Thermochemistry of aqueous solutions of alkylated nucleic acid bases. III. Enthalpies of hydration of uracil, thymine and their derivatives

Enthalpies of sublimation DeltaH(0)(subl) crystalline uracil, thymine and their methylated derivatives as well as of N,N-diethylthymine were determinated by the quartz-resonator method and mass spectrometry. Enthalpies of solution at infinite dilution DeltaH(0)(sol) in water of aBcylated compounds were obtained calorimetrically. Hence the calculated enthalpies of hydration: DeltaH(0)(hydrsubal) = DeltaH(0)(sol) - DeltaH(0)(subl), were corrected for energies of cavity formation in pure liquid water to yield enthalpies of interaction DeltaH(0)(sint) of the solutes with their hydration shells. For uracil DeltaH(0)(int) = -59.8 kJ mole(-1) was obtained in this way. This value decreased linearly on N-methyl substitution with a mean increment of about 6.5 kJ mole CH2(-1). After C(5) or C(6) ring substitution it increased by about 3 kJ. These results are discussed in connection with heat of dilution data and theoretical schemes of hydration.     

Thermodynamics of hexokinase catalyzed reactions. II. Measurement and calculation of enthalpies of reaction as a function of magnesium ion concentration.

Enthalpies of phosphorylation of glucose by adenosine 5'-triphosphate have been measured as a function of concentrations of magnesium chloride in TRIS/TRIS-HCl buffer in the pH range 8.64 to 8.98. These measurements are compared with the results of calculations of these enthalpies that use a coupled equilibrium formalism with equilibrium data and enthalpy values selected from the literature. The experimental results span the range of magnesium ion concentrations 1 X 10(-6) to 0.3 mol alpha-1 and show a total variation in the enthalpy of reaction of almost 10 kJ mol-1, with the most exothermic reaction occurring at a magnesium ion concentration of 6.0 X 10(-4) mol alpha-1. The calculated enthalpies of reaction, except for the magnesium ion concentration range 4 X 10(-6) to 5 X 10(-4) mol alpha-1, are, within estimated uncertainty intervals (0.8 to 10.2 kJ mol-1), in agreement with the measured values.     

Enthalpies of transfer for proteins from aqueous to water-alcohol solutions: a test for models of residues exposed to solvent

The enthalpies of transfer of proteins from aqueous solution to alcohol–water solutions are used as probes of solvent-accessible surface for these proteins. Enthalpies of transfer to 10 wt% ethanol solutions are determined calorimetrically for the native proteins ribonuclease A, lysozyme, and ovalbumin. Ribonuclease A and lysozyme are reduced and carboxamidomethylated to produce configurations in which interior residues of the native protein are exposed to the solvent; enthalpies of transfer are determined for these species. These data are then compared with enthalpies of transfer for the constituent amino acids of the proteins. The enthalpies of transfer for the residues are used to generate a maximal enthalpy of transfer that can be compared with the enthalpies of transfer for the reduced, carboxamidomethylated proteins. The residue amino acid enthalpies are coupled with probabilities that each residue is an exterior residue to predict an enthalpy of transfer for the native protein that can be compared with the observed enthalpy. The probabilities developed by Wertz and Scheraga and Lee and Richards, and Chothia are then compared on their ability to predict the native enthalpies of transfer for the protein. The Wertz–Scheraga model gives the better fit of this data in all cases.     

A phenomenological model for lipid-protein bilayers with critical mixing

A Landau expansion of free energy in terms of area/lipid has been used to obtain protein-lipid phase diagrams with critical mixing and a maximum phase separation concentration. Simulations using this model indicate that differential scanning calorimetry scan shapes and transition enthalpies observed for lipid-synthetic peptide mixtures are consistent with this type of phase diagram. The critical mixing point and the homogeneous mixture critical point are distinguished.     

Equilibrium of Bowman-Birk inhibitor association with trypsin and alpha-chymotrypsin.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. VI. Enthalpies of hydration of 2-alkyl-9-methyladenines

Enthalpies of solution in water, delta H0sol, and vant'Hoff enthalpies of sublimation, delta H0subl, were determined experimentally for a number of crystalline 2-alkyl derivatives of 9-methyladenine: m2(2,9)Ade, e2m9Ade, pr2m9Ade and but2m9Ade. Standard enthalpies of hydration, delta H0hydr derived from these data were corrected for the calculated cavity terms, delta H0cav, to yield enthalpies of interaction, delta H0int, of the solutes with their hydration shells. The apparent residual contribution of alkyl groups, R, to the enthalpy of interaction delta delta H0int (R) was found to increase linearly with the number of CH2 groups added upon alkyl substitution, whereas this contribution calculated per unit area of the water-accessible molecular surface, SB, of alkyl residues delta delta H0int (R): delta SB(R) appeared constant over the whole series of the compounds investigated. This indicates that alkyl groups substituted at the C(2) carbon atom of the adenine contribute additively to the van der Waals' part of the enthalpy of interaction and do not affect the electrostatic part of the energy of interaction of the solutes with their hydration shells.     

Enthalpy changes for inositol hexaphosphate binding to hemoglobins A and M Iwate.

Enthalpies of inositol hexaphosphate (IHP) binding to deoxy and carbonmonoxy (CO) HbA and HbM Iwate have been determined calorimetrically and compared as functions of pH. Values for deoxy HbA and for deoxy HbM Iwate are similar with CO HbM Iwate yielding slightly less heat of reaction. The results support the existence of both deoxy and CO HbM Iwate in T-like structures with only minor modifications occurring upon CO binding. For HbA observed heats of IHP binding have been corrected for heats of extraction of reacting protons from buffer. The resulting intrinsic IHP binding enthalpies show consistent values of ?7 to ?11 kcal/mol proton absorbed in binding. We suggest that a major driving force for organic phosphate binding is the exothermic protonation of histidine and/or a α-amino nitrogens induced by proximity of phosphate negative charges.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. IV. Enthalpies of hydration of 5-alkyluracils

Enthalpies of sublimation, DeltaH degrees (subl) and of solution in water, DeltaH degrees (sol) were determined for a series of crystalline 1,3-dimethyl-uracil derivatives substituted at the C5-ring carbon atom with alkyl groups (-C(n)H(2n+1), n = 2-4) and some of their C(5.6)-cyclooligomethylene analogues (-(CH2)(n)-, n = 3-5). From these data. enthalpies of hydration DeltaH degrees (hydr)= DeltaH degrees (sol) - DeltaH degrees (subl) were calculated and corrected for energies of cavity formation in pure liquid water in order to obtain enthalpies of interaction, DeltaH degrees (int) of the solutes with their hydration shells. The latter are discussed together with the recalculated DeltaH degrees (int) for variously methylated uracils, obtained previously according to a simplified correction procedure, in terms of perturbations in the energy and scheme of hydration of the diketopyrimidine ring brought about by alkyl substitution. It was found that each -CH2-group added with an alkyl substitution contributes favorably about -20 kJ mol(-1) toDeltaH degrees (int).This contribution is partially cancelled by the unfavorable contribution to DeltaH degrees (int) connected with removal of some water molecules bound in the first and subsequent hydration layers by an alkyl substituent. This is particularly evident on substitution at the polar side of the diketopyrimidine ring on which water molecules are expected to be bound specifically.     

Simultaneous modeling of phase and calorimetric behavior in an amphiphilic peptide/phospholipid model membrane

Differential scanning calorimetry (DSC) experiments have been performed on the amphiphilic peptide/1,2-bis(perdeuteriopalmitoyl)-sn-glycero-3-phosphocholine system for which partial phase diagrams have been measured by deuterium nuclear magnetic resonance. The solute concentration dependence of the transition enthalpy in such systems is often interpreted in terms of an annulus of lipid withdrawn, by the solvent, from participation in the transition while the bulk lipid melts with its fully enthalpy. This idea is equivalent to postulating ideal mixing between the lipid and the peptide/lipid complex, and there is little justification for such an assumption. Adaptation of regular solution theory to this system demonstrates that the peptide concentration dependence of the transition enthalpies can be incorporated into a thermodynamic model which reproduces the observed phase behavior fairly well without postulating that a complexing annulus of lipid around the peptide be withdrawn from participating in the chain-melting transition. The model parameters determined by simultaneous fitting of the phase behavior and transition enthalpies are used to simulate the DSC scan shapes. The asymmetry of the calorimetric scans for chi 2 less than or equal to 0.02 is reproduced by the model, but a broad component observed for higher concentration is not. In light of the results presented here, previous analyses of the calorimetric behavior of two-component systems in terms of symmetric transitions which do not account for the possible extent of a region of two-phase equilibrium must be questioned.     

Energy storage in the primary photochemical events of rhodopsin and isorhodopsin

The energetics associated with the photoequilibrium (Formula: see text) are measured at 77 K by using pulsed-laser photocalorimetry and a range of excitation wavelengths and relative starting concentrations. Enthalpies for the photochemical transformations R hv----B and I hv----B are measured to be delta HRB = 32.2 +/- 0.9 kcal mol-1 and delta HIB = 27.1 +/- 3.2 kcal mol-1, respectively. Although the value of delta HRB is slightly lower than that reported previously by Cooper of 34.7 +/- 2.2 kcal mol-1 [Cooper, A. (1979) Nature (London) 282, 531-533], the two values are in agreement within experimental error. The energy difference delta HRB - delta HIB = 5.1 +/- 3.3 kcal mol-1 is identical within experimental error with the difference in enthalpies of isorhodopsin and rhodopsin [5.2 +/- 2.3; Cooper, A. (1979) FEBS Lett. 100, 382-384]. We suggest that this result is consistent with the theory that bathorhodopsin is a single, common photochemical intermediate connecting rhodopsin and isorhodopsin.     

Hydration of complementary 9-methyladenine.1-methyluracil pairs at low temperatures according to data from field mass spectrometry

The hydration of nucleotide bases of m9Ade(A), m1Ura(U) and a complementary pair A.U was studied by field ionization mass-spectrometry at room and low (170 K) temperatures in vacuum. Enthalpies of A.U-pair formation and its monohydrate A.U(H2O) were measured using temperature dependences of association constants. From the analysis of intensities of mass-spectrum peaks, corresponding to monohydrates U(H2O), A(H2O), A.U(H2O), A.U-pair and initial components A, U, and also measured enthalpies it is supposed that monohydration of bases A and U essentially prevents the formation of the coplanar pair A.U. A qualitative information about the structure and energetics of hydrate clusters A(H2O)n, U(H2O)n and A.U(H2O)n for n = 1 divided by 7 was obtained from low temperature mass-spectra. The observed peculiarities in hydrate structures A(n = 5), U(n = 4), A.U(n = 4) are treated as a consequence of cyclization of water molecules around bases.     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

In silico screening of dicarboxylic acids for cocrystallization with phenylpiperazine derivatives based on both cocrystallization propensity and solubility advantage

In silico screening was performed to search for binary solids in which a phenylpiperazine-derivative drug was cocrystallized with a dicarboxylic acid. The phenylpiperazine derivative could be any of 61 such drugs, while the dicarboxylic acid could be any of nine such acids. The uniqueness of this approach was that two criteria had to be fulfilled simultaneously, namely a high propensity for cocrystallization and a sufficient solubility advantage. Using the mixing enthalpies of selected pairs of crystal formers with high affinities for one another permitted the classification of candidates with a high probability of cocrystallization. Further modeling of the solubility advantage allowed the identification of many binary solids that potentially exhibit significantly enhanced solubility in water. Based on the computed values for the mixing enthalpies and solubility advantage factors, it was concluded that dicarboxylic acids are both excellent coformers for cocrystallization with phenylpiperazines and very good solubility enhancers; indeed, the use of dicarboxylic acids as coformers would allow the degree of dissolution to be tuned for many of the studied drugs. The observed similarities of the cocrystallization landscapes of the studied drugs and excipients were also explored.     

Periodical changes of amino acid reactivity within the genetic code.

Enthalpies (delta H++) and entropies (delta S++) of activation for the reaction of 18 N'-hydroxysuccinimide esters of N-protected proteinaceous amino acids with p-anisidine were measured and free enthalpies of activation (delta G++) at 25 degrees C were calculated on this basis. A regular correlation between delta G++s and the corresponding amino acid codons was found. To obtain this correlation all the codons had to be arranged in a closed ring in which the consecutive codons were connected by one-step mutational changes. One-step mutations appeared as a regular series: 2,3,3,3,1,3,3,3,1,3,3,3,1,3,3,3,2,3,3,3. (the numbers denote a codon position in which a change took place). There were three such 'one-step mutation periods' in the ring, each containing 20 codons (in each block of 16 codons with A, U and C, in the central position and 4 codons containing G in the central position). The end of the third period (UG) and the beginning of the first period were bridged by the four codons of glycine with G in the second position. The values of delta G++ change similarly in each period, increasing upon approaching Lys, Pro, and Ile. The periodical relation between the chemical reactivities of the coded amino acids (reflected by delta G++s) and the structure of their codons could be of importance for the origin of the genetic code i.e. for selection of proper codons for the definite amino acids.     

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.     

Crystallization and phase behavior of 1,3-propanediol esters II. 1,3-propanediol distearate/1,3-propanediol dipalmitate (SS/PP) and 1,3-propanediol distearate/1,3-propanediol dimyristate (SS/MM) binary systems

Polymorphic influences on the phase behavior of two types of binary mixtures of saturated monoacid 1,3-propanediol esters (PADEs), dipalmitate/distearate (PP/SS) and dimyristate/distearate (MM/SS) were examined by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and by solid fat content (SFC), hardness and microscopy measurements. Three stacking modes have been found in the PP/SS binary system. Mixed SS-PP bilayers were detected in all mixtures, SS-SS bilayers in x(PP)=0.0-0.4 mixtures and PP-PP bilayers in x(PP)=0.6-0.1 mixtures. Two different but close beta polymorphs and one beta' polymorph were detected for this system. beta' was only detected in x(PP)=0.5-0.9 mixtures for the mixed bilayers. For the MM/SS binary system, only MM-MM and SS-SS bilayers were detected and both solid phases crystallized in two different beta forms. XRD data evidenced clearly that the MM and SS components were completely immiscible in the solid state. The phase diagrams constructed using DSC data, exhibited a typical eutectic-type phase boundary. The presence of eutectics, the shape of the solidus lines as well as the analysis of the individual enthalpies of melting indicated typical phase separation for both systems. A thermodynamic study based on the Hildebrand equation and using the Bragg-Williams approximation for non-ideality of mixing confirmed the phase separation in the solid phase and suggested that the PP and SS were miscible in the liquid phase and that SS formed an ideal mixing with MM. Avrami analysis of SFC vs. time curves indicated heterogeneous nucleation and spherulitic crystal development from sporadic nuclei, and suggested that the nucleation rate was higher for the mixture at the eutectic composition. The relative hardness was correlated with the enthalpies, the final SFC and the microscopy measurements.     

Thermal stability of DNA.

Tij and Delta Hij for stacking of pair i upon j in DNA have been obtained over the range 0.034-0.114 M Na+from high-resolution melting curves of well-behaved synthetic tandemly repeating inserts in recombinant pN/MCS plasmids. Results are consistent with neighbor-pair thermodynamic additivity, where the stability constant, sij , for different domains of length N depend quantitatively on the product of stability constants for each individual pair in domains, sijN . Unit transition enthalpies with average errors less than +/-5%, were determined by analysis of two-state equilibria associated with the melting of internal domains and verified from variations of Tij with [Na+]. Enthalpies increase with Tij , in close agreement with the empirical function: Delta Hij = 52.78@ Tij - 9489, and in parallel with a smaller increase in Delta Sij . Delta Hij and Delta Sij are in good agreement with the results of an extensive compilation of published Delta Hcal and Delta Scal for synthetic and natural DNAs. Neighbor-pair additivity was also observed for (dA@dT)-tracts at melting temperatures; no evidence could be detected of the familiar and unusual structural features that characterize tracts at lower temperatures. The energetic effects of loops were determined from the melting behavior of repeating inserts installed between (G+C)-rich barrier domains in the pN/MCS plasmids. A unique set of values for the cooperativity, loop exponent and stiffness parameters were found applicable to internal domains of all sizes and sequences. Statistical mechanical curves calculated with values of Tij([Na+]) , Delta Hij and these loop parameters are in good agreement with observation.     

Thermodynamics of the interaction of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in aqueous solutions at 298.15K

The Gibbs energy interaction parameters of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in water, g(ES), were obtained from electromotive force (emf) measurements of the electrochemical cell without liquid junction and containing two ion-selective electrodes (ISE): K-ISEmid R:RbCl(m(E))mid R:ISE-Cl and K-ISEmid R:RbCl(m(E)),saccharide (m(S))mid R:ISE-Cl, at 298.15K. The enthalpy interaction parameters of RbCl with these monosaccharides in water, h(ES), are determined according to the McMillan-Mayer theory from the measurements of the enthalpies of mixing of aqueous RbCl solutions with aqueous monosaccharide solutions, as well as the enthalpies of dilution of RbCl and monosaccharide solutions in pure water at 298.15K by a calorimetric method. Furthermore, the entropy interaction parameters, s(ES), can be evaluated through g(ES) and h(ES). The results suggest that the electrostatic interactions of these monosaccharides with RbCl in water are predominant compared with structural interactions, and these parameters are controlled primarily by the stereochemical structure of the monosaccharides in water.     

Kinetic studies on the unfolding and refolding of pepsinogen in urea. The nature of the rate-limiting step

Kinetic studies on the unfolding of pepsinogen by urea showed that changes in absorbance and potential pepsin activity followed simple first order kinetics. Changes in these variables on refolding were more complex. A large part of the absorbance was recovered within the mixing time of these experiments, whereas the appearance of activity was a slow sigmoidal function of time. The results were interpreted to show that pepsinogen can rapidly regain a globular form, but that its activatable form is produced by a slow conformational change in the folded protein. The enthalpies of activation of this change are similar to those of the cis-trans isomerization of proline residues. If the latter reaction is involved in the folding of pepsinogen, it must occur after extensive folding has already occurred.