Chemiluminescence detection coupled to high-performance frontal analysis for the determination of unbound concentrations of drugs in protein binding equilibrium

High-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL) was developed for the determination of unbound oxacillin concentration in human serum albumin solution. The HPFA system consisted of an ISRP column and a mobile phase of 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17. The luminol-H2O2-Co2+ system was used in the chemiluminescence detection. An enhancement of luminol chemiluminescence by oxacillin was investigated and employed for determining the concentration of oxacillin in the HPFA eluate. Sample solutions were directly injected onto the column; the drug was eluted as a zonal peak with a plateau region. The unbound drug concentrations were determined by using the height of the plateau. The results agreed with those obtained with conventional ultrafiltration-HPLC method. Good reproducibility was confirmed by the within run and between run RSD < or = 7.4%. HPFA-CL provided a selective method for determination of unbound drug concentration in protein binding equilibrium.     

高效迎头分析法测定药物-人血清白蛋白混合液中游离药物浓度

 用高效迎头分析法 (HPFA)测定了药物 人血清白蛋白 (HSA)混合液中游离药物的浓度。样品溶液不经任何处理直接进样到装有内表面反相固定相的色谱柱中 ,用 67mmol/L磷酸盐缓冲液 ( pH 7 4 ,I =0 17mol/L)作流动相。当进样体积足够大时 ,游离药物以平顶峰的形式被洗脱出来 ,平顶峰区域洗脱液中的药物浓度等于样品溶液中游离药物的浓度。收集平顶峰区域的洗脱液 ,然后将一定体积的洗脱液注入到反相色谱柱中 ,测定游离药物的浓度。用该法测定酮基布洛芬 HSA和头孢哌酮 HSA两种混合液中游离药物的浓度。     

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.     

Fibronectin conformation switch induced by coadsorption with human serum albumin

The dynamic adsorption of human serum albumin (HSA) and plasma fibronectin (Fn) onto hydrophobic poly(hydroxymethylsiloxane) (PHMS) and the structures of adsorbed protein layers from single and binary protein solutions were studied. Spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D) together with atomic force microscopy (AFM) were used to measure the effective mass, thickness, viscoelastic properties, and morphology of the adsorbed protein films. Adsorbed HSA formed a rigid, tightly bound monolayer of deformed protein, and Fn adsorption yielded a thick, very viscoelastic layer that was firmly bound to the substrate. The mixed protein layers obtained from the coadsorption of binary equimolecular HSA-Fn solutions were found to be almost exclusively dominated by Fn molecules. Further sequential adsorption experiments showed little evidence of HSA adsorbed onto the predeposited Fn layer (denoted as Fn ? HSA), and Fn was not adsorbed onto predeposited HSA (HSA ? Fn). The conformational arrangement of the adsorbed Fn was analyzed in terms of the relative availability of two Fn domains. In particular, (4)F(1)·(5)F(1) binding domains in the Hep I fragment, close to the amino terminal of Fn, were targeted using a polyclonal antifibronectin antibody (anti-Fn), and the RGD sequence in the 10th segment, in the central region of the molecule, was tested by cell culture experiments. The results suggested that coadsorption with HSA induced the Fn switch from an open conformation, with the amino terminal subunit oriented toward the solution, to a close conformation, with the Fn central region oriented toward the solution.     

The fluorescent probe prodan characterizes the warfarin binding site on human serum albumin

Abstract— The fluorescent probe Prodan (6-propionyl-2-dimethyl-aminonaphthalene) binds with high affinity to human serum albumin (HSA). The spectral characteristics of the Prodan bound to the protein are very different from the free Prodan in solution. These differences allowed the spectra to be deconvoluted into log-normal bands in order to quantify the bound and unbound ligand and to calculate the binding constant at different temperatures. From such temperature dependence, we found the binding to be exothermic with a van't Hoff enthalpy of -22.8 kJ mol^-1. Thermodynamic analysis suggests that the in teraction may be mainly caused by hydrophobic forces and electrostatic interactions. The above analysis of the spectra and the measures of the fluorescence polarization during the successive presence of six specific drugs suggest that the Prodan binding site corresponds with the warfarin binding site on HSA, whereas under the present experimental conditions the other characteristic binding sites of HSA were not affected. Thus, this fluorescent probe provides a rapid and simple means for the characterization of a specific binding site on HSA and also for detecting potential or nonspecific drug-protein interactions.     

Study on the protein binding of ketoprofen using capillary electrophoresis frontal analysis compared with liquid chromatography frontal analysis

A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high-performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm x 75- micro m i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association constant (K(2)) of 0.24 x 10(6)M(-1) and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 x 10(6)M(-1) and 0.94 for K(1) and n(1), respectively, and 0.12 x 10(6)M(-1) and 3.16 for K(2) and n(2), respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.     

Studies on the interaction of gallic acid with human serum albumin in membrane mimetic environments

In this study the interaction between gallic acid and human serum albumin (HSA) in AOT/isooctane/water microemulsions was characterized for the first time using fluorescence quenching technique in combination with UV absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique. In water-surfactant molar ratio (omega(o))=20 microemulsions fluorescence data revealed the presence of one binding site of gallic acid on HSA and its binding constants (K) were (1.18+/-0.02)x10(4), (1.13+/-0.02)x10(4), (1.03+/-0.02)x10(4), (0.95+/-0.02)x10(4), (0.87+/-0.02)x10(4) and (0.82+/-0.03)x10(4)M(-1) at 282, 289, 296, 303, 310 and 317 K, respectively. The affinities in microemulsions were much higher than that in buffer solution. FT-IR and CD data suggested that the protein conformations were altered with the reductions of alpha-helices from 54-56% for free HSA in buffer to 40-41% for free HSA in microemulsion. After binding with gallic acid, the alpha-helices of HSA in microemulsion increased 2-7% for different drug-protein molar ratio. The thermodynamic functions standard enthalpy (Delta H(0)) and standard entropy (DeltaS(0)) for the reaction were calculated to be -8.10 kJ mol(-1) and 49.42 J mol(-1)K(-1). These results indicated that gallic acid bound to HSA mainly by hydrophobic interaction and electrostatic interaction in microemulsions. In addition, the displacement experiments confirmed that gallic acid could bind to the site I of HSA, which was approved by the molecular modeling study. Furthermore, the DLS data suggested that HSA may locate at the interface of the microemulsion and gallic acid could interact with them.     

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basie drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-colunm capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of drug enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral separation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in the LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex media, particularly the matrix of protein coexisting with a variety of drugs.     

Osmotic Pressure, Small-Angle X-Ray, and Dynamic Light Scattering Studies of Human Serum Albumin in Aqueous Solutions

Osmotic pressure measurements of human serum albumin (HSA) dissolved in water and in 0.01, 0.1, and 1.0 M phosphate buffer are reported as a function of the protein concentration. Two different forms of the protein were studied: defatted HSA (HSA1) and HSA with fatty acids (HSA2). The measured values of the osmotic coefficient were well below 1, indicating large deviations from ideality even for dilute protein solutions. The measured values increased with increasing HSA concentration and the increase was a function of pH. For higher concentrations of added phosphate buffer, the pH of solution had less influence on the measured osmotic pressure. The osmotic pressure of HSA1 in water was found to be considerably lower than that of the HSA2 modification. This effect was ascribed to formation of dimers in the HSA1 solution. The osmotic measurements were complemented by the small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) studies of dilute HSA solutions in water. The SAXS and DLS data confirmed the dimerization of HSA1 molecules under these conditions. Detailed analysis of the SAXS data suggested a parallel orientation of two protein molecules in a dimer. Copyright 2001 Academic Press.     

Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.     

A novel chemiluminescence technique for quantitative measurement of low concentration human serum albumin.

An efficient and highly sensitive chemiluminescence (CL) technique is proposed in the current study for detection of low levels of human serum albumin (HSA). Chemiluminescence (CL) produced during interaction between fluoresceinyl cypridina luciferin analog (FCLA)-1O2 can be modified with the presence of HSA. The conventional CL technique uses a quenching effect of HSA for its quantitative measurement. We are reporting here that the CL intensity can be enhanced, rather than quenched, by the addition of HSA. The CL signal can be linearly correlated with the HSA concentration over a clinically interesting range of 5 x 10(-9) - 8 x 10(-8) mol L(-1), with a detection limit of 2.5 x 10(-9) mol L(-1). The determination result was consistent with that obtained from conventional methods. One possible mechanism of HSA detection technique using CL enhancement approach is discussed. Intermolecular energy transfer in chemiluminescence systems and changes of microenvironment are likely to be contributors of the CL enhancement with HSA.     

Single run measurements of drug-protein binding by high-performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.     

Study on the interaction of tamiflu and oseltamivir carboxylate with human serum albumin

Oseltamivir phosphate (OP; tamiflu) is an antiviral pro-drug, which is hydrolyzed hepatically to the active metabolite oseltamivir carboxylate (OC). It is the first orally neuraminidase inhibitor that was used in the treatment and prophylaxis of influenza virus A and B infection. Human serum albumin (HSA) is the most abundant of the proteins in the blood plasma and is major transporter for delivering several drugs in vivo. This study was designed to examine the interaction of HSA with oseltamivir phosphate (OP) and oseltamivir carboxylate (OC) in aqueous solution at physiological conditions, using a constant protein concentration and various drug contents. FTIR, UV-Vis spectroscopic methods were used to determine the drugs binding mode, the binding constant and the effects of drug complexation on protein secondary structure. Structural analysis showed that OP and OC bind HSA via polypeptide polar groups with overall binding constants of K(OP-HSA)=3.86(± 1.05)× 10(3)M(-1) and K(OC-HSA)=1.5(±0.45) × 10(2)M(-1). The alterations of protein secondary structure are attributed to a partial destabilization of HSA on drug complexation. The protein secondary structure showed no major alterations at low drugs concentrations (50 μM), whereas at higher content (1mM), decrease of α-helix from 58% (free HSA) to 38% (OP-HSA)-48% (OC-HSA), decrease of random coil from 15% (free HSA) to 2% (OP-HSA)-3% (OC-HSA), increase of β-sheet from 6% (free HSA) to 20% (OC-HSA)-29% (OP-HSA) and turn from 8% (free HSA) to 17% (OC-HSA)-19% (OP-HSA) occurred in the drug-HSA complexes. These observations indicated that low drug content induced protein stabilization, whereas at high drug concentration, a partial protein destabilization occurred in these drug-HSA complexes.     

New insights into the interactions of serum proteins with bis(maltolato)oxovanadium(IV): transport and biotransformation of insulin-enhancing vanadium pharmaceuticals

Significant new insights into the interactions of the potent insulin-enhancing compound bis(maltolato)oxovanadium(IV) (BMOV) with the serum proteins, apo-transferrin and albumin, are presented. Identical reaction products are observed by electron paramagnetic resonance (EPR) with either BMOV or vanadyl sulfate (VOSO4) in solutions of human serum apo-transferrin. Further detailed study rules out the presence of a ternary ligand-vanadyl-transferrin complex proposed previously. By contrast, differences in reaction products are observed for the interactions of BMOV and VOSO4 with human serum albumin (HSA), wherein adduct formation between albumin and BMOV is detected. In BMOV-albumin solutions, vanadyl ions are bound in a unique manner not observed in comparable solutions of VOSO4 and albumin. Presentation of chelated vanadyl ions precludes binding at the numerous nonspecific sites and produces a unique EPR spectrum which is assigned to a BMOV-HSA adduct. The adduct species cannot be produced, however, from a solution of VOSO4 and HSA titrated with maltol. Addition of maltol to a VOSO4-HSA solution instead results in formation of a different end product which has been assigned as a ternary complex, VO(ma)(HSA). Furthermore, analysis of solution equilibria using a model system of BMOV with 1-methylimidazole (formation constant log K1 = 4.5(1), by difference electronic absorption spectroscopy) lends support to an adduct binding mode (VO(ma)2-HSA) proposed herein for BMOV and HSA. This detailed report of an in vitro reactivity difference between VOSO4 and BMOV may have bearing on the form of active vanadium metabolites delivered to target tissues. Albumin binding of vanadium chelates is seen to have a potentially dramatic effect on pharmacokinetics, transport, and efficacy of these antidiabetic chelates.     

Spectrophotometric determination of urinary protein with o-sulfophenylfluorone-metal complex.

A simple and sensitive spectrophotometric method for the determination of human serum albumin (HSA) was established based on the ternary complex-formation reaction of HSA with o-sulfophenylfluorone (SPF) as a xanthene dye and metal ion (niobium(V) and bismuth(III)) in the presence of a dispersion agent. This new method enabled the determination of HSA in the range of 1 - 15 microg/ml HSA by measuring the difference of the absorbance at 530 nm between HSA-SPF-metal ion and SPF-metal ion solutions. In the determination of HSA, this method is about 2-times more sensitive than the Pyrogallol Red-molybdenum(VI) method (PR method), which accounts for more than 80% of the quantification methods for urinary protein assays in Japan. There was no significant difference between the results obtained by the present method and the PR method for human urine samples. The binding process between the SPF-metal complex and HSA was studied by determining the binding parameters and the thermodynamic parameters.     

Protein binding of quinolonecarboxylic acids. II. Spectral changes on the interaction of cinoxacin, nalidixic acid and pipemidic acid with human and rat albumins

The interaction of cinoxacin (CINX), nalidixic acid (NA), and pipemidic acid (PPA) with human and rat serum albumins (HSA and RSA) was studied by UV difference absorption and circular dichroism (CD) spectroscopy. CINX and NA bound to the albumins and generated difference absorption and induced CD (ICD) spectra. The difference absorption spectral data explained reasonably our previous observations that CINX bound to HSA more weakly than NA, but to RSA as strongly as NA. We used a quantity delta epsilon/epsilon, designated as relative molar difference absorbance, at positions corresponding to the longest wavelength peaks in the difference spectra. The quantity was found to correlate linearly with percent bound to both HSA and RSA, but with different slopes, from which the binding site for CINX and NA in RSA was supposed to provide a much more nonpolar environment than that in HSA. The magnitude of ICD bands observed at 371 nm for CINX and at 342-348 nm for NA corresponded to the binding degrees of these drugs to both albumins. Anisotropy factors for the ICD bands at 350-271 nm for CINX and 320-348 nm for NA were approximately similar between HSA and RSA, suggesting a similar ability to generate the ICD spectra in these wavelength regions upon binding to the albumins. Spectral results for PPA in albumin solutions showed little or no binding of this drug to HSA and RSA. PPA existed as a betaine form in neutral solution and its positively charged group acted as an unfavorable factor for binding to both albumins.     

Platinum metallodrug-protein binding studies by capillary electrophoresis-inductively coupled plasma-mass spectrometry: characterization of interactions between Pt(II) complexes and human serum albumin

Characterizing how platinum metallocomplexes bind to human serum albumin (HSA) is essential in evaluating anticancer drug candidates. Using cisplatin as a reference complex, the application of capillary electrophoresis (CE) to reliably assess drug/HSA interactions was validated. Since this complex is small compared to the size of the protein, the binding response could only be recognized when applying CE coupled to a (platinum) metal-specific mode of detection, namely inductively coupled plasma-mass spectrometry (ICP-MS). This coupling allowed for confirmation of a specific affinity of cisplatin and novel Pt complexes to HSA, measurement of the kinetics of binding reactions, and determination of the number of drug molecules attached to the protein. As the cisplatin/HSA molar ratio increased, the reaction rate became faster with a maximum on the kinetic curve appearing at about 50 h of incubation at 20 times excess of cisplatin. The reaction was characterized as a pseudo-first order reaction with the rate constant k = 0.003 min(-1) at 37 degrees C. When incubated with a 20-fold excess of cisplatin, HSA bound up to 10 mol of Pt per mol of the protein. This is indicative for a strong metal-protein coordination occurring at several HSA sites other than the only protein cysteine residue. Structural analogs of cisplatin, bearing aminoalcohol ligands, showed comparable protein binding reactivity and stoichiometry but a common equilibrium was not reached even after one week of incubation. Also apparent was a two-step mechanism of the binding reaction. Results demonstrated the suitability of CE-ICP-MS as a rapid assay for high-throughput studying of drug/HSA interactions.     

Microdetermination of Proteins by Enhanced Rayleigh Light Scattering Spectroscopy with Thorin

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Studies on the interaction of caffeic acid with human serum albumin in membrane mimetic environments

In this work, fluorescence quenching technique, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique were used to gain the binding information of caffeic acid and human serum albumin (HSA) in AOT/isooctane/water microemulsions. The interaction of HSA with caffeic acid at 296, 303, and 310 K in omega(0) 20 microemulsions was characterized by one binding site with the affinity constant K at (3.23+/-0.01) x 10(4), (3.06+/-0.03) x 10(4) and (2.82+/-0.05) x 10(4)M(-1), respectively. The affinities in microemulsions are much higher than that in buffer solution. The CD spectra and FT-IR spectra with qualitative and quantitative results proved that the protein secondary structure changed in the microemulsions in the absence and presence of caffeic acid compared with the free form of HSA in buffer. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses. These data indicated that hydrophobic interaction played a major role in the binding of caffeic acid to HSA in microemulsions and electrostatic interaction can not be excluded. The displacement experiments confirmed that caffeic acid could bind to the site I of HSA, which was in agreement with the result of the molecular modeling study. Furthermore, the DLS data suggested that HSA may locate at the interface of the microemulsion and caffeic acid could interact with them.     

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.