Volume and compressibility changes accompanying thermally-induced native-to-unfolded and molten globule-to-unfolded transitions of cytochrome c: a high pressure study

We have measured the transition temperatures, T(M), and van't Hoff enthalpies, DeltaH(M), of the thermally induced native-to-unfolded (N-to-U) and molten globule-to-unfolded (MG-to-U) transitions of cytochrome c at pressures between 50 and 2200 bar. We have used the pressure dependence of T(M) to evaluate the changes in volume, Delta(v), accompanying each protein transition event as a function of temperature and pressure. From analysis of the temperature and pressure dependences of Delta(v), we have additionally calculated the changes in expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), associated with the thermally induced conformational transitions of cytochrome c. Specifically, if extrapolated to 25 degrees C, the native-to-unfolded (N-to-U) transition is accompanied by changes in volume, Delta(v), expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), of -(5 +/- 3) x 10(-3) cm(3) g(-1), (1.8 +/- 0.3) x 10(-4) cm(3) g(-1) K(-1), and approximately 0 cm(3) g(-1) bar(-1), respectively. The molten globule-to-unfolded (MG-to-U) transition is accompanied by changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), of -(2.9 +/- 0.3) x 10(-3) cm(3) g(-1) at 40 degrees C and -(1.9 +/- 0.3) x 10(-6) cm(3) g(-1) bar(-1) at 35 degrees C, respectively. By comparing the volumetric properties of the N-to-U and N-to-MG transitions of cytochrome c, we have estimated the properties of the native-to-molten globule (N-to-MG) transition. For the latter transition, the changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), are approximately 0 cm(3) g(-1) at 40 degrees C and 1.9 cm(3) g(-1) bar(-1) at 35 degrees C, respectively. Our estimate for the change in expansibility, Delta(e), upon the N-to-MG is negative and equal to -(5 +/- 3) x 10(-4) cm(3) g(-1) K(-1). This finding contrasts with the results of previous studies all of which report positive changes in expansibility associated with protein denaturation. In general, our volumetric data permit us to assess the combined effect of temperature and pressure on the stability of various conformational states of cytochrome c.     

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.     

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.     

The pressure-temperature free energy-landscape of staphylococcal nuclease monitored by (1)H NMR

The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing (1)H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % (2)H(2)O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 degrees C-35 degrees C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Deltabeta), the volume change at 1 bar (DeltaV degrees ) and the expansivity change (Deltaalpha) upon unfolding among other thermodynamic parameters: Deltabeta=0.02(+/-0.003) ml mol(-1) bar(-1); Deltaalpha=1.33(+/-0.2) ml mol(-1) K(-1); DeltaV degrees =-41.9(+/-6. 3) ml mol(-1) (at 24 degrees C); DeltaG degrees =13.18(+/-2) kJ mol(-1) (at 24 degrees C); DeltaC(p)=13.12(+/-2) kJ mol(-1) K(-1); DeltaS degrees =0.32(+/-0.05) kJ mol(-1) K(-1 )(at 24 degrees C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Deltabeta and Deltaalpha values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.     

The pH threshold in the dissolution of beta-lactoglobulin gels and aggregates in alkali

The existence of a practical minimum pH for the dissolution of heat-induced whey gels in alkaline solutions has been studied using beta-lactoglobulin (betaLg) as a model protein. A sharp transition in solubility was observed between pH 11 and 12; this transition shifts to higher pHs for gels formed at higher temperatures and for longer gelling times. The breakdown reactions of heat-induced aggregates in alkali were monitored with size exclusion chromatography. The destruction of large aggregates was faster at higher pH and also showed a transition between pH 11 and 12. Using tryptophan fluorescence and near- and far-UV circular dichroism, this transition was assigned to the base-induced denaturation observed in solutions of aggregates (pK 11.53). It is suggested that the high protein repulsion caused by the large number of charges at pH > 11.5 drives the unfolding of the protein and the disruption of the intermolecular noncovalent bonds. Concentrated urea and GuHCl were found to be less effective than a pH 12 solution in destroying large aggregates. Aggregates formed for a long time (80 degrees C for 24 h) contained a larger number of intermolecular disulfide bonds that hinder the dissolution process. Gels formed at low temperatures (65 degrees C for 60 min), with fewer intermolecular noncovalent bonds, showed a similar solubility-pH profile to that observed for the base-induced denaturation of unheated beta-lactoglobulin (betaLg) (pK 10.63).     

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.     

Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico

Physiological responses of Dunaliella salina and Dunaliella viridis, isolated from solar saltworks on the Yucatan Peninsula, were studied. Optimal growth temperature for D. salina was 22 degrees C (3.06 x 10(6) cells mL(-1)) and 26 degrees C for D. viridis (4.04 x 10(6)cells mL(-1)). Total carotenoid content in D. salina increased with temperature to a maximum of 35.14 pg cell(-1) at 38 degrees C. Dunaliella salina alpha-carotene and beta-carotene content was 0.083+/-0.003 and 0.598+/-0.020 mg 100g dry wt(-1) respectively, whereas lower values were found in D. viridis cultured under same experimental conditions (0.018+/-0.002 and 0.136+/-0.012 mg 100g dry wt(-1) respectively). The highest specific growth rate in D. salina was obtained at 10% NaCl (0.28 d(-1)), while its cell volume increased from 524 to 2066.93 microm(3) when cultured from 10% to 35% NaCl. Maximum photosynthetic rates were attained when increasing from optimal growing temperature to 30 degrees C for D. viridis (108 n mol O(2)microg chl alpha h(-1)) and D. salina (139 n mol O(2)microg chl alpha h(-1)). Photosynthetic responses to temperature variations indicated physiological adjustments in both species, with higher acclimation in D. salina. Evaluation of physiological attributes of these species will be used for to carry out mass cultivation.     

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.     

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.     

红壤丘陵景观单元土壤有机碳和微生物生物量碳含量特征

为了探讨我国亚热带红壤丘陵区不同利用方式下土壤有机碳(SOC)和土壤微生物生物量碳(SMB-C)含量的特征,在湖南省桃源县选取典型样区,通过密集取样,分析了红壤丘陵景观单元内水田、旱地、林地、果园4种典型利用方式下表层土壤(0～20 cm)SOC和SMB-C含量.结果表明,典型红壤丘陵景观单元中SOC含量高低的顺序为水田(16.0 g·kg^-1)＞旱地(11.2 g·kg^-1) ＞果园(9.5 g·kg^-1)＞林地(8.4 g·kg^-1),SMB-C含量则为水田(830 mg·kg^-1)＞旱地(361 mg·kg^-1)＞林地(200 mg·kg^-1)＞果园(186 mg·kg^-1),且在不同利用方式下SOC与SMB-C均呈极显著正相关(P＜0.01),说明本研究区内各土地利用类型的土壤SMB-C含量变化可以敏感地指示SOC的动态.研究结果还表明,将我国亚热带红壤丘陵林地开垦为果园或耕地后,表层土壤 SOC含量不可能降低.     

Acid- and base-induced conformational transitions of equinatoxin II

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD-spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry, gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional alpha-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50 degrees C, the acid-induced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH II and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more alpha-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.     

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.     

Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

Effects of high and fluctuating pressure on microbial abundance and activity in a water hydraulic system

The effects of high and fluctuating pressure up to 220 bar on microbial growth and activity were determined in a pilot-scale water hydraulic system. An increase in the pipeline pressure from 70 to 220 bar decreased the total and the viable cell number in the pressure medium from 2.2(+/-0.5)x10(5) to 4.9(+/-1.5)x10(4) cells/ml and from 5.7(+/-2.8)x10(4) to 1.3(+/-0.7)x10(4) cfu/ml, respectively. Microbial attachment in the non-pressurised tank of the hydraulic system increased with increasing pipeline pressure [from 1.0(+/-0.3) to 3.8(+/-2.7)x10(5) cells/cm(2) on stainless steel]. The phosphatase, aminopeptidase and beta-glucosidase activities in the pressurised medium were between 0.02 and 1.4 micromol/lh ( V(max)) and decreased in response to increasing pipeline pressure. The alpha-glucosidase activity was detected only at 70 bar and the glucuronidase activity only occasionally. Based on principal component and cluster analyses, both the pressure applied and the original filling water quality affected substrate utilisation patterns. This study demonstrated the capability of freshwater bacteria to tolerate high and fluctuating pressure in a technical water system. Microbial survival was due to attachment and growth on the surfaces of the non-pressurised components and the nutrient flux released by cell lysis in the pressurised components. In summary, high pressures in water hydraulic systems do not prevent potential microbiologically related operational problems.     

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.     

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.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

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.     

Temperature-jump studies on hemoglobin. Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhemoglobin

Temperature jumps on mixtures of hemoglobin and pH indicators give rise to relaxation signals in the microsecond range. The pH and concentration dependences of the reciprocal relaxation time, 1/tau, may be rationalized on the basis of a reaction scheme in which a slow isomerization process in the protein moiety is coupled to a rapid co-operative ionization of two protons. At 11 degrees C the rate constants of the isomerization are kr = 4.2(+/- 1.8) x 10(4) s-1 and kf = 1.3(+/- 0.1) x 10(4) s-1 in deoxyhemoglobin; in carbonmonoxyhemoglobin they are kr = 3.9(+/- 1.3) x 10(4) s-1 and kf = 5.3(+/- 1.8) x 10(3) s-1. The pKa values of the coupled ionizing groups are 5.3 in deoxy- and 6.0 in carbonmonoxyhemoglobin. Modification of the CysF9(93) beta sulfhydryl group with iodoacetamide abolishes the pH dependence of 1/tau, suggesting that this sulfhydryl is involved in the isomerization process. Consideration of the X-ray structure of oxyhemoglobin allows a structural interpretation of the results. It is concluded that the isomerization may be important for the physiological function of hemoglobin, but that it is not identical with the quaternary structure transition.     

Raman study of the thermal behaviour and conformational stability of basic pancreatic trypsin inhibitor

We have studied the thermal denaturation of native basic pancreatic trypsin inhibitor (BPTI) by monitoring the Raman bands in the 4000-400 cm(-1) range. In agreement with results obtained by calorimetry, a cooperative melting transition is observed starting at 75 degrees C. This transition is found to involve predominantly the unfolding of helical structures accompanied by beta-aggregation, loss of hydrophobic interactions between side chains and changes in CSSC dihedral angles. However, salt bridge breaking starts near 40 degrees C, as deduced from the nu(s)(COO(-)) band and from the bands close to 1320 and 1345 cm(-1) which for the first time have been shown to be due largely to vibrations of the arginine guanidyl group in BPTI. The thermal stability is, hence, attributable to cooperative contributions from hydrophobic and backbone hydrogen bond interactions as well as from disulfide bonds.