Macromolecule-ligand binding studied by the Hummel and Dreyer method: current state of the methodology

The use of the Hummel and Dreyer method to measure binding parameters of ligand-macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.     

Hydrophylic surfactant–imidazole derivative association studied by RPLC using a hydroorganic solution

A high-performance liquid chromatographic (HPLC) method for an association study of imidazole derivatives in surfactant micellars using a hydrophilic detergent, i.e. Montanox DF 80 was presented. The thermodynamic results obtained showed that imidazole association in the surfactant micelles was effective over a concentration of surfactant equal to approximatively 4·10⁻⁴ mol/l. In addition, an enthalpy–entropy compensation study revealed that the type of interaction between the solute and the RP18 stationary phase was independent of the molecular structure. The thermodynamic variations observed were considered to be the result of equilibrium displacement between the solute and free ethanol (respectively free surfactant) and its clusters (respectively to micelles) created in the mobile phase.     

用气相色谱-质谱联用比较牛耳草代谢物的提取方法

通过比较不同的提取方法对牛耳草新鲜和脱水叶片中代谢物的提取效率,旨在建立一种可以有效鉴定并分析牛耳草脱水过程中关键小分子代谢物的种类和含量变化的方法,为研究植物耐脱水分子机制提供技术方法。本研究以气相色谱-质谱联用(GC-MS)为分析方法,对复苏植物牛耳草代谢物提取方法进行比较。从提取总色谱峰数目、提取效率、代谢物保留时间和提取效率稳定性等方面比较甲醇溶液(A法)和甲醇-氯仿-水溶液(B法)两种提取方法的提取效果。对牛耳草新鲜样品提取结果表明,B法提取的总色谱峰数目多于A法;对9种共有代谢物的提取效率比较结果表明,B法的提取效率高于A法;对10种色谱峰的保留时间和提取效率的方法学考察结果表明,两者保留时间RSD(相对标准偏差)值均小于1%,A法提取效率的RSD值≤10%的比例为50%,B法的为100%。A法对干样的提取色谱峰数目远少于鲜样,而B法对干样的提取色谱峰数目和鲜样没有显著差异,保留时间RSD值均小于1%,提取效率的RSD值与鲜样没有差异,稳定性良好。     

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues.The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method.Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.     

Analytical determination of orthophosphate in water

Methods for orthophosphate determination and the problems of interferences are reviewed.An important group of methods utilizes the phosphomolybdate complex. The complexation step, the reduction step and the extraction step are treated separately and alternative procedures compared.Another group of methods uses ion association complexes; they are primarily used in physiology and not commonly used in water analyses today.Enzymatic methods for orthophosphate analysis in natural waters have been developed lately and are ready for application in selected waterbodies.Flame spectroscopic, fluorometric, gas chromatographic, ion exclusion chromatographic, inductively coupled plasma and other methods are also shortly presented.Radiobiological bioassays for orthophosphate are also available.In conclusion it was emphasized that the most common and reliable technique still is the molybdenum blue method as modified by Murphy & Riley (1962).The need for more specific and sensitive methods is particularly strong in investigations of phosphorus turnover and phosphorus limitation in natural waters. For these purposes the enzymatic phosphatase methods has advantages due to their specificity for orthophosphate and they might offer an alternative to the molybdenum blue method.     

Liposome/saline partition coefficients of low-molecular-weight solutes by gel chromatography

A chromatographic method for the determination of association constants of rapidly dissociable complexes is described and applied to quantification of liposome/saline partition coefficients using gel chromatography. The approach allows for estimation of the free solute concentration in the sample by simple manual processing of the intact right-hand part of the solute peak deformed due to gradual diffusion of the accumulated solute from the liposomes along the separation column. Validity of the procedure was confirmed by both reasonable agreement with equilibrium dialysis data and model-based deconvolution of the distorted peak into its two components corresponding to initially unbound compound and to that escaped from the liposomes during the separation process.     

Determination of PHB in activated sludge by a gas chromatographic method

Summary In this study the gas chromatographic method of Poly-β-hydroxy butyric acid determination of Braunegg et al. (1978), was optimised for activated sludge samples. The poly-β-hydroxy butyric acid was extracted and quantified gravimetrically to confirm the accuracy of the gas chromatographic method. The authenticity of the extracted material was confirmed by several methods. It was also confirmed that the mixed liquor of an activated sludge process did not interfere with the esterification. The sample size required was 25 ml of mix liquor, or 50 mg of freeze-dried sludge.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

Schiff base copper(II) chelate as a tool for intermolecular cross-linking and immobilization of protein

A new method for intermolecular cross-linking or bridging of protein has been proposed. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde, alpha-amino acid residue and copper(II). Reliability of the process as a tool for protein cross-linking was evaluated by chromatographic procedures. Behavior of salicylaldehyde in a column packed with Sepharose attached alpha-amino acid residue showed that salicylaldehyde was bound tightly to the gel in the presence of copper(II) ion and was eluted by the addition of EDTA. The association was shown strong enough to be applied for the purpose of cross-linking of proteins. It was also proved that BSA salicylaldehyde conjugate was immobilized specifically to the column, and the process was reversed by the addition of EDTA as well. The method is proposed to be useful not only for immobilization of enzyme but also for cross-linking of proteins since the method is free from unexpected random coupling products which are unavoidable with bifunctional cross-linking reagents.     

Determination of chlorhexidine in saliva using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of chlorhexidine in saliva is reported. The method developed includes a simple and short sample preparation with a one-step extraction procedure and a short total chromatographic run time of 5 min. In a preliminary pharmacokinetic study with a healthy volunteer the chlorhexidine concentration found in saliva after 12 h was 0.8 μg/ml.     

A rapid and convenient method for purification and isolation of chlorophyll-A from Porphyra yezoensis.

A rapid and convenient method for purification and isolation of chlorophyll-a from Porphyra yezoensis without any chromatographic procedures has been developed. Thin-layer chromatographic and high-performance liquid chromatographic tests revealed that chlorophyll-a preparations in this study did not contain any other photosynthetic pigments and their degradation products.     

Quantitative colorimetric and gas chromatoographic determination of arecaidine propargyl ester

Arecaidine propargyl ester (APE) is a potent muscarinic agonist often used in pharmacological studies. To date, no sensitive quantitative analytical method for APE has been published. In this study, two methods for the quantitative determination of APE are compared: a colorimetric assay, based on the formation of the corresponding ferric(III)-hydroxamic acid complex, and a direct gas chromatographic method, using arecoline as the internal standard. The latter method was found to be more precise. The utility of the gas chromatographic assay was further demonstrated in a stability study of the drug in the biological fluid aqueous humor of rabbits.     

Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.     

Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes

A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions.     

Comparative analysis of chromatography peaks by computer and disc-integrator

A reliability study was conducted to compare a new, computerized method of measuring the areas under chromatographic curves with the traditional hand method of determining the areas. Three runs of 30 separate injections of standard inorganic phosphate were made using the chromatographic and dry-ashing procedure developed in the laboratory of the Department of Pharmacology of the USC School of Medicine. Results indicate that there is no statistically significant difference between the traditional method and the new computer method for amounts greater than 10 nm, and the computer method is more precise for amounts less than 10 nm. Hence, great savings in time and an increase in precision can be made by using a computer for these analyses.     

Amino acids-nucleotides biomolecular recognition: from biological occurrence to affinity chromatography

In this review, the protein-DNA interactions are discussed considering different perspectives, and the biological occurrence of this interaction is explained at atomic level. The evaluation of the amino acid-nucleotide recognition has been investigated analysing datasets for predicting the association preferences and the geometry that favours the interaction. Based on this knowledge, an affinity chromatographic method was developed also exploiting this biological favoured contact. In fact, the implementation of this technique brings the possibility to apply the concept of molecular interactions to the development of new purification methodologies. In addition, the integration of the information recovered by all the different perspectives can bring new insights about some biological mechanisms, though not totally clarified.     

Plasma membrane Ca(2+)-pump ATPase is not a substrate for cGMP-dependent protein kinase.

A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.     

Analysis of protein-protein interaction by simulation of small-zone size-exclusion chromatography: application to an antibody-antigen association

The association of two or more macromolecules results in the formation of a complex characterized by a larger Stokes radius than that of its components. Therefore, analytical procedures such as ultracentrifugation and size-exclusion gel chromatography that resolve molecules on the basis of size have been used to characterize the association. In this paper we describe an iterative computer simulation of small-zone size-exclusion gel filtration. The simulation describes univalent and bivalent interactions of proteins of equal and nonequal molecular weight and appears to have both qualitative and quantitative application to the evaluation of protein-protein interaction as revealed by alteration of chromatographic elution profiles. To test the validity of the simulation, the model was applied to an antibody-antigen interaction by determining the association constant (Ka) for the interaction between the binding fragment derived from a human immunoglobulin A rheumatoid factor and the antigenic fragment obtained from a human myeloma immunoglobulin G. The self-consistency of the estimated Ka values obtained with a valence value of 2 in contrast to the lack of self-consistency if an antigenic valence of 1 was assumed was taken to support the ability of the algorithm to reasonably emulate the chromatographic processes of interacting proteins. In conjunction with the computer simulation, a sensitive microcomputer-interfaced chromatography system was assembled, which is capable of analyzing 300 ng of protein in less than 1 h. This combination of rapid reagent-conservative chromatography and simulation analysis may contribute to the usefulness of small-zone gel filtration in studies of protein-protein interaction.     

Gas chromatographic determination of methamphetamine and its metabolite amphetamine in human plasma and urine following conversion to N-propyl derivatives

A gas chromatographic method for the simultaneous determination of methamphetamine and its metabolite amphetamine in human plasma and urine is described. The method utilizes reductive alkylation with propionaldehyde and sodium borohydride to produce N-propyl derivatives, which have excellent chromatographic properties. Structural analogs of the analytes, p-methylmethamphetamine and p-methylamphetamine, are used as internal standards. The method has good precision and accuracy for concentrations ranging from less than 10 ng/ml to 5000 ng/ml and has been used to measure plasma concentrations as part of a pharmacokinetic/pharmacodynamic study of methamphetamine in humans.     

Identification of affinity ligands for protein purification from synthetic chemical combinatorial libraries

A method to screen combinatorial libraries for the development of selective ligands for protein affinity chromatographic purification is described. The method is based on the application of parallel combinatorial libraries, and it has several potential advantages. The screening procedure is simple and straightforward, and it does not require the chemical derivatization of the target proteins or even that the target protein be pure. The experiment can also be designed to select binders that are less likely to cause protein denaturation. Feasibility of this approach is demonstrated with a model study of the chromatographic purification of bovine albumin serum (BSA) and Avidin.