Klotz Plot Research Articles

Flow Injection on Line Oxidizing Fluorometry Coupled to Microdialysis Sampling for Studying Phentolamine-Bovine Serum Albumin Interaction

Determination of phentolamine-bovine serum albumin (BSA) interaction based on a flow-injection microdialysis sampling fluorescence system (FI-MD-FL) was proposed. The analytical procedure was based on the oxidation of phentolamine by acidic Ce(IV) and monitoring of the fluorescence intensity of the formed Ce(III). The drug of phentolamine and BSA were mixed in different molar ratios in Ringer’s solution and incubated in a water bath. The microdialysate samples were on-line merged with acidic Ce(IV) solution by putting a microdialysis probe into the mixed solution and perfused with Ringer’s solution at 10 μL min-1. Then the sample was reached the flow cell, excited and monitored at 256/355nm (λex/λem). The dialytic efficiency under the experimental conditions was 38.8±2.2% (n=3). The data obtained was analyzed with Scatchard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scatchard analysis were 1.78×105 L mol-1 and 0.69, respectively. The proposed method has been applied to the study of the binding of phentolamine to bovine serum albumin (BSA) in vitro. The method provided a fast and simple technique for the study of drug-protein interactions.

Binding Study of Phenformin with Bovine Serum Album Using a Combined Technique of Equilibrium Dialysis with Flow-Injection Chemiluminescence Detection

ABSTRACT The interaction between phenformin hydrochloride and bovine serum albumin (BSA) was investigated by the methods of chemiluminescence combined with equilibrium dialysis technique. A novel N-bromosuccinimide (NBS)–eosin Y (EY) chemiluminescence (CL) method was established for the determination of phenformin. The mechanism of this chemiluminescence system was proposed. Optimization studies were performed to determine the phenformin. Under the optimal conditions, the CL intensity was linear for a phenformin concentration over the range of 4.6 × 10−8 to 5.0 × 10−5 g/mL. The detection limit was 1.5 × 10−8 g/mL. The data obtained by the present equilibrium dialysis–CL system were analyzed using the Klotz plot and the Scatchard analysis. The results showed that the Klotz plot and the Scatchard plot are linear with good correlation coefficient, indicating that the phenformin has only one type of binding site on BSA. The binding parameters were the number of the binding sites n (1.02) and the estimated association constant K (2.66 × 104 L/mol). The chemiluminescence system combined with equilibrium dialysis developed in this work demonstrated its use for determination of interaction between drug and protein by using relatively simple instrument.

Chemiluminescence determination of cefotaxime sodium with flow-injection analysis of cerium (IV)–rhodamine 6G system and its application to the binding study of cefotaxime sodium to protein with on-line microdialysis sampling

A simple, sensitive and rapid flow-injection chemiluminescence (CL) method has been developed for the determination of cefotaxime sodium based on the chemiluminescence reaction of cefotaxime sodium with ceric sulfate and rhodamine 6G in nitric acid solution. The concentration of cefotaxime sodium was proportional with the CL intensity in the range of 4 × 10 −8 – 8 × 10 −6 mol L −1. The detection limit (signal-to-noise ratio = 3) was 1 × 10 −8 mol L −1. Coupled to the technique of on-line microdialysis sampling, this method was successfully applied to study cefotaxime sodium–protein interaction. The drug and protein were mixed in different molar ratios in Ringer's solution, pH 7.4, and incubated at 37 °C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 μL min −1 and the recovery of cefotaxime sodium under experimental condition was 16.2%. The data obtained by the present Microdialysis-Flow Injection Analysis-CL method was analyzed with the Scatchard analysis and Klotz plot. The estimated association constant ( K) and the number of the binding sites ( n) on one of BSA molecule were 5.94 × 10 4 M −1 and 1.29 (Klotz equation), respectively.

Preparation of poly(ethylene glycol)-modified poly(amidoamine) dendrimers with a shell of hydrophobic amino acid residues and their function as a nanocontainer

We studied the use of poly(ethylene glycol) (PEG)-modified dendrimers as a nanocapsule with a biocompatible surface. We designed PEG-modified dendrimers having a shell of hydrophobic amino acid residues in the peripheral moiety of the dendrimer to increase their encapsulation ability. Subsequently, l-phenylalanine or γ-benzyl-l-glutamate residues were introduced to all chain ends of the poly(amidoamine) G4 dendrimers. Furthermore, PEG (MW 2000) chains were attached to the amino acid residues. These hydrophobic amino acid residues rendered the PEG-modified dendrimers as more compact. After binding of Rose Bengal (RB) guest molecules to dendrimers, an assay using the Klotz plot showed that the hydrophobic amino acid layer slightly affected the guest site number, but significantly increased intrinsic binding of the dendrimers to guest molecules. The PEG-modified dendrimers with the hydrophobic amino acid layer were better able to retain guest molecules than the dendrimer without the layer: they are therefore useful for drug delivery.

A Study on the Interaction of the DAS‐K with Bovine Serum Albumin by On‐line Ultrafiltration and Chemiluminescence

AbstractPotassium dehydroandrographolide succinate (DAS‐K) has antibacterial and antiviral effects. It has been used widely for the treatment of virus pneumonia, malaria and respiratory infections. In this work, a novel flow‐injection chemiluminescence (CL) method for the determination of DAS‐K was proposed. The method is based on the reaction between DAS‐K and hexacyanoferrate(III) in alkaline solution to give weak CL signal, which is enhanced by rhodamine B. The experimental conditions for the CL reaction were optimized and the possible reaction mechanism was discussed. Under the optimum conditions, the concentration of DAS‐K is proportional to the CL intensity in the range of 0.1–80 μmol·L−1 with a detection limit of 0.05 μmol·L−1. The interaction of the DAS‐K with bovine serum albumin by on‐line ultrafiltration and flow‐injection chemiluminescence was studied. The concentrations of unbound DAS‐K from ultra filter tube were determined by the flow‐injection CL method. The binding parameters were estimated by the Scatchard plot and Klotz plot. The proposed system proved that FIA‐CL coupled with on‐line ultrafiltration sampling was a fast and simple technique for the study of drug‐protein interaction.

Coupling microdialysis with flow-injection chemiluminescence detection for a protein–drug interaction study

The interaction of metronidazole (MTZ) and human serum albumin (HSA) was studied using the coupling system of on-line microdialysis sampling with flow-injection chemiluminescence detection (FI-MD-CL). The interested drug and HSA were mixed in different molar ratios in 0.067 mol L −1 phosphate buffer (pH 7.4) and incubated at 37 °C in a water-bath. Then the microdialysis probe was put into the MTZ–HSA mixed solution and sampled at a perfusion rate of 5 μL min −1. The microdialysates was determined using flow-injection chemiluminescence. In vitro recovery ( R) of MTZ under experimental conditions was approximately 25.2% with a R.S.D. of about 3.2%. The values estimated for the binding constant ( K) and the number of the binding sites ( n) were found to be 1.50 × 10 3 L mol −1 and 1.89, respectively. The values of nK obtained using Scatchard analysis and Klotz plot were found to be quite similar. The method provided a reliable and simple technique for the study of drug–protein interaction.

Inhibition of gentamicin binding to rat renal brush-border membrane by megalin ligands and basic peptides

Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181–200 (NISHTKEKKKGKAKKKRLTK, p I = 10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180–200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181–200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181–200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181–200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the α-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181–200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181–200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the α-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.

Determination of naproxen with flow injection chemiluminescence of Ru(bpy) 32+–PbO 2 system and its application for the binding study of naproxen to protein

A flow injection method based on Ru(bpy) 3 2+–PbO 2 chemiluminescence system was developed for the determination of naproxen. The factors affecting the CL intensity were performed including the amount of PbO 2 and H 2SO 4, pH of buffer and concentration of Ru(bpy) 3 2+. Under the optimum conditions, naproxen has a linear calibration graph in the range of 2 × 10 −8–6 × 10 −6 mol/L with a limit of detection ( S/ N = 3) of l.0 × 10 −8 mol/L, The correlation coefficient was 0.9976 ( n = 12) with a relative standard deviation of 3.4% for 15 determinations of l × 10 −6 mol/L of naproxen. Combining with ultrafiltration, this method was successfully applied to study naproxen–protein interaction. The binding constants ( K) to bovine serum albumen (BSA) were K 1 = 2.06 × 10 6L/mol and K 2 = 2.18 × 10 7 L/mol, and to human serum albumen (HSA) were K 1 = 4.80 × 10 6 L/mol and K 2 = l.25 × 10 7 L/mol, respectively. The correlation coefficients given by Scatchard plot and Klotz plot were satisfied, indicating that studied drug has two types of binding site in the molar ratio range studied. The number of the binding site ( n) on one molecule of BSA were n 1 = 6.79 and n 2 = 1.88, and of HSA were n 1 = 1.22 and n 2 = 1.80, respectively. The proposed method provided a sensitive, reliable and simple technique for studying naproxen–protein interaction and was for the first time applied to the determination of the unbound fraction of naproxen in its human serum albumin equilibrated solution.

Binding study of drug with bovine serum album using a combined technique of microdialysis with flow-injection chemiluminescent detection

Microdialysis coupled with flow-injection chemiluminescence (FI–CL) has been developed to determine the binding parameters of a drug binding to protein by using antibiotic tetracycline hydrochloride binding to bovine serum albumin as a model system. The drug and protein were mixed in different molar ratios in 0.067 M phosphate buffer, pH 7.4, and incubated at 37 °C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 μL/min. The concentration of unbound tetracycline hydrochloride in the microdialysate was determined by FIA–CL. In vitro recovery of tetracycline hydrochloride under experimental conditions was 30.0%. The data obtained by the present microdialysis–FI–CL system was analyzed using the Scatchard analysis and Klotz plot. The results show that the Scatchard plot and Klotz plot are linear with good correlation coefficient, indicating a good agreement of the experimental data and to the theoretical equation. The FIA chemiluminescence system combined with microdialysis developed in this work demonstrated its use for determination of interaction between drug and protein by using relatively simple instrument.

Flow-injection chemiluminescence detection for studying protein binding of terbutaline sulfate with on-line microdialysis sampling

The binding of terbutaline sulfate to bovine serum albumin was studied in vitro using the technique of microdialysis sampling combined with flow-injection chemiluminescence analysis (FIA-CL). In the presence of formaldehyde, terbutaline sulfate can be oxidized by KMnO 4 to produce high chemiluminescence emission in sulfate acid media. The concentration of terbutaline sulfate is proportional with the CL intensity in the range of 1×10 −7–2×10 −5 mol l −1 with a detection limit of 3×10 −8 mol l −1. The drug and protein were mixed in different molar ratios in 0.067 mol l −1 phosphate buffer, pH 7.4, and incubated at 37 °C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 μl min −1 and the dialytic efficiency of terbutaline sulfate under the experimental conditions was 26.3%. The data obtained by proposed microdialysis flow-injection chemiluminescence method was analyzed with Scrathard analysis and Klotz plot. The estimated association constant ( K) and the number of the binding site ( n) on one molecule of BSA by Scrathard analysis were 4.11×10 4 l mol −1 and 1.06, respectively. The proposed system proved that FIA-CL coupled with on-line microdialysis sampling is a simple and reliable technique for the study of drug–protein interaction.

A flow-injection ultrafiltration sampling chemiluminescence system for on-line determination of drug-protein interaction.

A flow-injection ultrafiltration sampling chemiluminescence system for on-line determination of cimetidine-bovine serum albumin (BSA) interaction is proposed in this paper. Cimetidine can be oxidized by N-bromosuccinimide (NBS) and sensitized by fluorescein to produce high chemiluminescence emission in basic media. The concentration of cimetidine is linear with the CL intensity in the range 3 x 10(-7) - 1 x 10(-4) mol L(-1) with a detection limit of 1 x 10(-7) mol L(-1) (3 sigma). The drug and protein were mixed in different molar ratios in 0.067 mol L(-1) phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water bath. The ultrafiltration probe was utilized to sample the mixed solution at a flow rate of 5 micro L min(-1). The data obtained by the proposed ultrafiltration flow-injection chemiluminescence method was analyzed with Scrathard analysis and a Klotz plot. The estimated association constant ( K) and the number of the binding site ( n) on one molecule of BSA by Scrathard analysis and Klotz plot were 3.15 x 10(4) L mol(-1) and 0.95, 3.25 x 10(4) L mol(-1) and 0.92, respectively. The proposed system proved that flow-injection chemiluminescence analysis coupled with on-line ultrafiltration sampling is a simple and reliable technique for the study of drug-protein interaction.

Ligand–receptor interaction. Klotz–Hunston problem for two classes of binding sites and its solution

The problem of the affinity and quantity determination of two classes of binding sites for ligand–receptor interaction using either Scatchard or Klotz plots was considered. Klotz and Hunston previously solved this problem only for the case of a representation of experimental data using the Scatchard plot. Since their publication, it was the common view that only the use of the Scatchard plot allows solving this problem. However, in some cases, using the Klotz plot is more convenient for a representation of experimental data concerning ligand–receptor interaction, though usually, this plot was used only for the evaluation of receptor affinity with one class of binding sites. In the present paper, it was demonstrated that Klotz plot also could be used for the evaluation affinity and quantity of two classes of binding sites.

Flow-injection analysis chemiluminescence detection combined with microdialysis sampling for studying protein binding of drug

The bovine serum album binding of streptomycin sulfate was studied in vitro using the technique of microdialysis combined with flow-injection analysis-chemiluminescence detection. The principle of the determination of streptomycin sulfate is that it increases the radiation emitted during the chemiluminescence oxidation of luminol by potassium hexacyanoferrate (III) in sodium hydroxide medium. The drug and protein were mixed in different molar ratios in 0.067 M phosphate buffer, pH 7.4, and incubated at 37°C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 μl min −1. The concentration of unbound streptomycin sulfate in the microdialysate was determined by FIA-CL. In vitro recovery of streptomycin sulfate under experimental conditions was 22%. The data obtained by the present microdialysis-FI-CL system was analyzed using the Scatchard analysis and Klotz plot. The results show that the Scatchard plot and Klotz plot are linear, showing that studied drug has only one type of binding sites. The estimated binding parameters agreed well with literature values.

Interaction of Vanillin with Soy and Dairy Proteins in Aqueous Model Systems: A Thermodynamic Study

ABSTRACT: Interactions of vanillin with soy, casein, and whey proteins were studied in aqueous model systems using a thermodynamic approach. Vanillin‐protein binding was examined at 12 and 4 °C using 3 proteins and 6 vanillin concentrations. The results were analyzed using a Klotz plot. Number of binding sites (n) and dissociation constants (Kd) were derived from the plots. The thermodynamic parameters (ΔG°, ΔH°, and ΔS°) for the bindings were calculated. Under identical experimental conditions, whey protein was found to have higher affinity to vanillin than the other 2 proteins. Bindings of vanillin with casein and whey protein were enthalpy driven, while the interaction of vanillin with soy protein was highly entropy driven. The results inferred that conformational changes of soy protein might be important in binding of vanillin.

Capillary electrophoresis study of human serum albumin binding to basic drugs

The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.

Bruceantin, a novel inhibitor of peptide bond formation

The effects of bruceantin on a number of steps of the protein synthesis process have been studied using resolved model systems from both yeast and reticulocytes. Bruceantin is a potent inhibitor of polyphenylalanine synthesis as directed by poly(U). However, inhibition is less pronounced on protein synthesis as directed by endogenous mRNA and the compound inhibits the poly(U) system only poorly if added after polyphenylalanine synthesis has been initiated. Peptide bond formation as assayed in both the fragment reaction and in the puromycin reaction with a preformed initiation complex containing ribosomes and [ 35S]Met-tRNA F is totally blocked by bruceantin. Neither the enzymic binding of Phe-tRNA to reticulocyte ribosomes nor the formation of the 35S-labeled tRNA · ribosome initiation complex is inhibited by bruceantin. The binding of [ 14C]trichodermin to yeast ribosomes is strongly inhibited by bruceantin. A Klotz plot shows that both these drugs bind to ribosomes in mutually exclusive fashion and it can be calculated that bruceantin binds to the peptidyltransferase center with K d = 0.34 μM. This high affinity is considerably lower for polyribosomes ( K d = 557 μM), which may explain the earlier finding that bruceantin only stabilizes polyribosomes at high drug concentrations.