), benzyladenine and ethylene (E) on mitotic activity and chromosomal aberrations in root tips of barley seeds (Hordeum vulgare L. cv. “Bülbül 89”) germinated under salt stress were investigated. It was determined that all of these plant growth regulators (PGRs) decreased mitotic index in root tips of barley seeds germinated at 20 °C and in distilled water. Furthermore, some of the PGRs studied increased significantly the frequency of chromosomal aberrations. The frequency of chromosomal aberrations in seeds treated with E and KIN was considerably higher than in the seeds germinated under nonstress conditions. The inhibitory effect of salt stress on mitotic index increased with increasing salt concentration (0.30, 0.35, 0.40 and 0.45 molal, m). GA₃ and KIN pretreatments showed a successful performance in ameliorating the negative effects of increasing salinity on mitotic activity. The number of chromosomal aberrations also increased with increasing NaCl concentration. However, most of the PGR pretreatments studied alleviated the detrimental effects of increasing salinity on chromosomal aberrations. KIN pretreatment at 0.30 and 0.35 m salinity could not rescued the cytogenetic activity of salt stress on this parameter.     

Solubility of soy lipophilic proteins: comparison with other soy protein fractions

Solubility of soy lipophilic proteins (LP) was studied as compared with that of other soy protein fractions. LP, β-conglycinin, glycinin, and soy protein isolate (N-SPI) were prepared under the condition to avoid heat denaturation. Solubility of LP was lower than that of other soy protein fractions under all the tested conditions varying in pH values and ionic strength. The solubility of LP was increased constantly by elevating temperature until 90 °C, whereas that of β-conglycinin and glycinin dropped at high temperature. Temperature-dependent change in solubility of N-SPI might reflect the balance among that of glycinin, β-conglycinin and LP. Based on the results of SDS-PAGE, determination of phospholipid content and Fourier Transform Infrared spectroscopy, we discussed the solubilization behavior of LP relating to its origin and composition.     

Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior. (c) 1992 John Wiley & Sons, Inc.     

Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements

The diffusion of hen egg-white lysozyme has been studied by dynamic light scattering in aqueous solutions of ammonium sulfate as a function of protein concentration to 30 g/liter. Experiments were conducted under the following conditions: pH 4-7 and ionic strength 0.05-5.0 M. Diffusivity data for ionic strengths up to 0.5 M were interpreted in the context of a two-body interaction model for monomers. From this analysis, two potential-of-mean-force parameters, the effective monomer charge, and the Hamaker constant were obtained. At higher ionic strength, the data were analyzed using a model that describes the diffusion coefficient of a polydisperse system of interacting protein aggregates in terms of an isodesmic, indefinite aggregation equilibrium constant. Data analysis incorporated multicomponent virial and hydrodynamic effects. The resulting equilibrium constants indicate that lysozyme does not aggregate significantly as ionic strength increases, even at salt concentrations near the point of salting-out precipitation.     

Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.