Scanning molecular sieve chromatography of interacting protein systems. III. Effect of kinetic parameters on the large zone boundary profiles for local equilibration between mobile and stationary phases.

Large zone reaction boundary profiles for molecular sieve chromatography as affected by kinetic parameters have been simulated for local equilibration between the mobile and stationary phases. Our studies of monomer-dimer and monomer-tetramer systems indicate that in a slowly equilibrating system, the kinetic controls operating between the mobile and stationary phases contribute most significantly to the overall boundary profile. In a rapidly equilibrating system, however, the kinetic parameters kij and kji operating in the mobile phase are the principal determinants of the reaction boundary, while the kinetic effects of kii and k-ii between the mobile and stationary phases are minimal.     

Scanning molecular sieve chromatography of interacting protein systems. Simulation of large zone behavior for self-associating solutes undergoing rapid chemical equilibration under kinetic control

Theoretical large zone reaction boundaries for molecular sieve chromatography have been simulated by computer for a self-associating solute undergoing rapid chemical equilibration under kinetic control. These patterns show that the kinetically-controlled reaction rate between the mobile and stationary phases is the principal determinant of the elution boundary profile in molecular sieve chromatography. The overall chemical reaction rate in the mobile phase was found to have a much greater role in a rapidly equilibrating system than did the effect of axial dispersion within the gel matrix.     

Simultaneous effect of organic modifier and physicochemical parameters of barbiturates on their retention on a narrow-bore PGC column

The retention time of 22 barbituric acid derivatives was measured on a narrow-bore porous graphitized carbon (PGC) column using water-dioxane mixtures as mobile phases. The capacity factor (k), theoretical plate number (N), and asymmetry factor (AF) were calculated for each solute in each mobile phase. The relationships between chromatographic characteristics and physicochemical parameters of solutes were elucidated by stepwise regression analysis (SRA). SRA indicated that the binding of barbiturates to the PGC surface is of mixed character electrostatic and apolar interactive forces are equally involved. Sterical correspondence between the surface of the stationary phase and the solutes also exert a significant influence on the retention behavior.     

Evaluation of the predictive power of calculation procedure for molecular hydrophobicity of some estradiol derivates

Several calculation procedures for log P values based on the fragmental and atomic contributions are compared with experimental reversed-phase liquid chromatography (RPLC) retention of estradiol derivates. The RPLC experiments were performed on HPTLC and HPLC commercially available stationary phases. Binary solvent mixtures of methanol-water and acetonitrile-water were used as mobile phases. The correlation between log P and various chromatographically obtained hydrophobicity parameters (R(M)0, log k(w) and phi0) are quantified. The R(M)0, i.e., log k(w) were obtained by linear extrapolation of retention to 0% organic modifier. Phi0 values were obtained from the slopes and intercepts of such linear relationship. The mutual relationship between phi(0,MeOH) and phi(0,ACN) values of the compounds were discussed. The obtained statistical results can be summarized in the following order of reliabilities for different log P calculation methods: Broto>ACD/logP>Crippen>Rekker>Viswanadhan.     

Structural and kinetic aspects of chemical reactions in DNA duplexes. Information on DNA local structure obtained from chemical ligation data.

Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.     

QRAR models for cardiovascular system drugs using biopartitioning micellar chromatography

The capability of biopartitioning micellar chromatography (BMC) to describe and estimate pharmacological parameters of cardiovascular system drugs has been studied. The retention of cardiovascular system drugs was studied using different pH of Brij-35 as micellar mobile phase in modified C(18) stationary phase. Quantitative retention-activity relationships (QRAR) in BMC were investigated for these compounds. An adequate correlation between the retention factors (log k) and the toxicity (LD(50)) of cardiovascular system drugs was obtained.     

Simultaneous HPLC analysis of hydrophobic prodrugs and their hydrophilic metabolites in biological media without sample preparation. Application to the biotransformation studies of pronucleotides derived from 5-fluorouracil

Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.     

Low density lipoprotein turnover in swine.

The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 +/- 3.7, 19.7 +/- 6.6, and 127 +/- 70 h (mean of four studies). Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h-1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 +/- 4.1 mg/h. Different kinetic models using two or three exponentials would provide different values for the synthetic rate of apo-LDL. However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.     

Kinetic studies on (N-formyltryptophyl)cytochrome c.

The formylation of the ring nitrogen atom of the tryptophan residue in cytochrome c was carried out and consequent changes in the kinetic properties of the protein were investigated. The reduction of formylated cytochrome c by Cr2+ was studied by stopped-flow techniques. At pH 6.5 the reduction process shows the presence of two phases. One phase (k = 4 X 10(4) M-1-s-1) is dependent on Cr2+ concentration and one phase (k = 5.0 s-1) is not. A study of the temperature dependence of the two phases yields values for their activation energies of 38.6kJ-mol-1 and 42.4kJ-mol-1 respectively. The reaction of the reduced formylated cytochrome c with CO was followed by means of both stopped-flow techniques and flash photolysis. The combination with CO at pH 6.8 measured in stopped-flow experiments shows two phases, both dependent on the concentration of CO (k1 = 1.8 X 10(2) M-1-s-1). If CO was dissociated from the protein by photolysis and then allowed to recombine with it, it was found to do so in a simple manner, at a rate which depended on the concentration of CO (k = 1.9 X 10(2) M-1-s-1). A tentative model which can accommodate these findings is proposed. The reaction of the oxidized form of formylated cytochrome c with NO was followed by means of stopped-flow techniques. The reaction was found to be biphasic with one phase dependent on the concentration of NO (k = 2.8 X 10(3) M-1-s-1) and one phase (k = 0.2x-1) independent of the concentration of NO. This behaviour is compared with that of the native molecule. A comparison of these kinetic observations with those on other tryptophan-specific modifications leads to the conclusion that the main alteration in kinetic properties is due, not to the nature of the modifying group, but rather to the disruption of the normal environment of the haem.     

SIMULTANEOUS HPLC ANALYSIS OF HYDROPHOBIC PRODRUGS AND THEIR HYDROPHILIC METABOLITES IN BIOLOGICAL MEDIA WITHOUT SAMPLE PREPARATION. APPLICATION TO THE BIOTRANSFORMATION STUDIES OF PRONUCLEOTIDES DERIVED FROM 5-FLUOROURACIL

Improvements of an “on-line cleaning” HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix; (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.     

Uphill transport of monosaccharides inCandida beverwijkii

The yeastCandida beverwijkii was found to transport several monosaccharides against a concentration gradient. The process is mediated by a mobile carrier and shows a pronounced pH dependence. It is tightly coupled with metabolism and only potassium sorbate uncoupled the equilibrating from the active transport component. Kinetic analysis of uptake and efflux at high monosaccharide concentrations indicates an active transport operating either into or out of cells. T. Deák carried out the work in Prague while supported by the Hungarian Academy of Sciences.     

Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C(18), have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH(2) (TAPP), H-Tyr-(D)Ala-Phe-NH(2) (TAP), H-Phe-Phe-NH(2) (PP) and H-Phe-NH(2) (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H(2)O as BGE, operating at 20 degrees C and 20 kV high voltage. For the C(18) stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 degrees C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.     

Chiral resolution of histidine using an anti-D-histidine L-RNA aptamer microbore column

In this paper, we report a new anti-amino acid aptamer chiral stationary phase (CSP). The enantiomers of histidine were separated using an immobilized histidine-specific L-RNA aptamer (40-mer) and an aqueous buffer as mobile phase. The effects of the variation of different operating parameters, including the mobile phase pH and the MgCl2 concentration as well as the column temperature, on the solute retention were assessed. The results suggested that (i) the protonated form of histidine was involved in the stereospecific RNA binding and (ii) Mg2+ was essential for the target enantiomer binding to the specific aptamer sites. From a practical point of view, it appeared that the baseline resolution in a minimum analysis time can be achieved at a column temperature of 35 degrees C for an eluent containing 10 mM of MgCl2, pH 5.5.     

Direct calculation and computer simulation of the enantiomerization barrier of oxazepam in dynamic HPLC experiments--a comparative study

Dynamic chromatographic methods constitute a versatile approach to the rapid and precise determination of enantiomerization barriers of stereolabile drugs. In the present study enantioselective dynamic high-performance liquid chromatography (DHPLC) was employed to determine the enantiomerization barrier of oxazepam. Dynamic elution profiles, exhibiting plateau formation and/or peak broadening between 20 and 60 degrees C at pH 2.6 and pH 8 were obtained in the presence of the chiral stationary phase (CSP) Nucleodex-beta-PM (permethylated beta-cyclodextrin chemically bonded to silica) using a 6:4 mixture of phosphate buffer and methanol as mobile phase. Evaluation of the experimental chromatograms was performed by the novel approximation function (AF) (without computer simulation), and by the stochastic model implemented in the ChromWin simulation software (with computer simulation) furnishing the respective apparent forward rate constants, k(1)(app)(T). From the rate constants, k(1)(app)(T), measured at variable temperatures, the kinetic Eyring activation parameters, deltaG(T)(#), deltaH(#) and deltaS(#), of the enantiomerization of oxazepam were obtained. By variation of the flow rate of the mobile phase, the expected independence of the enantiomerization barrier from the chromatographic time scale was demonstrated for the first time.     

氮杂18—冠—6键合硅胶固定相上小分子肽的高效液相色谱研究

报导了一种新型单氮杂 18—冠— 6键合硅胶固定相 ( BCN 18— C— 6)在小分子肽分析中的应用。研究了流动相 p H值、Cu2 +浓度、缓冲溶液浓度、甲醇体积比、氯化钠浓度等因素对容量因子 ( k)的影响 ,并分析了保留机理。在优化条件下 ,成功地分离了 4种小分子肽。肽的色谱峰面积与进样量间有良好的线性关系 ( r均大于 0 .99) ,各肽的最小检出量 ( mmin)均可达到 10 - 10 ～ 10 - 11m ol。 BCN 18— C— 6柱对小分子肽的分离选择性优于 ODS柱和硅胶柱     

A new rapid and simple method to determine the kinetics of electrode reactions of biologically relevant compounds from the half-peak width of the square-wave voltammograms

A new method is introduced to determine the kinetic parameters of electron transfer reactions of biologically important compounds, based on the measurements of the half-peak width (DeltaE(p/2)) of the square-wave voltammograms. A simple surface (diffusionless) redox reaction, and a simple electrode reaction occurring from dissolved state are considered as model systems. In the region of quasireversible electron transfer, the half-peak widths of theoretical square-wave voltammograms are linear functions of the logarithm of the dimensionless kinetic parameter ln(K) that characterizes the rate of the electron transfer reaction. The dimensionless kinetic parameter K is defined as K=k(s)(fD)(-0.5) for the redox reaction taking place from dissolved state, whereas for the surface redox reaction K is defined as K=k(s)/f (k(s) is the standard rate constant of electron transfer, f is the SW frequency, and D is the diffusion coefficient). A set of linear regression equations for the dependences DeltaE(p/2)vs. ln(K) are derived, which can be used for rapid and precise determination of the charge-transfer kinetic parameters. The estimated values for the standard rate constants of various biologically relevant redox systems using this approach are in very good agreement with the experimental values determined by other square-wave voltammetric methods. The square-wave voltammetric half-peak width method can be used as a simple and reliable alternative to other voltammetric methods developed for the kinetic characterization of electron transfer rates.     

Parameters of enzyme reactions in microcirculation

It has been shown that time-dependent change in the concentration of enzymic reaction substrate in a microcirculatory channel cell, as well as its steady spatial distribution contain information both about the structure of the microcirculatory channel and about the reaction kinetic parameters. In terms of the hypothesis about the stationary state of the enzyme-substrate complex at selective values of hydrodynamic parameters of the substrate, enzyme, channel correlation between the change in the substrate concentration, its stationary distribution, kinetic parameters and the microcirculation cell structure were estimated.     

Carrier facilitated diffusion

The concept of a mobile carrier combining reversibly with a substrate is considered as a possible mechanism for facilitated transport across biological membranes. The mathematical model is a system of three reaction diffusion equations with certain boundary conditions. Two limiting cases are discussed in detail: The case of a "thin" membrane where the diffusion of bound and unbound carrier from one surface to the other may be simulated by a single jump. If the diffusion rate of the substance to be transported is small, then an approximate stationary solution is derived using singular perturbation theory. Finally, the results of numerical simulations are presented for a wide range of parameters.