Standard Volume Change Research Articles

Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20.

Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (KD) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process. According to this study, delta V of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of F-actin induced by hydrostatic pressure.

F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.

Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure.

Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.

Reversible inhibition of EcoRI with elevated pressure

The endonuclease activity of EcoRI is completely inhibited at 200 MPa, 37°C using the plasmid pBR 322 as a substrate. When assayed at 133 MPa approximately half the activity at atmospheric pressure was observed; from these data the standard molar volume change is estimated to be −80 cm 3 mol −1. Upon return to atmospheric pressure the enzyme reacted in a standard manner with its restriction site in pBR 322. Pressurization did not decrease the specificity of the endonuclease activity of the enzyme for its canonical site. These results are discussed in terms of the role of ionic interactions in protein-DNA interactions.

Partial molar volumes of sulfur dioxide in organic solvents: formation of charge-transfer complexes

New apparatus for measuring the apparent molar volumes of gases in liquids is described. The apparatus has been used to obtain the limiting partial molar volume of sulfur dioxide in 17 organic solvents. The limiting partial molar volume of sulfur dioxide is found to be significantly smaller in electron-donating solvents that in non-electron-donating solvents, with this difference being interpreted in terms of charge-transfer complex formation. We propose two methods for estimating the standard volume change of the complexation reaction and illustrate the use of these methods by application to the sulfur dioxide + benzene system.

The first ionization constant from 25 to 200�C and 2000 bar for orthophosphoric acid

The ionization constant of orthophosphoric acid, determined by conductivity measurements, decreased from 7.11×10−3 at 25°C to 6.2×10−4 mol-kg−1 at 200°C. The pressure effect to 2000 bar was also measured and the ratio K2000/K1 is 2.7 at 25°C and 3.7 at 200°C. The standard partial molar volume change for the ionization at 1 bar, $$\Delta \overline V _{_1^o }$$ , changes from −16.1 at 25°C to −33.3 cm3-mol−1 at 200°C. The partial molar compressibility change for the ionization, $$\Delta \overline x ^\circ$$ , varies from −3.8×10−3 to −8.3×10−3 cm3-mol−1 bar−1 over the same temperature range.

The origin of celestite in deep-sea carbonate sediments

Several celestite nodules were recovered on DSDP Leg 90 from four drilling sites on the Lord Howe Rise, southwest Pacific Ocean. The sediments at these sites are predominantly very pure calcareous nannofossil oozes and chalks. As a result of a higher-than average accumulation rate, they undergo relatively rapid burial diagenesis, which causes the expulsion of Sr from the biogenic calcite, to the interstitial waters. Another result of the high accumulation rate is the occurrence of microbial sulfate reduction in the interstitial waters. The downcore Sr increase is proportionately greater than the sulfate decrease, and celestite precipitates below about 100 m subbottom at each of these sites. The celestite contains high concentrations of many substituent cations: 4.3–7.8 mole% BaSO 4, 1.7–6.4 mole% CaSO 4, 1100–2600 ppm Al, and 400–750 ppm K. Ion activity products of Sr and sulfate at each site were calculated from the Pitzer equations and the measured concentrations of porewater constituents, and are in close agreement with the celestite solubility product corrected for in situ temperatures and pressures. Strontium concentrations of the porewaters are nearly at equilibrium with respect to celestite, and are controlled by the extent of microbial sulfate reduction. Celestite solubility increases with increasing water depth, in excellent agreement with values of the standard state partial molal volume change.

Relationship among Entropies,Volumes and Internal Pressures of Liquid Water and Aqueous Prototropic Ions

According to the Webb theory of ionic hydration modified by the Onsager reaction field, an internal pressure of liquid water p in (H 2 O) is calculated by using exclusively the physical properties of water at the temperature T =298 K and the external pressure 1 atm; p in (H 2 O)=11.6 kbar, which is the pressure of liquid water as an elastic fluid equated to an electrostatic force per unit area at the surface of an ionic cavity. The cavity has been charged up to the elementary unit of e , and takes the same radius as that of the Onsager cavity for water, 1.93 Å. The calculated value agrees exactly with the value of T S 0 / V 0 in terms of the standard molar entropy and volume of water, S 0 and V 0 , and also favorably with T Δ S i 0 / Δ V i 0 =10.9 kbar in terms of the standard entropy change and volume change in the self-ionization of liquid water, Δ S i 0 and Δ V i 0 . This relationship may be extended to the individual aqueous ions of H + and OH - .

Effects of Some Additives on Proton-Transfer Reaction Studied by Ultrasonic Methods

Abstract Ultrasonic absorption and velocity were measured in aqueous solutions of propylamine at 25 °C both in the absence and in the presence of 2 g dm−3 PVA(poly(vinyl alcohol)), 5 mol dm−3 urea and 1 mol dm−3 NaCl. No excess absorption was observed in these solutions without propylamine. A single relaxational absorption was observed in the concentration range from 0.0196 to 0.719 mol dm−3 of propylamine both in the presence and absence of the additives. The ultrasonic parameters have been found to be quite dependent on the concentration of propylamine and also on the structure of additives. This relaxational absorption has been attributed to the perturbation of an equilibrium associated with a proton-transfer reaction. The rate constants and the standard volume change of the reaction have been determined from the concentration dependences of the relaxation frequency and the maximum excess absorption per wavelength. As a result, it was discovered that the forward rate in a solution with PVA increased and those with urea and NaCl, on the other hand, decreased; also, the standard volume change associated with the reaction increased in a series of the solutions with urea, no additive, NaCl, and PVA. The data have been interpreted by means of the structural changes of the solvent water.

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase.

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.

Analysis of Proton-Transfer Reaction in Aqueous Solutions of Butylamines by Ultrasonic Methods

Abstract Ultrasonic absorption has been measured in an aqueous solution of s-butylamine over the frequency range 6.5–220 MHz and the concentration range 0.0115–3.18 mol dm−3 at 25 °C. The single relaxational phenomenon observed in a range less than 1 mol dm−3 was found to be associated with a perturbation of the equilibrium; R-NH3++OH− (Remark: Graphics omitted.) R-NH3+···OH−. The rate constants of the forward and backward reactions, k12 and k21, have been determined to be 2.1×1010 s−1 mol−1 dm−3 and 1.8×108 s−1, respectively. It was also determined that the next step, R-NH3+···OH− (Remark: Graphics omitted.) R-NH2+H2O, which had been thought to have a considerable influence on the first step, was not very effective regarding the ultrasonic absorption observed in the MHz frequency range. The equilibrium constant in the second step, K32, has been determined to be 3.5×10−2. The standard volume change of the reaction has been calculated to be 26 cm3 mol−1 from the concentration dependence of the maximum excess absorption per wavelength. The relaxation observed in a range greater that 1 mol dm−3 has been estimted to be associated with a molecular aggregation reaction. An ultrasonic study in an aqueous solution of butylamine has also been performed in order to determine the rate constants associated with a proton-transfer reaction at 25 °C.

Relaxation spectra of gramicidin dimerization in a lipid bilayer membrane

The kinetics of formation and dissociation of gramicidin dimers in a lipid bilayer membrane have been studied by pressure-jump and electric field-jump methods. The traditional AC-coupled pressure-jump apparatus has been modified so that a known DC-voltage drop is maintained across a Teflon cell divided by a septum with a hole for membrane formation. From the response of the amplified output voltage after the pressure release, information about the kinetics of channel (dimer) formation is obtained. In addition, using the same apparatus, electric field-jump measurements were performed on the gramicidin / membrane system. In asolectin / 7-dehydrocholesterol (5:1) membranes at 25 ± 0.1°C, the best fit to the pressure-jump data gives a dimer dissociation rate constant of 0.5 ± 0.3 s −1. The standard volume change for dimerization determined from the amplitude of the pressure-jump experiments is −66 ± 35 cm 3 / mol. Rate data determined by the electric field-jump method are consistent with the pressure-jump values; results obtained with either technique are compatible with other determinations of the kinetics of dimerization on gramicidin / membrane systems.

Ultrasonic absorption of the oxyethylene group in aqueous nonelectrolyte solutions

The ultrasonic absorption from 6.5 to 220 MHz and velocities at 2.5 MHz have been measured in aqueous solution of triethylene glycol monobutyl ether as a function of concentration at 25°C. A single relaxational excess absorption, observed from 0.60 to 2.5 mol-dm−3, is attributed to a perturbation of an equilibrium associated with solute-solvent interaction. Rate constants for the forward and reverse processes have been determined from the concentration dependence of the relaxation frequency, and the influence on the water structure by an addition of the solute has been considered. Solution densities were also measured in order to obtain the expansivity of the solution. The standard volume and enthalpy changes of the reaction have been estimated from the concentration dependence of the maximum excess absorption per wave length. A linear relationship between the number of oxyethylene groups and the free energy change between bonded water and nonbonded water is established.

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg).

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic study of the interaction between diols and water by ultrasonic methods. Part 3.—2-methylpentane-2,4-diol solution

Ultrasonic absorption, velocity, density and viscosity have been measured in aqueous solutions of 2-methylpentane-2,4-diol at 25 °C as a function of the concentration. A single relaxational absorption has been found whose cause has been attributed to the perturbation of an equilibrium associated with the solute–solvent interaction AB⇌A + B, where A is the solute and B the solvent. The forward and backward rate constants for this interaction have been evaluated from the concentration dependence of the relaxation frequency as kf= 1.1 × 108 S–1 and kb= 1.1 × 108 dm3 mol–1 S–1. The standard volume and enthalpy changes of the reaction have also been determined from the concentration dependence of the maximum excess absorption per wavelength as ΔV= 0.070 cm3 mol–1 and ΔH= 1.7 kJ mol–1. At high concentrations (above 5.5 mol dm–3) another relaxation, which might be due to the interconversion of rotational isomers, has been also found in the same frequency range. This is confirmed from ultrasonic-absorption measurements in toluene mixtures.

Kinetics of Solute-Solvent Interaction in Aqueous Solution of Butyl Carbitol by Ultrasonic Methods

Abstract Ultrasonic absorptions in the frequency range 4.5–220 MHz and sound velocities at 2.5 MHz were measured at 25 °C with aqueous solutions of Butyl Carbitol (diethylene glycol monobutyl ether) over the concentration range 0.497–3.00 mol dm−3. Only a single relaxational process was observed and attributed to a perturbation of the equilibrium AB\oversetk12\undersetk21↔A+B, where A is Butyl Carbitol as the solute and B is water as the solvent participating in the reaction. A data analysis has led to the estimates k12=3.4×107 s−1, k21=2.0×108 dm3 mol−1 s−1, the standard volume change of reaction ΔV°=0.32 cm3 mol−1, and the standard enthalpy change ΔH°=5.4 kJ mol−1. The effect of the hydrophobicity of solute molecules on the water structure is discussed by comparing the present results with previous ones for other aqueous nonelectrolyte solutions.

Ionization constants of aqueous ammonia from 25 to 250�C and to 2000 bar

The ionization constant of ammonia has been determined by conductivity measurements and found to vary from 1.77 x 10/sup -5/ at 25/sup 0/C to 1.3 x 10/sup -6/ mol-kg/sup -1/ at 250/sup 0/C. The pressure effect to 2000 bar has been measured and the ratio K/sub 2000//K/sub 1/ is 6.8 at 25/sup 0/C. The standard molar volume change for the ionization at 1 bar, ..delta.. V/sub 1//sup 0/, changes from -28.8 at 25/sup 0/C to -67 cm/sup 3/-mol/sup -1/ at 250/sup 0/C. 6 figures, 9 tables.

A study on the micelle formation of surfactants in aqueous solutions under high pressure by laser light-scattering technique. II

The constant aggregation numbers of micelles, which form above certain concentrations in aqueous solutions of polyoxyethylene (penta-, hepta-, and octaoxyethylene) dodecyl ethers (C12E5, C12E7, and C12E8, respectively), have been determined by means of a laser light-scattering apparatus at 298.15°K and various pressures up to 160 MPa. The aggregation numbers decreased with increasing pressure, in a similar manner as is the case for hexaoxyethylene dodecyl ether (C12E6, Part I). By exploiting Tanford's expressions about the restricted length and the volume of a hydrocarbon chain in the micellar core of liquid, and the compressibility data for the micellar core, the critical micellization concentration (CMC) under pressure has been estimated for each of C12E7 (n = 5, 6, 7,and 8) from the micellar size vs pressure. From the relation of CMC vs pressure, the standard volume change on micellization was calculated for each surfactant, as a function of pressure. The calculated volume changes are larger than those for ionic surfactants of similar length of hydrocarbon chain, irrespective of pressure. We assigned the volume change to the hydrocarbon chain and an oxyethylene (OE) group, and the contribution of the latter was estimated as 0.3–0.4 cm3 mole−1 nearly independent of pressure. This positive volume change is attributed to the partial dehydration of OE groups during micellization, which characterizes the micellar properties of nonionic surfactants under pressure in aqueous solutions.

Ionization constants of benzoic acid from 25 to 250�C and to 2000 bar

The ionization constant of benzoic acid has been determined by conductivity measurements of dilute aqueous solutions and found to vary from 6.27×10−5 at 25°C to 0.39×10−5 at 250°C. The pressure effect to 2000 bar has been measured, and the ratio of ionization constants K2000/K1 is 2.26 at 25°C and 7.3 at 250°C. ΔV°1, the standard partial molar volume change for the ionization at 1 bar, varies from −11.7 cm3-mol−1 at 25°C to −60 cm3-mol−1 at 250°C. The volume changes are smaller at higher pressures.

Pressure-induced reversible dissociation of enolase.

A study of the polarization of the intrinsic fluorescence and the fluorescence of dansyl conjugates of enolase shows that an increase in hydrostatic pressure, in the range of 1 bar-3 kbar, promotes the dissociation of this protein into dimers. The dissociation of oligomeric proteins under pressure is predicted to be a general phenomenon by a model that assumes the existence of small "free volumes" at the intersubunit boundaries. The same model predicts a dependence of the standard volume change in the dissociation reaction upon the pressure, owing to the additional surface compressibility of the monomers, and numerical analysis of the results clearly shows that dependence for enolase. For a midpoint dissociation pressure of 1.5 kbar the standard volume change in the dissociation reaction is delta V p0 = -65 +/- 8 mL mol-1 and the dependence of the volume change upon pressure (dVp0/dp) is approximately -30 mL mol-1 kbar-1. The reversibility of the pressure effects is shown to be better than 95% by either polarization or fluorescence spectrum recovery. The pressure perturbation of the fluorescence polarization is a method of general applicability to studies of protein aggregation, and it can be also of value in characterizing the effect of ligands on the aggregation of oligomeric proteins.