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1.
Trtić-Petrović T Liu JF Jönsson JA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,826(1-2):169-176
The determination of drug-protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug-protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug-protein binding was investigated. The brief theoretical background for determination of the drug-protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug-protein binding. 相似文献
2.
David S. Hage Stacey A. Tweed 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,699(1-2)
Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent. 相似文献
3.
Equilibrium dialysis is often used to study the binding of steroids to proteins. With this technique it is customary to determine the percent bound and unbound steroid in the sample, the affinity constant for the steroid-protein binding reaction, and the concentration of binding sites on the protein. Investigators have used many different ratios of dialysis buffer to sample volumes in their experiments assuming that the equilibrium in the post-dialysis sample was the same as existed before dialysis. Chemical equilibrium expressions for the system before and after dialysis indicate that during dialysis the concentration of steroid in the sample decreases resulting in a new equilibrium in which the percent bound and unbound are different from the original sample. The magnitude of the difference between the pre- and post-dialysis systems is proportional to the ratio of dialysis buffer to sample volumes. Accurate values for the affinity constant and binding site can be obtained only if this change in the equilibrium is considered.Experimental verification of the application of these principles was made in an equilibrium dialysis study of testosterone-albumin binding. 相似文献
4.
Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes. 总被引:4,自引:1,他引:3
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Ammonium ion and proton concentration profiles near the surface of a planar bilayer lipid membrane (BLM) generated by an ammonium ion gradient across the BLM are studied by means of microelectrodes. If the concentration of the weak base is small compared with the buffer capacity of the medium, the experimental results are well described by the standard physiological model in which the transmembrane transport is assumed to be limited by diffusion across unstirred layers (USLs) adjacent to the membrane at basic pH values (pH > pKa) and by the permeation across the membrane itself at acidic pH values. In a poorly buffered medium, however, these predictions are not fulfilled. A pH gradient that develops within the USL must be taken into account under these conditions. From the concentration distribution of ammonium ions recorded at both sides of the BLM, the membrane permeability for ammonia is determined for BLMs of different lipid composition (48 x 10(-3) cm/s in the case of diphytanoyl phosphatidylcholine). A theoretical model of weak electrolyte transport that is based on the knowledge of reaction and diffusion rates is found to describe well the experimental profiles under any conditions. The microelectrode technique can be applied for the study of the membrane permeability of other weak acids or bases, even if no microsensor for the substance under study is available, because with the help of the theoretical model the membrane permeability values can be estimated from pH profiles alone. The accuracy of such measurements is limited, however, because small changes in the equilibrium constants, diffusion coefficients, or concentrations used for computations create a systematic error. 相似文献
5.
Neil Arthur Sparrow Allan Edward Russell Leslie Glasser 《Analytical biochemistry》1982,123(2):255-264
An automated, continuous-flow dynamic dialysis technique has been developed to investigate protein-ligand binding. The method depends on a comparison of the diffusion of the low molecular mass ligand, in the presence and absence of protein, through a semipermeable membrane. The ligand passes from the sample compartment of a dialysis cell into the sink compartment through which a constant flow of eluting buffer is maintained. Digitized spectrophotometric determinations of the ligand concentration in the eluting buffer at successive, equally spaced time intervals, punched onto paper tape, provide the primary data (normally about 1000 data points). A mathematical treatment of the data based on a model of the diffusion system, whereby the protein-ligand binding isotherm may be evaluated, is discussed. The validity of the method is demonstrated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15, 20, and 25°C. The method yields a large number of points on the binding isotherm (usually several hundred) which, in terms of a Scatchard model, provide values for the number of binding sites on the BSA molecule and binding constants for the phenol red-BSA interaction. The results obtained are consistent with values reported in the chemical literature but which are based on much scantier data. 相似文献
6.
Reza Hosseinzadeh Khatereh Khorsandi Vahid Sheikh-Hasani Ali Khatibi 《Journal of biomolecular structure & dynamics》2016,34(10):2146-2154
The binding of thiamine (vitamin B1) on lysozyme has been examined at various ionic strengths of phosphate buffer (pH 6.9), various pH values, and various protein concentrations at 25°C using thiamine selective membrane electrode. This method is faster and more precise than equilibrium dialysis technique which can obtain sufficient and accurate data for binding analysis. The values of Hill equation parameters were estimated for each set using binding capacity concept and used for calculation of intrinsic binding affinity. The results represent two binding sets for thiamine on lysozyme at various experimental conditions. 相似文献
7.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,677(1):1-28
In the last few years, continuous progress in instrumental analytical methodology has been achieved with a substantial increase in the number of new, more specific and more flexible methods for ligand-protein assays. In general, the methods used for drug-protein binding studies can be divided into two main groups: separation methods (enabling the calculation of binding parameters, i.e. the number of binding sites and their respective affinity constants) and non-separation methods (describing predominantly qualitative parameters of the ligand-protein complex). This review will be focussed particularly on recent trends in the development of drug-protein binding methods including stereoselective and non-stereoselective aspects using chromatography, capillary electrophoresis and microdialysis as compared to the “conventional approach” using equilibrium dialysis, ultrafiltration or size exclusion chromatography. The advantages and limitations of various methods will be discussed including a focus on “optimal” experimental strategies taking into account in vitro, ex vivo and/or in vivo studies. Furthermore, the importance of some particular aspects concerning the drug binding to proteins (covalent binding of drugs and their metabolites, stereoselective interactions and evaluation of binding data) will be outlined in more detail. 相似文献
8.
J Vallières M Drummond G I Drummond 《Canadian journal of physiology and pharmacology》1975,53(3):458-469
Binding of [3H]epinephrine to plasma membrane enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H]Epinephrine and [3H]norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H]epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at O degrees C. Binding was antagonized by the unlabeled beta-agonists, isopropylnorepinephrine, epinephrine, and norepinephrine but all were equipotent. A variety of catechol compounds were as effective antagonists of binding as the catecholamines. The beta-adrenergic antagonists propranolol, pronethalol, and dichloroisoproterenol were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equi-effective in antagonizing binding of [3H]norephinephrine to skeletal muscle membranes. It was concluded that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique does not represent interaction with the specific beta-adrenergic receptor, but more likely reflects a less specific binding of compounds having one or more hydroxyl groups on a ring. 相似文献
9.
A method is reported for measuring the stoichiometry of complex formation between actinomycin and a series of deoxynucleotides. The amount of bound actinomycin is measured by distribution of the drug between two liquid phases, a buffer phase containing deoxynucleotide and an organic phase in which the nucleotide is insoluble. Using simple statistical mechanical analysis, the equilibrium equations for several models of actinomycin-deoxynucleotide complexes have been derived: actinomycin with one binding site, with two equivalent independent binding sites, and with two sites which must be occupied together. The binding of actinomycin C3 with dpG, dpApG, dpA, and dpGpC has been examined compared with these models. It is found that binding to dpG and dpApG involves two independent binding sites of nearly equal affinity for nucleotides, whereas binding of dpGpC to the two binding sites on actinomycin is a cooperative process. Binding of dpA tp actinomycin is partially cooperative and weaker than binding of dpG. The dimerization constant of actinomycin was also determined by the phase separation technique, and found in agreement with other values, including the results of kinetic measurements reported here. 相似文献
10.
The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors. 相似文献
11.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,666(1):127-137
The potential of on-line dialysis as a sample preparation procedure for compounds highly bound to plasma proteins is evaluated, using non-steroidal anti-inflammatory drugs as model compounds and column liquid chromatography as the separation technique. Different strategies to reduce the degree of drug-protein binding and so increase the analyte recovery are systematically explored and discussed: alteration of the conformation of the binding protein by changing the pH of the sample or by adding an organic solvent, addition of several displacing compounds and combinations of such approaches. A fully automated method is presented for the determination of ketoprofen, ibuprofen, flurbiprofen, fenoprofen and naproxen in human plasma, in which the absolute analyte recoveries are increased from 0–1% (untreated samples) to 40–65%. Relevant analytical data are given to demonstrate the reliability of the proposed procedure. 相似文献
12.
13.
Estimation of drug-protein binding parameters on assuming the validity of thermodynamic equilibrium 总被引:1,自引:0,他引:1
Soltés L Mach M 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,768(1):113-119
This contribution focuses the reader's attention on the pitfalls usually emerging during the phase of evaluation of experimental data of drug-protein binding studies. To overcome the occurrence of problem(s) apparently defying solution, the concept of "affinity spectra" is recommended to be implemented for data evaluation. A (general) "binding study protocol" is also suggested, which can prevent the formation of inadequate conclusions and the generation of unrealistic drug-protein binding parameters. 相似文献
14.
Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37°C were found to be 2.8 · 105 M−1 and 1.9 · 106 M−1 for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 · 103 M−1 and 4.1 · 103 M−1 at neutral pH and 37°C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase. 相似文献
15.
M Esmann 《Biochimica et biophysica acta》1992,1110(1):20-28
A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP. 相似文献
16.
Using an equilibrium dialysis technique, moles (Gamma(2)(1)) of cationic and anionic surfactants bound per kilogram of proteins of various types in aqueous media have been measured previously in this laboratory under different physicochemical conditions. From a thermodynamic analysis in the present paper, Gamma(2)(1) has been shown to be equal to the Gibbs relative excess of surfactant per kilogram of protein at a measured value of solute activity, a(2). The values of relative solvent excesses, Gamma(2)(1) (which are negative for surfactants) can be estimated from values of Gamma(2)(1) and a(2). Using the Gibbs-Duhem relationship for protein solution inside the dialysis bag and dialysate solutions respectively at equilibrium, an integrated expression for the standard free energy change, DeltaG(o) (in kilojoules per kilogram of protein for binding with ligand as a result of the change of a(2) from zero to unity) can be calculated from experimental data. The isopiestic vapour pressure technique was used extensively for evaluation of negative binding (-Gamma(2)(1)) of inorganic salts to proteins of different types for various values of a(2) of salts present in the bulk media. With some modifications of our derived equations for free energy of binding in such a system, DeltaG(o) has been evaluated for the change of mean activity of electrolyte from zero to unity in the rational scale. DeltaG(o) is positive since Gamma(2)(1) is negative and Gamma(2)(1) is positive for such ionic systems. DeltaG(o) in all cases, however, are expressed in terms of the standard state of reference of unit activity so that their magnitudes and sign can be related to the relative affinities of a solute for binding with proteins in aqueous media. 相似文献
17.
The activity of many biomolecules and drugs crucially depends on whether they bind to biological membranes and whether they translocate to the opposite lipid leaflet and trans aqueous compartment. A general strategy to measure membrane binding and permeation is the uptake and release assay, which compares two apparent equilibrium situations established either by the addition or by the extraction of the solute of interest. Only solutes that permeate the membrane sufficiently fast do not show any dependence on the history of sample preparation. This strategy can be pursued for virtually all membrane-binding solutes, using any method suitable for detecting binding. Here, we present in detail one example that is particularly well developed, namely the nonspecific membrane partitioning and flip-flop of small, nonionic solutes as characterized by isothermal titration calorimetry. A complete set of experiments, including all sample preparation procedures, can typically be accomplished within 2 days. Analogous protocols for studying charged solutes, virtually water-insoluble, hydrophobic compounds or specific ligands are also considered. 相似文献
18.
O A Schmid 《Biomedizinische Technik》2005,50(3):38-44
This study tested the accuracy of a new 3-D calibration technique under dynamic situations. The technique was firstly introduced in 1998 for biomechanical human tests and calibrates 3-D volumes in an easy way. It revealed superior in static tests to others. In order to disclose dynamic accuracy two different tests were performed. With this technique it does not matter whether redundant information from multiple camera views is available or not. The mean error for distances measured at 0.018% for redundant information and at 0.012% for the non-redundant test in contrast to other procedures found in literature, which attain values of 0.09% and 0.04% respectively. The maximum error ranged there between 5.5% and 17.9%, whereas the presented data reached values of 0.33% and 0.48%. The more important angle error was at maximum 0.055% (9 times less than the most accurate in literature) and nearly zero for the mean error value. The level of noise was the same in the test with redundancy and 7.4 times lower in the present study than other commercial available systems for non-redundant video information. The new procedure revealed as a stable and very accurate 3-D reconstruction technique for a variety of application not limited to biomedical applications. 相似文献
19.
Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain 总被引:5,自引:4,他引:1
J. Simon M. Szücs S. Benyhe A. Borsodi P. Zeman M. Wollemann 《Journal of neurochemistry》1984,43(4):957-963
Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, an Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane-bound and the solubilized receptors showed weak binding of enkephalin and mu-specific drugs, suggesting that they are predominantly of the kappa-type. The membrane-bound and the soluble receptors showed the same distribution of subtypes, i.e., 70% kappa, 13% mu, and 17% delta for the membrane-bound and 71% kappa, 17% mu, and 12% delta for the soluble receptors. 相似文献
20.
The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and =4.5 in the E(1) conformation; 6.7 and =2 in the P-E(2) conformation. The equilibrium dissociation constants for K(+) binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K(+) concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites. 相似文献