Compressibility changes accompanying conformational transitions of apomyoglobin

We used high-precision density and ultrasonic velocity measurements to characterize the native (N), molten globule (MG), and unfolded (U) conformations of apomyoglobin. The molten globule states that were studied in this work include the MG(pH4)(NaCl) state observed at pH 4 and 20 mM NaCl, the MG(pH4)(NaTCA) state observed at pH 4 and 20 mM sodium trichloracetate (NaTCA), the MG(pH2)(NaCl) state observed at pH 2 and 200 mM NaCl, and the MG(pH2)(NaTCA) state observed at pH 2 and 20 mM NaTCA. We used our densimetric and acoustic data to evaluate changes in adiabatic compressibility associated with the acid- or salt-induced N-to-MG, MG-to-U, MG-to-MG, and U-to-MG transitions of the protein. The N-to-MG(pH4)(NaCl) and N-to-MG(pH4)(NaTCA) transitions are accompanied by decreases in compressibility of -(3.0 +/- 0.6) x 10(-6) and -(2.0 +/- 0.6) x 10(-6) cm3 g(-1)bar(-1), respectively. The N-to-MG(pH2)(NaCl) and N-to-MG(pH2)(NaTCA) transitions are associated with compressibility changes of -(4.9 +/- 1.1) x 10(-6) and (0.7 +/- 0.9) x 10(-6) cm3 g(-1) bar(-1), respectively. We interpret these data in terms of the degree of unfolding of the various molten globule forms of apomyoglobin. In general, our compressibility data reveal significant disparities between the various equilibrium molten globule states of apomyoglobin while also quantitatively characterizing each of these states. Volumetric insights provided by our data facilitate gaining a better understanding of the folding pathways, intermediates, and kinetics of apomyoglobin folding.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

Some taste molecules and their solution properties.

The solution properties of a variety of different sapid substances from all four basic taste modalities, namely, sweet (n = 24), salty (n = 7), sour (n = 11) and bitter (n = 2), have been investigated. Some multisapophoric molecules, i.e. molecules exhibiting more than one taste, have also been included in the study in an attempt to define their properties in relation to the tastes they exhibit; eight sweet-bitter and three salty-bitter molecules were used. The density and sound velocity of their solutions in water have been measured and their apparent volumes, apparent compressibilities and compressibility hydration numbers calculated and compared. Apparent molar volumes (phi(v)) and apparent specific volumes (ASV) reflect the state of hydration of the molecules, and thus their extent of interaction with water structure. The range of ASVs reported are 0.13-0.49 cm3/g for salty molecules, 0.55-0.68 cm3/g for sweet molecules, 0.53-0.88 cm3/g for sweet-bitter molecules and a much wider range (0.16-0.85 cm3/g) for sour molecules. Isentropic apparent specific compressibilities range from -2.33 x 10(-5) to -8.06 x 10(-5) cm3/g x bar for salty molecules, -3.38 x 10(-7) to -2.34 x 10(-5) cm3/g x bar for sweet molecules, +6.35 x 10(-6) to -2.22 x 10(-5) cm3/g x bar for sweet-bitter molecules and +6.131 x 10(-6) to -2.99 x 10(-5) cm3/g x bar for sour molecules. Compressibility hydration numbers are also determinable from the measurements of isentropic compressibilities and these reflect the number of water molecules that are disturbed by the presence of the solutes in solution. This study also shows that it is possible to group isentropic apparent molar compressibility values by the taste quality exhibited by the molecules in the same order as for ASV.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Thermodynamic and structural properties of the acid molten globule state of horse cytochrome C

To understand the stabilization, folding, and functional mechanisms of proteins, it is very important to understand the structural and thermodynamic properties of the molten globule state. In this study, the global structure of the acid molten globule state, which we call MG1, of horse cytochrome c at low pH and high salt concentrations was evaluated by solution X-ray scattering (SXS), dynamic light scattering, and circular dichroism measurements. MG1 was globular and slightly (3%) larger than the native state, N. Calorimetric methods, such as differential scanning calorimetry and isothermal acid-titration calorimetry, were used to evaluate the thermodynamic parameters in the transitions of N to MG1 and MG1 to denatured state D of horse cytochrome c. The heat capacity change, ΔC(p), in the N-to-MG1 transition was determined to be 2.56 kJ K(-1) mol(-1), indicating the increase in the level of hydration in the MG1 state. Moreover, the intermediate state on the thermal N-to-D transition of horse cytochrome c at pH 4 under low-salt conditions showed the same structural and thermodynamic properties of the MG1 state in both SXS and calorimetric measurements. The Gibbs free energy changes (ΔG) for the N-to-MG1 and N-to-D transitions at 15 °C were 10.9 and 42.2 kJ mol(-1), respectively.     

Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase

To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C. These mutations induced large changes in v(o) (0.710-0.733 cm(3). g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3). g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities. The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large beta(s)(o) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K(m)), despite the fact that the mutation sites are far from the catalytic site. These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.     

Changes in the adiabatic compressibility of mono- and polyclonal antibodies during interaction with antigens

The values of apparent adiabatic compressibility of free and antigen-bound antibodies were determined by means of precise density and ultrasound velocity measurements. It was shown that during the formation of soluble immune complexes (insulin--monoclonal antibodies to insulin and alpha-amylase--monovalent Fab-fragments of antibodies to alpha-amylase), the apparent compressibility of antibodies decreased by (0.3 divided by 0.9).10(-6) cm3/g.bar. During the formation of large insoluble aggregates (alpha-amylase--polyclonal antibodies to alpha-amylase), the apparent compressibility decreased by (5.5 +/- 0.7).10(-6) cm3/g.bar. It is suggested that the decrease in the magnitude of thermal fluctuations of the molecular volume of antibodies during antigen binding, manifesting itself by the decrease in their compressibility and strengthened several-fold by precipitate formation, may favour the activation of the effectory functions of antibodies.     

Inner-sphere complexes of divalent cations with single-stranded poly(rA) and poly(rU)

A combination of ultrasound velocimetry, density, and UV spectroscopy has been employed to study the hydration effects of binding of Mn(2+) and alkaline-earth cations to poly(rA) and poly(rU) single strands. The hydration effects, obtained from volume and compressibility measurements, are positive due to overlapping the hydration shells of interacting molecules and consequently releasing the water molecules to bulk state. The volume effects of the binding to poly(rA), calculated per mole of cations, range from 30.6 to 40.6 cm(3) mol(-1) and the compressibility effects range from 59.2 x 10(-4) to 73.6 x 10(-4) cm(3) mol(-1) bar(-1). The volume and compressibility effects for poly(rU) are approximately 17 cm(3) mol(-1) and approximately 50 x 10(-4) cm(3) mol(-1) bar(-1), respectively. The comparative analysis of the dehydration effects suggests that the divalent cations bind to the polynucleotides in inner-sphere manner. In the case of poly(rU) the dehydration effects correspond to two direct coordination, probably between adjacent phosphate groups. The optical study did not reveal any effects of cation on the secondary structure or aggregation of poly(rU). In the case of single-helical poly(rA) binding is more specific: dehydration effects correspond to three to five direct contacts and must involve atomic groups of adenines, and the divalent cations stabilize and aggregate the polynucleotide.     

Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility

The partial specific volume, v, and adiabatic compressibility, beta(s), of Escherichia coli dihydrofolate reductase were measured at 30 degrees C in the presence of various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP, methotrexate, and KCl). Binding of these ligands (binary and ternary complexes) brought about large changes of v (0.734-0.754 cm(3) g(-1)) and beta(s) (6. 6x10(-6)-9.8x10(-6) bar(-1)), keeping a linear relationship between the two parameters. The values of v and beta(s) increased with an increase in internal cavity, V(cav), and a decrease in accessible surface area, ASA, which were calculated from the X-ray crystal structures of the complexes. A large variation of V(cav) relative to ASA by ligand binding suggested that the cavity is a dominant factor and the effect of hydration might be small for the ligand-induced changes of v and beta(s). The beta(s) values of the binary and ternary complexes suggested a characteristic conformational flexibility of the kinetic intermediates in the enzyme reaction coordinate. Comparison of beta(s) with the cavity distribution in the crystal structures revealed that the flexibility of the intermediates was mainly determined by the total cavity volume with minor contributions of the number, position, and size of cavities. These results demonstrate that the compressibility is a useful measure of the conformational flexibility of the intermediates in the enzyme reaction and that the combined study of compressibility and X-ray crystallography gives new insight into the protein dynamics through the behavior of the cavities.     

High pressure effect on the main transition from the ripple gel P'beta phase to the liquid crystal (Lalpha) phase in dipalmitoylphosphatidylcholine. Microcalorimetric study

Scanning microcalorimetry has been used to study the high pressure effect on the main transition from the ripple gel P'(beta) phase to the liquid crystal (L(alpha)) phase in DPPC (dipalmitoylphosphatidylcholine). It has been demonstrated that an increase of the pressure by 200 MPa shifts the transition to higher temperatures by 36.4 degrees. The pressure increase does not affect the cooperativity of transition but reduces noticeably its enthalpy. The changes of the molar partial volume, isothermal compressibility as well as volume thermal expansibility during transition in DPPC suspension have been estimated. It has been shown that monovalent ions (Na(+), Cl(-)) in solution slightly affect the main thermodynamic parameters of the transition. Calcium ions significantly decrease distinction in compressibility and thermal expansibility between liquid-crystal and ripple gel phases of lipid suspension, which in its turn reflects less difference in their volume fluctuations.     

Effects of active and passive hyperthermia on heat shock protein 70 (HSP70)

Summary. The purpose of this study was to delineate the effects of hyperthermia and physical exercise on the heat shock protein 70 (HSP70) response in circulating peripheral blood mononuclear cells (PBMCs). Six healthy, young (age: 24 ± 3 yrs), moderately trained males (VO_2max: 48.9 ± 2.7 ml · kg · min⁻¹) undertook two experimental trials in a randomised fashion in which the core temperature (T _c) was increased and then maintained at 39 °C during a 90 min bout by either active (AH) or passive (PH) means. AH involved subjects cycling at 90% of their lactate threshold in attire designed to impede heat loss mechanisms. In the PH trial, subjects were immersed up to the neck in a hot bath (40.2 ± 0.4 °C), once the critical T _c was achieved, intermittent cycling and water immersions were prescribed for the AH and PH conditions, respectively, to maintain the T _c at 39 °C. HSP70 was measured intracellularly pre, post and 4 h after trials, from circulating PBMCs using an ELISA technique. T _c reached 39 °C quicker in PH than during AH trials (PH: 21 ± 4 min vs. AH: 39 ± 6 min; P < 0.01), thereafter T _c was maintained around 39 °C (PH: 39.1 ± 0.2 °C; AH: 38.8 ± 0.3 °C; P > 0.05). AH induced a marked leukocytosis in all sub-sets (P < 0.05). PH generated significant monocytosis and granulocytosis (P < 0.05), without changes in lymphocyte counts (P > 0.05). There were no significant increases in intracellular HSP70 at 0 h (AH: Δ − 21.1 ± 44.8; PH: Δ + 12.5 ± 32.4 ng/mg TP/10³/μl PBMCs; P > 0.05) and 4 h (AH: Δ − 30.0 ± 40.1; PH: Δ + 36.3 ± 70.4 ng/mg TP/10³/μl PBMCs; P > 0.05) post active and passive heating. Peak HSP70 expressed as a fold-change from rest was also not increased by AH (1.1 ± 0.9; P > 0.05) or PH (3.2 ± 4.8; P > 0.05). There were no significant differences between the AH and PH trials at any time-point, and the HSP70 response appeared to be individual specific. These results did not allow us to delineate the effects of hyperthermia and other exercise associated stressors on the heat shock response and therefore further work is warranted. Authors’ address: Ric Lovell, Department of Sport, Health and Exercise Science, University of Hull, Hull HU6 7RX, U.K.     

Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes

In the present study, mitochondrial ribosomes of the nematode Ascaris suum were isolated and their physiochemical properties were compared to ribosomes of Escherichia coli. The sedimentation coefficient and buoyant density of A. suum mitochondrial ribosomes were determined. The sedimentation coefficient of the intact monosome was about 55 S. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.40 g/cm(3), which suggests that the nematode mitoribosomes have a much higher protein composition than other mitoribosomes. The diffusion coefficients obtained from dynamic light scattering measurements were (1.48 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for 55 S mitoribosomes and (1.74 +/- 0.04) x 10(-)(7) cm(2) s(-)(1) for the 70 S E. coli monosome. The diameter of mitoribosomes was measured by dynamic light-scattering analysis and electron microscopy. Though the nematode mitoribosome has a larger size than the bacterial ribosome, it does not differ significantly in size from mammalian mitoribosomes.     

Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

Spin probe mobility in the whole blood of white rats]

By means of ESR-method the rotary mobility of a tanol spin probe is studied in the whole blood of white rats at the temperatures 5.20 and 37 degrees C. It is shown that at all the temperatures the spin probe is localized in the blood plasma and has a value HFS a = (17.1 +/- 0.1) G. By means of linear anamorphism method it is shown on the example of the spectrum central line that the contour is lorenz, i. e. the superposition of the spectra of different sample regions is absent. The spin probe rotation frequency v is a stable blood parameter, the same for 11 rats investigated and dependent only on the blood temperature. For T = 5.20 and 37 degrees C the values have been received v = (86 +/- 2) x 10(8) s-1, (98 +/- 2) x 10(8) s-1 and (107 +/- 3) x 10(8) s-1, subsequently, which compared to v value in water-glycerin system (1:1) (WGS) allow one to calculate the blood microviscosity values (7.2 +/- 0.4), (6.3 +/- 0.4) and (5.8 +/- 0.4) mPds, subsequently. For the mentioned temperatures the non-sphericity parameter epsilon of the spin probe rotation has the values 0.19 +/- 0.03, 0.22 +/- 0.04 and 0.21 +/- 0.05, subsequently that is close to this parameter value for WCS (epsilon = 0.21 +/- 0.02; v = (6 divided by 20) x 10(9) s-1).     

Osmotic behavior of red blood cell as seen with an ultrasonic method

We have measured the density and ultrasonic velocity (usv) of swine red blood cell (RBC) suspensions in the wide osmolarity range from 300 mOsm to 1400 mOsm in saline solution. The cellular density and compressibility of RBC at each osmolarity were obtained using the fact that the density and the compressibility are additive by volume. The osmolarity dependence of hematocrit was also measured at a constant number concentration of RBC in the range of 300 mOsm to 1700 mOsm. The cellular density and the cellular compressibility of RBC as well as the inverse of hematocrit were expressed well into one unique exponential type equation f (pi) = a [1 - b exp (-c pi)] with a common value for the coefficient c = 0.0025 against the osmolarity pi. The results were analyzed with a simple consideration based only upon the contribution of free water inside the erythrocyte through the volume concentration phi of the free water in it. According to this theoretical analysis, the density and the compressibility of the free water were found to be 0.990 g/cm3 and 4.59 x 10(-11) cm2/dyne which agree closely with 0.998 g/cm3 and 4.59 x 10(-11) cm2/dyn of pure water at 20 degrees C within the experimental error.     

Rapid diffusion of spectrin bound to a lipid surface.

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

Influence of the local anesthetic tetracaine on the phase behavior and the thermodynamic properties of phospholipid bilayers.

We investigated the influence of the local anesthetic tetracaine on the thermodynamic properties and the temperature- and pressure-dependent phase behavior of the model biomembrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine by using volumetric measurements at temperatures ranging from 0 degrees to 40 degrees C and at pressures from ambient up to 1000 bar. The pVT measurements were complemented by temperature-dependent differential scanning calorimetric measurements. Information about the influence of different concentrations of the local anesthetic on the thermodynamic changes accompanying the lipid phase transitions, and on the thermal expansion coefficient, the isothermal compressibility, and the volume fluctuations of the lipids in their different phases, could be obtained from these experiments. The incorporation of tetracaine leads to an overall disordering of the membrane, as can be inferred from the depression of the main transition temperature and the reduction of the volume change at the main lipid phase transition. The expansion coefficient alpha p and the isothermal compressibility chi T of the lipid bilayer are enhanced by the addition of tetracaine and strongly enhanced values of alpha p and chi T, and the lipid volume fluctuations are found in the direct neighborhood of the main phase transition region. As tetracaine can be viewed as a model system for amphiphilic molecules, these results also provide insight into the general understanding of the physicochemical action of amphiphilic molecules on membranes. The experimental results are compared with recent theoretical predictions for the phase behavior of anesthetic-lipid systems, and the biological relevance of this study is discussed.     

广东省桉树人工林土壤有机碳密度及其影响因子

研究了广东省粤北、粤东、粤西和珠江三角洲4个地区桉树人工林土壤有机碳含量和有机碳密度,以及土壤有机碳密度的主要影响因子.结果表明:土壤A层和B层有机碳含量分别为(23.94±2.97)g·kg-1和(9.68±1.05)g·kg-1,二者差异显著;A层和B层有机碳密度分别为(27.64±7.72)t·hm-2和(108.36±9.37)t·hm-2,有显著差异;0～50 cm土层有机碳密度为(66.72±6.53)t·hm-2,略高于同一地区马尾松和杉木人工林.有机碳密度与海拔、A层和B层土壤总孔隙度、毛管孔隙度、毛管持水量和全氮含量呈显著正相关;土壤毛管孔隙度、毛管持水量和pH值是影响有机碳密度的主导因子.     

High pressure effect on the main transition from the ripple gel P′β phase to the liquid crystal (Lα) phase in dipalmitoylphosphatidylcholine. Microcalorimetric study

Scanning microcalorimetry has been used to study the high pressure effect on the main transition from the ripple gel P′_β phase to the liquid crystal (L_α) phase in DPPC (dipalmitoylphosphatidylcholine). It has been demonstrated that an increase of the pressure by 200 MPa shifts the transition to higher temperatures by 36.4 degrees. The pressure increase does not affect the cooperativity of transition but reduces noticeably its enthalpy. The changes of the molar partial volume, isothermal compressibility as well as volume thermal expansibility during transition in DPPC suspension have been estimated. It has been shown that monovalent ions (Na⁺, Cl⁻) in solution slightly affect the main thermodynamic parameters of the transition. Calcium ions significantly decrease distinction in compressibility and thermal expansibility between liquid-crystal and ripple gel phases of lipid suspension, which in its turn reflects less difference in their volume fluctuations.