A pulsed field gradient NMR diffusion investigation of enkephalin peptide-sodium dodecyl sulfate micelle association

Pulsed field gradient NMR (PFG-NMR) diffusion experiments were used to investigate the binding of leucine and methionine enkephalin peptides to anionic sodium dodecyl sulfate (SDS) micelles. The study was undertaken to investigate the mechanism of interaction between enkephalin peptides and SDS micelles and to determine if NMR-derived association constants, K(eq), can predict the elution order in electrokinetic chromatography (EKC). In EKC, peptides are separated on the basis of their interactions with micelles. The Leu-enkephalin peptide-micelle association constant increased from 130 +/- 8 to 1459 +/- 57 and 1744 +/- 64 M(-1), respectively, when an Arg or Lys was added to the C-terminus. The association constant of Leu-enkephalinamide was approximately equal to that of Leu-enkephalin-Arg. Substitution of Phe4 with a Trp or Gly2 with an Ala in the Leu-enkephalin peptides also increased the micelle binding affinity. These results confirm that the interaction of Leu-enkephalin peptides with SDS micelles is largely electrostatic and that the non-polar amino acid side chains interact with the hydrophobic micelle core. The peptide-micelle association constants for the cationic Met-enkephalin peptides were also greater than their zwitterionic counterparts. For example, the Met-enkephalin K(eq) value was 162 +/- 9 M(-1), while the association constants for Met-enkephalin-Arg, Met-enkephalin-Lys, and Met-enkephalinamide were, respectively, 674 +/- 31, 426 +/- 23, and 453 +/- 27 M(-1). In both Met-enkephalin and Met-enkephalinamide, replacing Gly2 with an Ala did not significantly increase the association constant. These results confirm that with the Met-enkephalin peptides, there was little or no interaction of the amino acid side chains with the micelle core. For both the Leu-enkephalin and Met-enkephalin peptides, the association constants were consistent with EKC results, in that the peptides with smaller K(eq) values were found to elute before those with larger association constants.     

Characterization of the antioxidative activity of novel nontoxic neuropeptides by using capillary electrophoresis

In the present study, we have monitored the oxidation process of novel nontoxic neuropeptides and determined its rate constants, which describe the antioxidative potential of the peptides. A capillary electrophoretic method was implemented which ensures the simultaneity of analysis of reactants and products in a short time of analysis. The rate constants of oxidation of the four novel peptides, 4-methoxy-L-tyrosinyl-gamma-L-glutamyl-L-cysteinyl-glycine (UPF1), D-serinyl-gamma-L-glutamyl-L-cysteinyl-glycine (UPF6), 4-methoxy-L-tyrosinyl-alpha-L-glutamyl-L-cysteinyl-glycine and D-serinyl-alpha-L-glutamyl-L-cysteinyl-glycine, designed by us, were compared with those of oxidation of glutathione (reduced glutathione) by using capillary electrophoresis. The second-order rate constants were similar for all peptides if the oxidation was carried out with hydrogen peroxide (k(II) = 0.208 - 0.236 x 10(3)/M.min). The rate constants were also determined for the mixtures of peptides. When the oxidation is caused by hydroxyl radical (OH*), the gamma-glutamate containing peptides (UPF1 and UPF6) exhibited two to four times higher antioxidative activity (k(II) = 4.428 and 2.152 x 10(3)/M.min, respectively). The results suggest that the antioxidative potential of the peptides studied is not determined by the formation of disulphide bridge alone.     

Complete resolution of the ionization equilibria of 5-deoxypyridoxal in water-dioxane mixtures

We have used a potentiometric method to determine the thermodynamic equilibrium constants for the macroscopic ionization processes of 5-deoxypyridoxal (DPL) in water-dioxane mixtures (0-70% weight fraction in dioxane) at temperatures ranging from 10°C to 50°C. These data, together with previously published equilibrium constants for the tautomerism and hydration processes, have allowed us to resolve the complete microconstant system. We have also calculated the microscopic ionization equilibrium constants under all the experimental conditions. The changes of standard thermodynamic function for the macroscopic and microscopic ionization processes were obtained in various water-dioxane mixtures at 25°C. The values of a given microscopic pK with different solvents and temperatures fit very well to an equation which relates this magnitude with the thermodynamic parameters, the solvation of the components of the reaction, and a solvent parameter. We have obtained an interesting linear correlation between the thermodynamic parameters corresponding to all the microscopic ionizations of DPL and the net change of the solvation during the process: enthalpies correlate linearly for all the microscopic ionizations, while entropies do so for the phenols and pyridinium ions separately.     

Prediction of peptide retention times in reversed-phases high-performance liquid chromatography during linear gradient elution

The retention behavior of 100 peptides was studied during high-performance liquid chromatography on a C₁₈ column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in a linear gradient elution system. Retention times of the peptides were linearly related to the logarithm of the sum of Rekker's constants (R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977, p. 301) for the constituent amino acid. Assuming this relationship, the best fit constants for this system were computed by non-linear multiple regression analysis. Using the new constants, it is possible to predict retention times for a wide variety of peptides at any slope of linear gradient, if the amino acid composition is known. It also enables accurate prediction of the retention time of peptides, whose amino acid composition in not known, after an analytical run with an alternate gradient.     

Prediction of microscopic protonation constants of polybasic molecules via computational methods: A complete microequilibrium analysis of spermine

For the first time, a simple methodology is reported for theoretical calculation of microscopic protonation constants of polybasic molecules in solution. Density functional theory study was used for complete microequilibrium analysis of spermine, H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂, a linear tetraamine with 16 known microspecies. A general thermodynamic cycle is proposed to calculate protonation microconstants of polybasic molecules using calculated micro‐ΔG values in aqueous solution. The microscopic protonation constants were determined with considering both the most abundant and most stable conformers for all microspecies. The results show that the microscopic protonation constants derived from the most abundant conformers (i.e., linear conformers in which the intramolecular hydrogen bonding does not exist) are in good agreement with the corresponding available experimental data. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Characterization of ester hydrolysis in terms of microscopic rate constants

Hydroxide-catalyzed ester hydrolysis for molecules of coexisting species is quantitated in terms of microscopic rate constants, a new, species-specific physicochemical parameter. Relationships between the overall and component reactions, as well as the macroscopic and microscopic rate constants are deduced. Experimental techniques, evaluation methods, and feasibility are discussed. Species-specific, pH-independent rate constants of four coexisting, differently hydrolyzing microspecies are determined for the first time. Protonation of an alpha-amino and beta-imidazolyl site in amino acid esters has been found to accelerate the hydroxide-catalyzed hydrolysis by factors of 120 and 7.5, respectively, whereas they jointly exert a nearly 3000-fold acceleration. A total of 20 microscopic protonation equilibrium constants, as component parameters in the rate equations, have also been determined. The species-specific rate constants have been found to correlate with the site- and species-specific basicity of the leaving group and the NMR chemical shift of an adjacent proton. Individual contributions of the various microforms to the overall hydrolysis rate are depicted in microscopic reaction fraction diagrams.     

Conformational analysis of XA and AX dipeptides in water by electronic circular dichroism and 1H NMR spectroscopy

We measured the temperature-dependent electronic circular dichroism (ECD) spectra of AX, XA, and XG dipeptides in D2O. The spectra of all XA and AX peptides indicate a substantial population of the polyproline II (PPII) conformation, while the ECD spectra of LG, KG, PG, and AG were found to be quantitatively different from the alanine-based dipeptides. Additional UV absorption data indicate that the ECD spectra of the XG peptides stem from electronic coupling between the peptide and the C-terminal group, and that spectral differences reflect different orientations of the latter. We also measured the 1H NMR spectra of the investigated dipeptides to determine the 3JHalphaNH coupling constants for the C-terminal residue. The observed temperature dependence of the ECD spectra and the respective room-temperature 3JHalphaNH coupling constants were analyzed by a two-state model encompassing PPII and a beta-like conformation. The PPII propensity of alanine in the XA series is only slightly modulated by the N-terminal side chain, and is larger than 50%. As compared to AA, XA peptides containing L, P, S, K V, E, T, and I all cause a relative stabilization of the extended beta-strand conformation. The PPII fractions of XA peptides varied between 0.64 for AA and 0.58 for DA, whereas the PPII fractions of AX peptides were much lower. From the investigated AX peptides, only AL and AQ showed the expected PPII propensity. We found that AT, AI, and AV clearly prefer an extended beta-strand conformation. A quantitative comparison of AA, AAA, and AAAA revealed a hierarchy AAAA > AAA approximately AA for the PPII population, in agreement with predictions from MD calculations and results from Raman optical activity studies (McColl et al. J. Am. Chem. Soc. 2004, 126, 5076).     

A theoretical form of the Martin-Hou equation of state

A new equation of state is derived from the Barker-Henderson hard-sphere perturbation theory. It has the form similar to the Martin-Hou equation of state. The numerical values of the characteristic constants in the equation can be calculated by the method of Martin and Hou. The equation can be used to predict P-V-T properties accurately for fluids when the critical parameters (T_c, P_c and V_c) and one point on the vapor pressure cure are given. By using the functional relationships between the characteristic constants and the microscopic parameters, the molecular microscopic parameters of the substance can be obtained.     

Microscopic acid dissociation constants of 3,4-dihydroxyphenylpropionic acid and related compounds, and 3,4-dihydroxyphenylalanine (DOPA)

The dissociation constants of 3,4-dihydroxyphenylpropionic acid and related compounds and of DOPA were determined by potentiometric titration and complementary tristimulus colorimetry at 25 degrees and mu = 0.1 (NaClO(4)) in aqueous solution. The thermodynamic parameters were calculated from the values of the dissociation constants at various temperatures. The dissociation constants and corresponding thermodynamic parameters for the first phenol group of the catechols showed almost the same values as those of the phenol derivatives. In the dissociation of the second phenol group of the catechols, formation of an intramolecular hydrogen bond was indicated. The microscopic acid dissociation constants of 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylpropionic acid were calculated by two different methods. In the physiological pH-region (pH 7.2-7.4), 3,4-hydroxyphenylpropionic acid is present in the carboxylate form, and the two possible monophenolate anions are present to the extent of about 45% and 40%, respectively, at pH 11.0. The twelve micro-constants for the eight chemical species from DOPA were similarly evaluated.     

Antisense peptide interactions studied by electrospray ionization mass spectrometry

Non-covalent interactions between met- and leu-enkephalins and their antisense peptides were studied by electrospray ionization mass spectrometry. Mixtures of sense and antisense peptides gave both the corresponding homodimers and heterodimers. The relative abundance ratios of the heterodimer to that of the homodimer of the sense peptide and the relative stability constants of the heterodimers were compared with the corresponding values from mixtures of the sense peptides and a control peptide. The results show that there is a preferential interaction between the sense and antisense peptides compared with that between the sense and control peptides.     

Aqueous Capillary Electrophoresis of Peptides Part 2: An Investigation of Quantitative Determinants of Hydration and Solvation Relationships

Summary From the published electrophoretic data for a set of 58 peptides at pH 2.5 we have determined the excess hydration required to correct the deviations of the 31 higher-charged peptides from the Offord plot for the 27 singly-charged peptides. These values of the excess hydration were then used to test physical models by regression analysis. The dominant variable is the total excess positive charge, but the number of excess single positive charges and the number of pairs of positive charges – both at the end and internally – were significant determinants. The effect of acidic side chains was ambiguous throughout the analyses and this was presumed to arise from the ability of carboxy groups to be the sites of additional hydration or to diminish hydration via internal bonding with amino groups. A hydrophobicity index was included in he analysis, but surprisingly this too had little effect. In the last stage of the regressions, we also included coding for secondary structure within the peptides. This increased R² for the plots of the excess hydration versus values calculated from the regression constants (Y) from 0.95 to 0.97. Some solvation relationships were particularly good at modelling the hydration of the smaller, lesser-charged peptides, whilst others excelled in the opposite direction. The final model appeared to deal equally well with both extremes, but is not sufficiently sensitive to allow for all of the variations between peptides. As a result, the excess hydration was modelled very well for many of the peptides (±1–2 waters over the range of excess hydration form 5–276), but poorly for a few (± 100–200%). It was not possible to find another set of peptides that could be independently analysed in a similar fashion and compared. However, the regression constants were applied to an alternative set. Although appearing to fall in a generally reasonable range, various tests of the resultant calculated values indicated that this application was not valid. The second set of peptides were much more hydrophilic and the number of amino acid residues with an extended secondary structure appeared to be a contributing factor.     

Statistical mechanical approach to competitive binding of metal ions to multi-center receptors

A microscopic site binding model to treat binding of several metal ions to multi-center receptors is proposed. The model introduces the appropriate parameterization in terms of microscopic complexation constants and metal-metal pair interaction energies. The model is solved with statistical mechanical techniques, including direct enumeration or transfer matrices. We obtain microscopic and macroscopic complexation constants, microstate probabilities, and binding isotherms for chain-like receptors, including the long-chain limit. Various examples to illustrate the usefulness of the model are given.     

Highly specific affinities of short peptides against synthetic polymers

We investigated polymer-binding 7-mer peptides that recognize differences in the polymer stereoregularity of all-purpose poly(methyl methacrylate)s (PMMAs) with simple chemical structures. Quantitative surface plasmon resonance measurements detected association/dissociation processes of the peptides against PMMA film surfaces, followed by an estimation of kinetic parameters such as association/dissociation rate constants and affinity constants. Greater association and smaller dissociation constants of the peptides were observed against a target isotactic PMMA than the structurally similar reference syndiotactic PMMA, followed by greater affinity constants against the target. A c02 peptide composed of the Glu-Leu-Trp-Arg-Pro-Thr-Arg sequence showed the greatest affinity constant (2.8x10(5) M(-1)) for the target, which was 41-fold greater than that for the reference, thus demonstrating extremely high peptide specificities. The substitution of each amino acid of the c02 peptide to Ala (Ala scanning) clearly revealed the essential amino acids for the affinity constants; the essential order was Pro5>Thr6>Arg7>Glu1>Arg4. In fact, the shorter 4-mer peptide composed of the C-terminal Arg-Pro-Thr-Arg sequence of the c02 peptide still demonstrated strong target specificity, although the N-terminal 4-mer peptide Glu-Leu-Trp-Arg completely lost its specificity. The possible conformations modeled with Molecular Mechanics supported the significance of the Arg-Pro-Thr-Arg sequence. The thermodynamic parameters of the c02 peptide suggested an induced fit mechanism for the specific affinity. The present affinity analyses of polymer-recognizing peptides revealed significant and general information that was essential for potential applications in peptidyl nanomaterials.     

羟氨苄青霉素的微观电离常数

测知酸碱性物质的微观电离常数, 对全面深入地了解该物质的化学行为及生物活性均很重要, 本文报道了用电位滴定法和分光光度法, 测求羟氨苄青霉素在20℃时的微观电离常数.     

Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level

We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.     

Competitive Cd2+/H+ complexation to polyacrylic acid described by the stepwise and intrinsic stability constants

Stepwise constants can be used to describe competitive proton and metal binding to macromolecules with a large number of sites. With the aim of accessing information on the microscopic binding model, we report an expression that connects the stepwise constants to the site-specific metal constants. This expression holds for a very general complexation model including heterogeneity, interactions, and chelate complexation. Assuming bidentate binding of the Cd ions to adjacent carboxylate groups in poly(acrylic acid), stepwise and intrinsic stability constants for proton and cadmium binding were estimated from the experimental data. Intrinsic values were split into specific and electrostatic contributions (by means of the Poisson-Boltzmann equation under cylindrical geometry). Free of the electrostatic contribution, the remaining Cd binding energy showed almost no dependence on the coverage and ionic strength, and the corresponding average values allowed for a reasonable reproduction of raw binding data. Small systematic discrepancies from the homogeneous behavior are critically discussed.     

Supramolecular parallel β-sheet and amyloid-like fibril forming peptides using δ-aminovaleric acid residue

Four terminally blocked tripeptides containing δ-aminovaleric acid residue self-assemble to form supramolecular β-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel β-sheet structures. Self-aggregation of these β-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils.     

Microscopic acid-base equilibria of arginine

Three and two protonation macroconstants as well as twelve and four microconstants have been determined for arginine and citrulline, respectively. These data include microscopic basicity values of the arginine guanidino group, for which no reliable constants have been reported earlier. The determinations were carried out by a combination of potentiometric and deductive methods. The guanidino basicity proved to be extremely high, and can be characterized by microconstants between 14.7 and 15.0 log k units. The interactivity parameters could also be determined from the microscopic protonation constants. These values for the guanidino—amino and the guanidino—carboxylate interactions are 0.2 and 0.1 log k units, respectively. The pH-dependent distribution of all eight arginine microspecies is made visual by a microspeciation diagram. The microspeciation diagram is the most useful tool to predict bidentate binding isomerism in metal complex formation and site-specific bioligand—bioligand associations.     

Thiyl radicals abstract hydrogen atoms from the (alpha)C-H bonds in model peptides: absolute rate constants and effect of amino acid structure

Thiyl radicals are important intermediates in biological oxidative stress and enzymatic reactions, for example, the ribonucleotide reductases. On the basis of the homolytic bond dissociation energies (BDEs) only, the (alpha)C-H bonds of peptides and proteins would present suitable targets for hydrogen abstraction by thiyl radicals. However, additional parameters such as polar and conformational effects may control such hydrogen-transfer processes. To evaluate the potential of thiyl radicals for hydrogen abstraction from (alpha)C-H bonds, we provide the first absolute rate constants for these reactions with model peptides. Thiyl radicals react with (alpha)C-H bonds with rate constants between 1.7 x 10(3) M(-1) s(-1) (N-acetylproline amide) and 4 x 10(5) M(-1) s(-1) (sarcosine anhydride). However, the correlation of rate constants with BDEs is poor. Rather, these reactions may be controlled by conformation and dynamic flexibility around the (alpha)C-H bonds.     

Effects of hydration on scale factors for ab initio force constants

Experimentally measured vibrational frequencies from the polar groups of peptides in aqueous solutions do not agree with frequencies calculated from scaled quantum mechanical force fields (SQMFF) using differential scale factors developed for molecules in the vapor phase. Measured stretching frequencies for carbonyl groups are more than 50 wavenumbers lower than the calculated values. On the other hand, frequencies for non-polar groups calculated using these scale factors are relatively accurate. Our goal is to develop a SQMFF that yields accurate calculated frequencies for peptides in aqueous solutions. To this end, we have calculated scale factors for ab initio force constants for formic acid, acetic acid, and acetone using a least squares fit of calculated and experimental frequencies. We compare these scale factors with changes observed in the ab initio force constants calculated for these molecules at various states of hydration. These force constants are calculated using fully optimized geometries for these hydrated molecules using the 4-31G basis. We present a comparison of the experimental and calculated frequencies, along with their potential energy distributions, for both vapor and aqueous phases. The results indicate that scale factors can simulate the effects of solvation on molecular force constants to yield accurate scaled ab initio force fields.