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Drug-receptor interaction plays an important role in a series of biological effects, such as cell pro-liferation, immune response, tumor metastasis, and drug delivery. Therefore, the research on drug-re-ceptor interaction is growing rapidly. The equilibrium dissociation constant (KD) is the basic parameter to evaluate the binding property of the drug-receptor. Thus, a variety of analytical methods have been established to determine the KD values, including radioligand binding assay, surface plasmon resonance method, fluorescence energy resonance transfer method, affinity chromatography, and isothermal ti-tration calorimetry. With the invention and innovation of new technology and analysis method, there is a deep exploration and comprehension about drug-receptor interaction. This review discusses the differ-ent methods of determining the KD values, and analyzes the applicability and the characteristic of each analytical method. Conclusively, the aim is to provide the guidance for researchers to utilize the most appropriate analytical tool to determine the KD values.
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This work aimed to develop an ab initio procedure for accurately calculating pKa values and applied it to study the acidity of asparagine and glycyl-asparagine. DFT methods with B3LYP composed by 6-31+G(d) basis set were applied for calculating the acidic dissociation constant of asparagine and glycyl-asparagine. The formation of intermolecular hydrogen bonds between the available species and water was analyzed using Tomasi,s method. Results showed that in alkaline solutions, the cation, anion and neutral species of asparagine and glycyl-asparagine were solvated with one, two, three and four molecules of water, respectively. There was an excellent similarity between the experimentally attained pKa values and the theoretically ones in this work.
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Objective: To investigate the specific molecular recognition ability for galangin molecular imprinting polymer (GMIP). Methods: GMIP was prepared through the method of thermal polymerization with galangin as template molecular, acrylamide (AM) as functional monomer, ethyleneglycol dimethacrylate (EGDMA) as cross-linker, and tetrahydrofuran, acetonitrile, and acetone as pore forming agents, respectively. The GMIP was characterized by infrared spectroscopy and scanning electron microscopy (SEM). In addition, the GMIP was investigated in equilibrium binding experiment to evaluate its adsorption property and selective recognition. Results: The Scathard model analysis showed that two kinds of binding sites existed in the GMIP. The dissociation constant (Kd) and apparent maximum binding constant (Qmax) were Kd1 = 0.961 mmol/L, Qmax1 = 19.79 μmol/g for high affinity binding sites and Kd2 = 0.101 mmol/L, Qmax2 = 51.09 μmol/g for low affinity binding sites, respectively. Conclusion: GMIP has the specific adsorption and recognition capabilities to the galangin molecules. It can be a novel material for the separation and purification of the active ingredients from natural products.
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OBJECTIVE: To explain that receptor-ligand binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide information about the relative binding affinity of ligand-receptor. METHODS: The correlative literature and abroad were collected and analyzed. RESULTS AND CONCLUSION: The concept of MS binding assay based on mass spectrometric quantification of nonlabeled markers represents a promising alternative to conventional radioligands binding without inherent drawbacks. The technique will play an important role in the design and screening of receptor-targeting pharmaceuticals.
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OBJECTIVE: To study the physical and chemical properties and transdermal delivery characteristics oi capsaicin. METHODS: Equilibrium solubility method, ultraviolet spectrophotometry, bottle-shaking method, differential scanning ealorimetry (DSC) and in vitro diffusion cell method were used to determine the apparent solubility, dissociation constant, apparent oil/water partition coefficient, melting point and percutaneous penetration of capsaicin, respectively. And the permeation characteristics were evaluated by lag time method. RESULTS: Capsaicin was slightly dissolved in water, and the apparent solubility was (22.85 ± 0.06) mg · L-1. It was a weak base with a dissociation constant of (10.25 ± 0.11). The apparent oil/water partition coefficient of capsaicin changed with the pH value, and increased when pH was greater than 8. Its melting point was 60.20°C. The residence time(ttag) was (2.437 ± 0.273) h, the permeability coefficient (P) (7.012 ± 0.341) × 10-2cm · h-1, and the percutaneous penetration rate (Js) (4.647 ± 0.226) μg · cm-2 · h-1. CONCLUSION: Capsaicin possesses appropriate physical and chemical properties for percutaneous penetration and exhibits excellent percutaneous penetration.
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Aim To study the fluorescence spectroscopy of human serum albumin(HSA)and the interaction of aspirin and HSA.Methods The quenching mechanism of the fluorescence of human serum albumin by aspirin was studied with the fluorescence.The interaction dissociation constants KD of human serum albumin and aspirin were determined at different temperatures according to double reciprocal Lineweaver-Burk plot and the main binding force was discussed by thermodynamic equations.The effect of aspirin on human serum albumin was also studied by synchronous fluorescence spectrometry.Results The quenching mechanism of aspirin to human serum albumin was static quenching.The interaction dissociation constants KD at 37℃,25℃ was 1.44?10-3 and 1.96?10-3 mol?L-1 respectively.The thermodynamic parameters of the reaction was-19.73 kJ?mol-1(?H),-16.21 kJ?mol-1(?G),-11.77 kJ?mol-1(?S).Conclusions The main binding force between aspirin and HSA was Van der Waals interaction.Aspirin binding on the human serum albumin could change the serum protein conformation.
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Aim To study the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor. Methods The mice sera were collected after peritoneal injection with big-dose TMP,low-dose TMP,protamine and NS. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum. The culture medium of ECV304 was treated with the mice sera in different groups. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(B_ max) and dissociation constant(K_d).Results The sera of big-dose TMP inhibited 125I-VEGF binding to its receptor, K_d=343.30?36.64 pmol?L-1,B_ max=46.26?5.85 fmol/2?10~5 cells(P0.05),but B_ max decreased(P
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We prepared two monoclonal antibodies-A-I30 and A-I4 to HDL apo A-I-with the ultimate goals of expressing and overproducing the valuable immunodiagnostic single chain Fv by phage display libraries in E, coli. Monoclonal antibodies were produced by immunizing mice with apolipoprotein A-I, and purified from ascitic fluid by affinity chromatography on a Protein A Sepharose CL-4B column. The specificity of A-I30 and A-I4 was confirmed by ELISA and Western blotting. The dissociation constants(Kd) of antigen-antibody complex obtained by enzyme linked immunosorbent assay(ELISA) method were 2.25 x 10(-8) M for A-I30 and 2.15 x 10(-8) M for A-I4. The experimental values of Kd are shown to be close to those obtained by immunoprecipitation of the radiolabeled antigen. From the above results, it was shown that A-I30 and A-I4 could be provided as excellent reagents for the determination of plasma HDL concentrations in clinical specimens using ELISA.
Subject(s)
Mice , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Apolipoprotein A-I/immunology , Blotting, Western , Hybridomas , Lipoproteins, HDL/immunology , Mice, Inbred BALB C , Tumor Cells, CulturedABSTRACT
Objective To detect normal rat jejunum epithelial cells to learn about the distribution of peptide YY(PYY)receptor and measured two important parameters of PYY receptor so as to explore the target that PYY binds to and display its physiological function.Methods Receptor radioligand assay was conducted by using 125I-PYY to detect normal rat jejunum epithelial cells.We also measured receptor dissociation constant(Kd),the affinity or the ability that the receptor could bind ligand;maximum binding capacity(Bmax),the concentration of binding site;and Hill coefficient.Results There were PYY receptors in normal rat jejunum epithelial cells.Dissociation constant(Kd)of PYY receptor was(386.69?129.95)pmol/L.Maximum binding capacity(Bmax)was(303.21?116.85)fmol/mg protein.Hill coefficient was 1.PYY receptor had high affinity.Conclusion There are distributions of PYY receptors in normal rat jejunum epithelial cells.Kd means that PYY receptor has high affinity.Bmax means that the number of receptors is limited and decides the characters of saturability.We think that PYY directly inhibits secretion by binding to PYY receptor in jejunum epithelial cells when PYY inhibiting jejunum secretion.
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Objective To investigate the effect of psychological stress on the function of dopamine receptor in the central nervous system in rats and the intervention effect of tyrosine. Methods Rats were continuously subjected to psychological stress by communication box model 30min a day for 14 days. They were fed with tyrosine 250mg/kg, 500mg/kg, or 1000mg/kg, respectively. The maximal bonding capacity (Bmax) and the balance dissociation constant (Kd) of D1, D2 receptor of DA at the site of ventral tegmental area (VTA), nucleus accumbens (Nac), mesoprefrontal cortex (mPFC) were determined by radioactive ligand bonding analysis. Results Bmax of the D1 receptors declined significantly in mPFC and Nac area of psychological stress treated group compared with that of control group (P