The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds

A post-column infusion system was developed in order to analyze suppression of electrospray ionization (ESI) tandem mass spectrometry response in the presence of endogenous plasma interferences. By enabling direct detection of these interfering components, this experimental system was used to analyze the ability of several common extraction procedures to remove endogenous plasma components that cause changes in the ESI response of model drug substances. Methyl-t-butyl ether (MTBE) liquid-liquid, Oasis and Empore solid-phase, and acetonitrile (ACN) protein precipitation sample preparation methods were tested using the post-column infusion system. In all cases, ACN protein precipitation samples showed the greatest amount of ESI response suppression while liquid-liquid extracts demonstrated the least. In addition, the three test compounds, phenacetin, caffeine, and a representative Merck compound, demonstrated that ESI response suppression is compound dependent. Suppression was greatest with caffeine, the most polar analyte, and the smallest for the Merck compound, the least polar analyte. Copyright 1999 John Wiley & Sons, Ltd.     

Hydrophobic hydration in cyclodextrin complexation

We report temperature-dependent acoustic and densimetric data on changes in volume, expansibility, and adiabatic compressibility accompanying the binding of 1-adamantanecarboxylic acid (AD) to beta-cyclodextrin (beta-CD). We interpret our volumetric results in terms of hydration. Based on our compressibility and expansibility data, we estimate that, at 25 degrees C, the binding of AD to beta-CD is accompanied by displacement of 20 to 25 water molecules from the hydration shells of the two interacting species. Comparison of the temperature-dependent compressibility changes accompanying the binding of AD to beta-CD with the compressibility contribution of aliphatic groups suggests that displaced water molecules predominantly come from the hydrophobic loci of AD and beta-CD. Thus, we conclude that hydrophobic interactions play a major role in stabilizing the AD-beta-CD complex. Our estimate of the number of water molecules released to the bulk is consistent with structural considerations. There is also good agreement between our volumetric data and osmotic stress results reported by Harries et al. (Harries, D.; Rau, D. C.; Parsegian, V. A. J. Am. Chem. Soc. 2005, 127, 2184). This observation is consistent with the picture in which the two techniques probe the same population of water molecules solvating AD and beta-CD.     

Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion

The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism. We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 degrees C. The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants. The exquisite sensitivity of the methodology to small changes in folding/unfolding rates facilitated the use of relatively low-pressure values (between 1 and 270 bar) so that pressure-induced changes to the unfolded state ensemble could be minimized. A volume change for unfolding of -81 mL/mol is measured (at 1 bar), a factor of 1.4 larger (in absolute value) than the volume difference between the transition state ensemble (TSE) and the unfolded state. Notably, the changes in the free energy difference between folded and unfolded states and in the activation free energy for folding were not linear with pressure. Thus, the difference in the isothermal compressibility upon unfolding (-0.11 mL mol(-1) bar(-1)) and, for the first time, the compressibility of the TSE relative to the unfolded state (0.15 mL mol(-1) bar(-1)) could be calculated. The results argue for a TSE that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding. The notion of a collapsed and hydrated TSE is consistent with expectations based on earlier temperature-dependent folding studies, showing that the barrier to folding at 45 degrees C is entropic (Choy, W. Y.; Zhou, Z.; Bai, Y.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 5066-5072).     

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.     

Hydration changes accompanying nucleic acid intercalation reactions:volumetric characterizations

We use high precision ultrasonic velocimetric and densimetric techniques to determine at 25 degrees C the changes in volume, deltaV, and adiabatic compressibility, deltaK(S), that accompany the binding of ethidium to the poly(rA)poly(rU), poly(dAdT)poly(dAdT), poly(dGdC)poly(dGdC), and poly(dIdC)poly(dIdC) duplexes, as well as to the poly(rU)poly(rA)poly(rU) triplex. The binding of ethidium to each of the duplexes and the triplex is accompanied by negative changes in volume, deltaV, and adiabatic compressibility, deltaK(S). We discuss the basis for relating macroscopic and microscopic properties, particularly, emphasizing how measured changes in volume and compressibility can be quantitatively interpreted in terms of the differential hydration properties of DNA and RNA structures in their ligand-free and ligand-bound states. We also estimate the entropic cost of intercalation-induced changes in hydration of each of the nucleic acid structures and the drug. In general, our results emphasize the vital role of hydration in modulating the energetics of drug-DNA binding, while also underscoring the fact that hydration must be carefully taken into account in analysis and prediction of the energetics of nucleic acid recognition.     

关于单位园周上阶梯函数的性质

Ｔ．Ｓｈｅｉｌ－Ｓｍａｌｌ［１］讨论了阶梯函数的Ｆｏｕｒｉｅｒ级数。本文对［１］中所涉及到一些阶梯函数的性质进行了详细讨论，并给出了详细证明。     

极限方程有奇性的一类六阶椭圆型方程混合边值问题解的渐近式

该文研究极限方程在部分边界上为退缩椭圆型(椭圆-抛物)的一类六阶椭圆型方程混合边值问题的奇摄动,在适当的假设下,应用改进了的多重尺度法,求得其解包括边界层和套层在内除了半圆域的两个角点外,在整个半圆域中有任意阶的一致有效的渐近展开式.     

Energetics of nucleic acid stability: the effect of DeltaCP

We report high-resolution differential scanning calorimetric data on the poly(dAdT)poly(dAdT), poly(dA)poly(dT), poly(dIdC)poly(dIdC), poly(dGdC)poly(dGdC), poly(rA)poly(rU), and poly(rI)poly(rC) nucleic acid duplexes. We use these data to evaluate the melting temperatures, TM, enthalpy changes, DeltaHM, and heat capacity changes, DeltaCP, accompanying helix-to-coil transitions of each polymeric duplex studied in this work at different NaCl concentrations. In agreement with previous reports, we have found that DeltaCP exhibits a positive, nonzero value, which, on average, equals 268 +/- 33 J mol(-1) K(-1). With DeltaCP, we have calculated the transition free energies, DeltaG, enthalpies, DeltaH, and entropies, DeltaS, for the duplexes as a function of temperature. Since, DeltaG, DeltaH, and DeltaS all strongly depend on temperature, the thermodynamic comparison between DNA and/or RNA duplexes (that may differ from one another with respect to sequence, composition, conformation, etc.) is physically meaningful only if extrapolated to a common temperature. We have performed such comparative analyses to derive differential thermodynamic parameters of formation of GC versus AT, AU, and IC base pairs as well as B' versus A and B helix conformations. We have proposed some general microscopic interpretations for the observed sequence-specific and conformation-specific thermodynamic differences between the duplexes.     

Theoretical analysis of an ultrasonic interferometer for precise measurements at high pressures

Expressions making it possible to calculate the transmission coefficient of an ultrasonic interferometer, its Q factor and errors of interferometric measurements are presented. These expressions are obtained on the basis of a one-dimensional model which takes into account the non-ideal reflection of acoustic waves from the transducers, diffraction losses and electromechanical properties of transducers. Fixed path ultrasonic interferometers which are widely used for high precision measurements of ultrasound absorption and velocity in liquids, include air-backed transducers. Air-backing limits the application of these interferometers for the measurements under the high pressures. The influence of non-gaseous backing of transducers on the characteristics of an interferometer is theoretically analysed by means of suggested expressions. Frequency dependences of main parameters of the interferometer are obtained. The possibility of high precision measurements of velocity and absorption of ultrasound in liquids under the high pressures by means of fixed path interferometers with liquid-backed transducers is shown.     

Volumetric characterization of sodium-induced G-quadruplex formation

Oligodeoxyribonucleotides (ODN) with repeats of the human telomeric sequence can adopt different tetrahelical conformations that exhibit similar energetic parameters. We studied the volumetric properties of the folded and unfolded states of an ODN with four repeats of the human telomeric sequence, d[A(GGGTTA)(3)GGG], by combining pressure-perturbation calorimetry (PPC), vibrating tube densimetry, ultrasonic velocimetry, and UV melting under high pressure. We carried out our volumetric measurements in aqueous buffers at pH 7 containing 20, 50, and 100 mM NaCl. All of the methods employed yielded volumetric parameters that were in excellent agreement. The molar volume changes, ΔV, of the conformational transition leading to formation of the folded state are large and positive. At 50 mM NaCl, the average transition volume, ΔV(tr), obtained from all the methods is 56.4 ± 3.5 cm(3) mol(-1) at the transition temperature of 47 °C, with ΔV(tr) decreasing with an increase in temperature. We carried out a molecular dynamics simulation of the change in the intrinsic geometric parameters of the ODN accompanying quadruplex formation. On the basis of the experimental and computational results, the folding transition of the ODN is accompanied by a release of 103 ± 44 water molecules from its hydration shell to the bulk. This number corresponds to ~18% of the net hydration of the coil conformation.