Flavonoid Interactions With Human Serum Albumin Biology Essay

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The study of flavonoids-protein binding is very important in clinical therapy as well as in pharmacokinetic and pharmacodynamic studies. The main aim of this paper is to study the characterization of interactions between human serum albumin ((HSA) and three structurally different flavonoids such as flavones, quercitrin and rutin under the physiological conditions (67 mM phosphate buffer pH 7.4, temperature 36.5 °C, HSA concentration (40 μM), flavonoids concentration (50-500 μM)) by capillary electrophoresis based on the principles of frontal analysis (CE-FA). The investigated flavonoids are found to shown reasonable affinity toward HSA with binding constants in the range 103−104 M−1. The order of binding constants of flavonoids: flavone (21.39Ã-103 M−1) > quercitrin (6.64Ã-103 M−1) > rutin (4.13Ã-103 M−1). The thermodynamic parameters such as the hydrophobicity and electrostatic interactions are the key driving forces between the HSA and flavonoids. According to displacement experiment studies, the main binding site of most flavonoids is site I of the HSA molecule and glycosylation of the flavonoids are not showing influence on the binding position. The short analysis time, high separation efficiency, low sample requirement, easy computerization, robustness and precision of results are the main benefit of the CE-FA and these are all used for the study of binding processes at the near-physiological conditions. Binding parameters between flavonoids and HSA are determined by CE-FA based on the principles of zone electrophoresis. In frontal analysis, the equilibrium is maintained during the electrophoresis where the zones overlap and in this method because of the variations in the mobility of the free ligand and protein/protein-ligand complex, the premixed protein-ligand sample were injected as a large plug and the separation takes place between them. Sudlow site I and II are the two binding sites identified on HSA and the greater binding capacity of HSA is due to the presence of two hydrophobic binding sites. The relationship between a flavonoid-rich diet and health benefits are still uncertain. In this paper, this study shows that the CE-FA is an excellent method when compared with the traditional methods, so it can be able to determine the different binding parameters and the characterization of physiological behaviours of bioactive compounds. The equilibrated (30 min) samples were injected hydro dynamically at 50 mbar, at two different temperatures 36.5 and 25oC into the anodic end of the uncoated fused silica capillary. When +8 kV voltages were applied, the separation takes place between the flavonoids and HSA. The height of the corresponding plateau peak on the electropherogram give the concentration of unbound flavonoids after incubation. The nonlinear regression equation was used for the calculation of the binding constant (K) and the number of sites (n). The main requirement for CE-FA analysis is the improved injection volume of sample and a difference among electrophoretic mobility of free ligand from the protein and the protein-ligand complex. Due to disappearance of the electro-osmotic flow, charge absence, stability in the weight of the protein and its electrophoretic mobility, the flavonoid-HSA complex and HSA would migrate as a single zone. In CE-FA, The results were compared to those obtained with validated method fluorescence spectroscopy and good agreement was achieved. (Knjazeva & Kaljurand, 2010)

Paper 1

Topic: Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis


Capillary electrophoresis-frontal analysis is used to study the interaction between human serum albumin, mixtures of human serum albumin, α1-acid glycoprotein and with the ten natural polyphenolic compounds such as chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin. The advantage is that this method requires very less amount of sample, short analysis time and it provide the better separation and flexible versatility to study multiple equilibria. The main aim of this paper is to characterize the interaction of ten natural polyphenolic compounds with human serum albumin (HSA) and the mixture HSA +AGP (α1-acid glycoprotein) under physiological conditions: pH=7.4, 67mM sodium phosphate buffer, 15 kV voltage, and the temperature 36.5oC and also to determine the binding properties between human serum albumin protein and the ten polyphenolic compounds and these binding affinities can be assessed by using ultrafiltration and capillary electrophoresis. This paper demonstrate the binding affinity between the presence of C(4)=O in the polyphenolic structure and HSA. The binding affinity with HSA identified were very small (<10%) for chlorogenic acid, epicatequin and vicenin-2, moderate for catechin and rutin, and maximum (>90%) apigenin, flavanone, flavone, quercetin, and vitexin. This is because of the absence of carbonyl group (C=O) at C4 position, low hydrophobicity and steric hindrance due to the presence of glycoside. The differences among the structures of the polyphenolic compounds were less but ten polyphenolic compounds shown greater variation with proteins in the binding affinities. The interactions between xenobiotic and serum protein would be the determining factor in the therapeutic, pharmacodynamic and toxicological properties of xenobiotics. By using hydrogen bonds or hydrophobic interaction, polyphenols form, a reversible noncovalent complexes with proteins under nonoxidative conditions. Denaturant agents were incapable to break the irreversible polyphenol-protein covalent bindings. The driving forces to plasmatic proteins- flavonoids grouping are hydrogen bonding, hydrophobic forces, steric hindrance, and spatial arrangement. The order of binding affinity between natural polyphenolic compounds and human serum albumin (HSA): apigenin > quercetin ≈ vitexin > flavanone > rutin > catechin > vicenin-2 and the binding of polyphenols with AGP was not of hydrophobic nature. (Diniz et al., 2008)

Paper 2

Topic: Determination of protein-drug binding constants by pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA)


The development of pressured-assisted capillary electrophoresis (PACE) is based on the principle of frontal analysis (FA) and this is used for the resolution of binding constants of drugs to human serum albumin (HSA) and α1-acid glycoprotein (AGP). The height of the frontal peak shows the free drug concentration build on the external drug standard in the lack of protein. When the concentration of HSA increases, the height of the free drug plateau decreases, due to maximum number of drugs bind to the protein. Electrostatic and hydrophobic interactions play the key role in determining the binding constant. HSA is mainly responsible for the binding of acidic drugs and basic, neutral drugs are binded with containing single binding site of the AGP. The percentage of drug binding constants to HSA or AGP is determined with a known concentration of total drug. The percentage of bound drug is used as a function of total protein concentration or total drug concentration could yield the binding constants of drug to HSA or AGP from non-linear curve fitting. The phosphate buffered saline (PBS; pH7.4; ionic strength 0.17 M) was used for the preparation of sample, in this solution, the sample was synthesised by the combination of known concentrations of drug and protein, and equilibrated for 30 min. The fused silica capillary was filled with phosphate buffered saline (PBS) solution and a huge volume of sample solution (~80 nL) was injected in this fused silica capillary at 1.0 psi for 40 s. When 15-25 kV voltage and 0.5-1.5 psi air pressure applied, the free drug was separated from the protein/protein-drug complex, because of the variation among charge and size ratio. For the better drug plateau, increasing the throughput, reduction in retention time and prevent protein loss could be obtained by applying external air pressure. An extensive range of binding constants was calculated by altering the experimental conditions. The CE method also facing some problems that are adsorption of protein and drug to the capillary wall, throughput, reproducibility, sensitivity, and the range of binding constants. The binding constants of the basic, neutral, and weakly acidic compounds were studied and determined by pressured-assisted capillary electrophoresis (PACE) based on the principle of frontal analysis (FA) and for strongly acidic drugs, it is very difficult due to poor separation and not suitable for non-UV absorbing or poorly soluble compounds. The PACE/FA method is suited for drugs with binding constants in the range of 102-106 M−1 and only a small amount of proteins and drugs needed. (Jia, Ramstad & Zhong, 2002)

Paper 3

Topic: Investigating noncovalent interactions of rutin - serum albumin by capillary electrophoresis - frontal analysis


Capillary electrophoresis-frontal analysis the low consumption of sample is done by the CE- frontal analysis one of the method in pharmaceutical analysis, this method is used to study noncovalent interaction amid rutin and serum albumin (bovine serum albumin, BSA and human serum albumin, HSA). CE-FA is the best method due to simple, rapid and low cost. Benefits of this method consist of low sample consumption, ease of automation, and analysis time. And this method the (CE-FA) method which is used to study small molecules binding to protein. CE-FA contains a huge sample plug and a pre- equilibrated mixture of analyte and protein. At pH=7.4 and in 67mM phosphate buffer solution, this mixture proteins (BSA and HSA) with rutin was injected into fused silica capillary, gave the square-shaped peak in the spectrum. The binding affinity was determined by the plateau height and this peak height based on the changes of analyte's migration time, EOF, capillary length and voltage. The pH=7.4, 67mM phosphate buffer, temperature 37oC and pre- equilibrium were the optimised conditions for this experiment. The variation in electrophoretic mobilities of the complex and free analyte was the key requirements of CE-FA. In this method, the binding parameter of proteins (BSA and HSA) with rutin and the number of primary binding sites per HSA or BSA molecules were determined by keeping at a fixed HSA or BSA concentration and increasing rutin concentration. The binding constants of rutin-HSA and rutin-BSA agreed well with those determined by conventional fluorescence methods. The CE-FA method shown that the noncovalent binding parameters for rutin-HSA and rutin-BSA were similar. The CE-FA method, which may be useful in further high-throughput protein binding studies of multi-components in traditional herbal medicines for pharmacological effect evaluations. (Lu, Ba & Chen, 2008)

Paper 4

Topic: Study of multiple binding constants of dexamethasone with human serum albumin by capillary electrophoresis-frontal analysis and multivariate regression


Capillary electrophoresis-frontal analysis is an efficient technique due to its low sample consumption, short analysis time and high separation efficiency. The main aim of this paper is to study the interactions amid drugs and human serum albumin (HSA) by using Capillary electrophoresis-frontal analysis (CE-FA). This study is very useful in drug discovery, due to HSA plays a major role and acts as a carrier for external drugs and internal biological molecules. This method was used to study the interaction among dexamethasone (DXM) and human serum albumin (HSA), to assess the pharmacokinetic and pharmacodynamic properties of dexamethasone. The properties, which were in turn related to the absorption, distribution, metabolism and excretion of the dexamethasone, as well as concentrations at the sites at which it function. A huge volume (60-200nL) of drug and protein mixture was used in CE-FA analysis. The separation among the free drug, the protein and the drug-protein complex takes place when an electric field applied. The peak height of the drug is relative to its concentration. In CE-FA, the Klotz equation was used for the calculation of the number of binding sites and binding constants. Four binding sites amid DXM and HSA, the average binding constant 1.05Ã-103 M−1 were acquired by the Klotz equation and the average binding constant 1.05Ã-103 M−1 confirmed that there was noncovalent interaction between DXM and HSA. In addition, the binding constant at one site 3.539Ã-103 M−1, and the average of the other three1.234Ã-103 M−1 were obtained by using the multiple equilibrium theory, based on the assumption that there were two types of binding site in HSA. Moreover, multivariate regression equation was used to study the complete binding information at each binding site, and the four binding constants were calculated by using multivariate regression equation between DXM and HSA , and binding constants were 5.558Ã-101 M−1, 2.158Ã-104 M−1, 7.312Ã-103 M−1 and 2.043Ã-103 M−1 . In addition, the different drug-protein complexes electrophoretic mobilities (μPL1, μPL2, μPL3 and μPL4) were determined by using Binding constants and the values were 9.22, 9.82, 10.24 and 10.38 cm2min−1kV−1 . The complete interactions between proteins with multiple binding sites and drugs are studied by using Binding constants. (Zhao et al., 2009)

Paper 5

Topic: Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions


Capillary electrophoresis frontal analysis was used for the study of the interaction between 12 low molecular weight and human serum albumin (HSA). The reaction between human serum albumin and drug displacement was studied by using CE-FA. In drug development, the characterization of plasma protein binding affinity is very important. Sudlow site I and II are the two binding sites identified on HSA. CE-FA is a simple method and this method offers many advantages when compared with conventional methods such as equilibrium dialysis and ultrafiltration methods. The CE-FA uses are small sample size, relatively short analysis time, ease of automation, and no errors due to protein leakage through membrane, no membrane adsorption of the drug, and no osmotic or Donnan effects. The 12 low molecular weight compounds including 8 drug compounds exhibit a variety of properties according to their binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. For the increasing in the separation of the drug and human serum albumin plateau peaks, dextran was added to the run buffer. The attained binding parameters such as binding constants and binding curves were similar to that of the literature values. In this experiment the results showing slight errors in mobility variations among free and complex human serum albumin irrespective of the charge of the investigated low molecular weight ligands. The low sensitivity of the UV-detection system was the major problem was found in the application of CE-FA for drug-HSA binding studies. This experiment was done at near physiological buffers condition. CE-FA is simple and automated and it is perfectly suitable for screening in early drug development. (Østergaard et al., 2002)

Paper 6

Topic: Protein binding study of clozapine by capillary electrophoresis in the frontal analysis mode


This can be observed that clozapine is found strongly in association with protein in human plasma (bound fraction about 84%), rabbit, and serum while rarely to human serum albumin (bound fraction 9.1%). The original unbound concentration of clozapine in human serum albumin, human plasma, rabbit serum and plasma can be found out by capillary electrophoresis in the frontal analysis mode (CE-FA). The height of the frontal peak is given an idea about the unbound clozapine concentration. The unbound clozapine concentration was determined by comparing with peak height of the equilibrated samples with that of neat clozapine solution. An uncoated fused silica capillary column (0.65m (LC) Ã-75µm i.d.; LE = 0.35 m) is used and samples are injected directly without any pre-treatment and separation occur within 11 min. The good separation takes place when a lower voltage (6 kV) applied and by addition of running phosphate buffer. The buffer used for the identification of the substances found to contain 1 mmol l−1 EDTA, 0.5 mol l−1 glycine, and 67 mmol l−1 phosphate with pH 7.4. Addition of glycine in the running buffer prevents the adsorption of proteins, to untreated fused silica capillary. The poor sensitivity is one of main limitation in this method. The advantages by this method is that it gives an exact concentration of unbounded drug concentration in multiple equilibrium system with very low volume of injections, and therefore most commonly beneficial for the protein binding study of biological fluids that may present in our body in very small amount. The main advantage of CE-FA is that the binding constants identification of basic drugs whose migration is similar to the clozapine. This method is very suitable in terms of lower time, reagent and the extreme simplification of the analytic procedure, and to applications in the therapeutic drug monitoring (TDM) laboratory in the future. (Zhou & Li, 2004)