High-performance affinity chromatography of human serum concanavalin A binding proteins

A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.     

Exploring binding properties of gliclazide with human serum albumin

Journal of Thermal Analysis and Calorimetry - The thermodynamic parameters of the binding of gliclazide (GL) with human serum albumin (HSA) have been discussed along with the insight into the...     

High-performance affinity chromatography system for the rapid, efficient assay of glycated albumin.

A high-performance affinity chromatographic system was constructed and shown to permit highly reproducible, rapid, automatic assays of serum glycated albumin (GA) by separation of albumin (Alb) on an anion-exchange column (Asahipak ES-502N) packed with a vinyl alcohol copolymer bearing diethylamino groups and consecutive separation of GA on a column packed with a vinyl alcohol copolymer bearing boronate groups. The first column selectively retained Alb free of other serum proteins and permitted at least a 95% recovery of sample Alb. The purity of the Alb peak was confirmed by two-dimensional electrophoresis. Chromatographic analyses of human serum Alb incubated with glucose on the second column showed that the peak area for GA increased in accordance with the incubation time and suggested selective adsorption of GA on the second column. Optimization of the conditions for the two-column system reduced the analysis time to 10 min. Analyses of human sera with the present system showed GA to be 16.1 +/- 1.1% (mean +/- S.D.) of total Alb in non-diabetic children and 39.9 +/- 9.1% (mean +/- S.D.) in diabetic children (0-17 years old).     

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and l-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1?×?10⁴–3?×?10⁵ M^?1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions.

Analysis of drug interactions with serum proteins and related binding agents by affinity capillary electrophoresis: A review

Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug–protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.     

Prediction of human serum albumin-drug binding affinity without albumin

A new liquid chromatographic system was developed to measure protein-drug binding affinity indirectly without albumin and was evaluated using log nK values of drugs measured by a modified Hummel-Dreyer method using purified human serum albumin. The retention factors of acidic and basic drugs were measured by reversed-phase and ion-exchange liquid chromatography in sodium phosphate buffer, pH 7.40, containing 50 vol.% methanol at 37 °C. The bonded phases were pentyl, guanidino and carboxyl phases. The combined retention factors were correlated with the log nK values measured by a modified Hummel-Dreyer method because glycosylation of human serum albumin did not significantly affect log nK value. The correlation coefficients were 0.949 (n=7) for acidic drugs and 0.978 (n=5) for basic drugs. The log nK values of 26 acidic and 18 basic drugs were predicted from their retention factors measured by reversed-phase and ion-exchange liquid chromatography.     

高效亲和色谱法测定丹皮酚与固定化人血清白蛋白结合域

利用亲和色谱,在模拟人体生理环境下(37 ℃、pH 7.4),采用竞争置换法研究了丹皮酚(PAE)与固定化人血清白蛋白(HSA)的相互作用。通过对PAE的自我竞争分析及PAE与HSA上结合位点的标记物间的竞争置换分析,得到了PAE和HSA间的结合常数、结合位点数和结合域。结果表明: PAE在HSA分子中仅存在一类结合位点,结合常数为4.84×103 L/mol,该结合位点为HSA上的Sudlow siteII;通过对PAE与HSA相互作用的热力学研究,推断出二者间的作用力类型为氢键或范德华力。     

High-performance liquid chromatographic determination of the binding of ceftriaxone to human serum albumin solution and albumin from diluted human serum

The binding of ceftriaxone to human serum albumin has been studied by high-performance liquid chromatography. The gel permeation method of Hummel and Dreyer was used. Ceftriaxone was tested with two sources of albumin (aqueous solution and diluted serum). After internal calibration the binding parameters were determined for each albumin, and results compared. These data are in agreement with those from classical methods for the determination of protein binding of ceftriaxone.     

High-performance liquid chromatography with electrochemical detection for analysis of gliclazide in plasma

A sensitive HPLC-electrochemical detection method was developed for the analysis of gliclazide (GL) in human plasma. After deproteination of 100 microL of plasma by acetonitrile, evaporation, and reconstitution, GL was separated on a C18 column (150 mm x 4.6 mm) by the mobile phase (70 mM disodium tetraborate, pH 7.5, containing 26.5% of acetonitrile). The regression equations were linear (r> 0.9990) over the range of 50 nM to 4.00 microM. The precision and accuracy of intra- and inter-day analysis were less than 5.3 and 0.93% for relative standard deviation and relative error, respectively. The limit of detection for plasma was 10 nM for GL (S/N = 3, 10 microL injection). This newly developed method was applied for monitoring blood levels with one healthy volunteer dosing with a GL tablet.     

Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns

Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.     

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.     

Betulinic acid binding to human serum albumin: a study of protein conformation and binding affinity

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K(BA)=1.685+/-0.01 x 10(6) M(-1), indicating a strong binding affinity. The secondary structure of the HSA-BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA+drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.     

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis‐frontal analysis

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis‐frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis‐frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.     

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK _a) of 1.9 (±0.1) × 10⁵ M⁻¹ for R-propranolol and 2.7 (±0.2) × 10⁵ M⁻¹ for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K _a) of 5.2 (±2.3) × 10⁵ M⁻¹ at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.     

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.