Thermally induced conformational transition of double-stranded xanthan in aqueous salt solutions.

The thermally induced conformational transition of double-stranded xanthans (degree of pyruvate substitution, DSp = 0.45) having Mw = 3.1, 5.7, and 20.3 x 10(5) has been studied in aqueous salt solutions by high-sensitivity differential scanning calorimetry (DSC). The double strandedness of these samples in the ordered conformation was ascertained by the value of mass per unit length, ML = 2090 +/- 270 g mol-1 nm-1, which was determined from the contour length obtained by electron microscopic observations and the molecular weight by light scattering measurements. The temperature at half completion of the transition T 1/2 for these samples increased linearly with the logarithm of the cation (Na+, K+) concentration. The plot of 1/T1/2 vs the natural logarithm of cation (Na+) concentration in mM for the sample with Mw = 5.7 x 10(5) (15-SX) yielded the equation 10(3)/T1/2 = 3.45-0.159 ln [Na+]. The specific enthalpy delta hcal for 15-SX, essentially independent of salt concentration above 20 mM, was 8.31 +/- 0.39 J/g (SD, n = 6). No systematic dependence of molecular weight on the transition temperature and the enthalpy was observed. Application of the Manning polyelectrolyte theory to the system using the DSC data suggested that the separation of the double strand of xanthan into two single chains was not completed at the temperature where the endothermic peak was finished. This suggestion is consistent with recent findings by light scattering measurements as a function of temperature. Our DSC study was extended to include four other samples from various sources. It was found that T1/2 and delta hcal depend on the pyruvate contents of the samples. For example, the t1/2 (t1/2/degrees C = T1/2/K - 237.15) values for samples with high pyruvate content (DSp = 0.9) and depyruvated (DSp = 0.14) in 20 mM aqueous NaCl were 48.8 and 85.3 degrees C, respectively. Two other samples showed relatively broad DSC curves having shoulders, which were resolved into two independent components. Thermodynamic parameters for each component were examined as a function of salt concentration, and the results obtained were interpreted in terms of the heterogeneity of the pyruvate content of the samples.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Ordering in aqueous polysaccharide solutions. II. Optical rotation and heat capacity of aqueous solutions of a triple-helical polysaccharide schizophyllan

Deuterium oxide solutions of schizophyllan, a triple-helical polysaccharide, undergoing an order-disorder transition centered at 17 degrees C, were studied by optical rotation (OR) and heat capacity (C(p)) to elucidate the molecular mechanism of the transition and water structure in the solution and frozen states. The ordered structure at low temperature consisted of the side chains and water in the vicinity forming an ordered hydrogen-bonded network surrounding the helix core and was disordered at higher temperature. In the solution state appeared clearly defined transition curves in both the OR and C(p) data. The results for three samples of different molecular weights were analyzed theoretically, treating this transition as a typical linear cooperative transition from the ordered to disordered states and explained quantitatively if the molecular weight polydispersity of the sample was considered. The excess heat capacity C(EX)(p) defined as the C(p) minus the contributions from schizophyllan and D(2)O was estimated. In the frozen state it increased with raising temperature above 150 K until the mixture melted. This was compared with the dielectric increment observed in this temperature range and ascribed to unfreezable water. From the heat capacity and dielectric data, unfreezable water is mobile but more ordered than free water. In the solution state, the excess heat capacity originates from the interactions of D(2)O molecules as bound water and structured water, and so forth. Thus the schizophyllan triple helix molds water into various structures of differing orders in solution and in the solid state.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturation of staphylococcal nuclease

The fully reversible thermal denaturation of staphylococcal nuclease in the absence and presence of Ca2+ and/or thymidine 3',5'-diphosphate (pdTp) from pH 4 to 8 has been studied by high-sensitivity differential scanning calorimetry. In the absence of ligands, the denaturation is accompanied by an enthalpy change of 4.25 cal g-1 and an increase in specific heat of 0.134 cal K-1 g-1, both of which are usual values for small globular proteins. The temperature (tm) of maximal excess specific heat is 53.4 degrees C. Each of the ligands, Ca2+ and pdTp, by itself has important effects on the unfolding of the protein which are enhanced when both ligands are present. Addition of saturating concentrations of these ligands raises the denaturational enthalpy to 5.74 cal g-1 in the case of Ca2+ and to 6.72 cal g-1 in the case of pdTp. The ligands raise the tm by as much as 11 degrees C depending on ligand concentration. From the variation of the denaturational enthalpies with ligand concentrations, binding constants at 53 degrees C equal to 950 M-1 and 1.4 X 10(4) M-1 are estimated for Ca2+ and pdTp, respectively, and from the enthalpies at ligand saturation, binding enthalpies at 53 degrees C of -15.0 and -19.3 kcal mol-1.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Conformational transitions of schizophyllan in aqueous alkaline solution

The conformational transitions of schizophyllan were studied in aqueous alkaline solutions by high-sensitivity differential scanning calorimetry (DSC) and optical rotation measurements. The temperature of half completion for reversible intramolecular conformational transition determined by DSC, centered at 7.4°C in water, increases to 37.2°C at 0.01M KOH with increasing alkaline concentration. The transition enthalpy per mole of the polysaccharide repeating unit is 2.62 ± 0.23 kJ mol⁻¹ independent of the alkaline concentration. The cooperative unit size for the transition decreases with increasing alkaline concentration. Optical rotation was measured as a function of pH at 25 and 60°C. A sharp decrease in optical rotation was observed at pH = 13, which is ascribed to the triple helix-coil transition. From data obtained by DSC and optical rotation measurements, in combination with results reported previously, a phase diagram for the conformation of schizophyllan as a function of temperature and pH is proposed. The irreversibility of the triple helix to single coil transition, induced by strong alkali, was investigated as a function of polymer concentration by gel permeation chromatography and electron microscopy. The renatured samples at polymer concentrations < 1.0 mg/mL, which are prepared by dissolution in 0.25M KOH followed by neutralization with HCl, are observed as a mixture of globular, linear, and circular structures, and larger aggregates with less-defined morphology by electron microscopy. Higher concentrations lead to increased proportions of multichain clusters (aggregates). Subsequent annealing of the renatured samples at 115–120°C increases the proportion of circular species. The change in molecular weight distribution of samples that accompanies the renaturation and annealing mentioned above can be well interpreted in terms of the proportion of species having different morphology as observed by electron microscopy. © 1996 John Wiley & Sons, Inc.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI Kunitz inhibitor) to human Lys77-plasmin has been investigated. Ka values decrease with decreasing pH, reflecting the acid-pK and -midpoint shifts, upon BPTI binding, of a single ionizable group, between pH 5 and 9, and of a three-proton transition, between pH 3 and 5. At pH 8.0, values of thermodynamic parameters for BPTI binding to human Lys77-plasmin are: Ka = 1.2 X 10(9) M-1, delta G degree = -12.2 kcal/mol, and delta S degree = +49 entropy units (at 21 degrees C); and delta H degree = +2.3 kcal/mol (temperature independent between 5 degrees C and 45 degrees C; 1 kcal = 4184 J). BPTI binding properties of human Lys77-plasmin have been analysed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates. Considering the known molecular structures of homologous serine (pro)enzymes, or Kunitz and Kazal-type inhibitors and of their complexes, the observed binding behaviour of BPTI to human Lys77-plasmin was related to the inferred stereochemistry of the enzyme-inhibitor contact region.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts.

The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.     

Physicochemical studies of processes involving potential photodynamic drugs on route to their targets: self-aggregation and membrane-binding of Zn-hematoporphyrin

The thermodynamics of zinc hematoporphyrin (ZnHP) dimerization and ZnHP-membrane binding were studied. The dimerization equilibrium was determined over the temperature range 19-40 degrees C, using fluorometric techniques. The dimerization constant obtained at 37 degrees C (neutral pH in phosphate-buffered saline) is 4.6 (+/- 0.6) X 10(4) M-1. The dimerization was found to decrease with temperature over the range 19-36 degrees C, the data allowing the extraction of the following thermodynamic parameters for the temperature range 19-31 degrees C: delta G0 = -9.3 kcal/mol, delta H0 = -7.4 kcal/mol, delta S0 = -6.4 eu. For temperatures above 36 degrees C the dimerization was found to be temperature independent, giving the following parameters: delta G0 = -6.6 kcal/mol, delta H0 = 0 kcal/mol, delta S0 = 21.2 eu. On the basis of the data the case is made for the existence of two types of ZnHP dimers, differing in the location of the fifth Zn2+ ligand and in the nature of the contribution of the solvent to the dimerization. For the membrane binding, large unilamellar liposomes served to model biological membranes. The binding of ZnHP to the liposomes was found to be similar, quantitatively, to the corresponding metal-free molecule, namely, fitting a case of one type of site and giving a binding constant of 1600 +/- 160 M (neutral pH and 37 degrees C) which is independent of the length of the porphyrin-liposome.