Kinetic and thermodynamic analysis of 9‐nitrocamptothecin hydrolysis at physiological pH in the presence and absence of human serum albumin

Kinetic and thermodynamic analysis of the 9‐nitrocamptothecin (9NC) hydrolysis reaction in the presence and absence of human serum albumin (HSA) in phosphate‐buffered saline (PBS) of pH 7.4 was carried out by first derivative absorption spectroscopy. The thermodynamic parameters determined in these studies provided a mechanistic explanation toward the endothermic but yet thermodynamically favorable hydrolysis of 9NC at physiological temperature and pH. In the presence of HSA, the apparent rate constant of 9NC hydrolysis was 3–3.5 times higher than in 9NC solutions alone, whereas the apparent equilibrium constant of 9NC hydrolysis was found to increase at a higher extent in the presence of HSA than in PBS with increasing temperature, reaching almost complete hydrolysis of the 9NC to the 9NC‐carboxylate at 315.15 K. Importantly, the E_a of the 9NC hydrolysis reaction in the presence of HSA was determined to be on average 17 kJ mol^?1 lower than the E_a determined in plain PBS. Moreover, analysis of binding isotherms constructed for the HSA interaction with 9NC, using infinitely cooperative and independent binding models, demonstrated an incredibly higher binding constant for the 9NC‐carboxylate form as compared to the very weak and concentration‐dependent binding for the 9NC‐lactone species at 310.15 K. Taken together, the preferential association of the carboxylate form with HSA and the lower E_a of 9NC hydrolysis in the presence of HSA provide a mechanistic explanation for the shift of lactone–carboxylate equilibria toward the carboxylate product under physiological conditions of pH and ionic strength. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 693–703, 2010     

Kinetic and thermodynamic analysis of 10-hydroxy-camptothecin hydrolysis at physiological pH

To derive accurately the thermodynamic parameters governing the hydrolysis of the lactone ring at physiological pH, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of the 10-hydroxy-camptothecin (10-HC). Validation of the analytical method was done with respect to reproducibility, percent recovery, and level of detection. Hydrolysis of the lactone ring of 10-HC followed a 1st order decay with a rate constant equal to (0.0281 ± 0.001) min⁻¹ in PBS at pH 7.4 and at a temperature of 310 K. The activation energy for the hydrolysis reaction as calculated from the Arrhenius equation was (79.41 ± 0.92) kJ · mol⁻¹, whereas the enthalpy and entropy of hydrolysis of 10-hydroxy-camptothecin were on average 12.45 kJ · mol⁻¹ and 52.37 J · K⁻¹ · mol⁻¹, respectively. The positive enthalpy and entropy values of the 10-HC-lactone hydrolysis indicate that the reaction is endothermic and entropically driven.     

Electrochemical and spectroscopic characterization of interaction between antimalarial drug cinchonine and human serum albumin at physiological pH

In the present study, the interaction between cinchonine (CCN) and human serum albumin (HSA) was investigated using differential pulse polarography (DPP), cyclic voltammetry (CV) and spectroscopic techniques in Britton-Robinson (B-R) buffer pH 7.4. CCN displayed a main cathodic peak at ?1.228 V (vs. Ag|AgCl|KCl_sat) on mercury working electrode. The addition of HSA into CCN sulfate solution resulted in the decrease of the main reduction peak current of CCN and no new peaks appeared. The decay in the peak current of CCN, after the addition of HSA, showed a decrease in free drug concentration and formation of a biocomplex. The peak current changes of CCN in the presence of HSA were followed by DPP to determine the binding parameters. The logarithm of binding constant and binding ratio between CCN and HSA were 6.128 and 1: 1, respectively. This interaction was also confirmed by UV-Vis and FTIR-ATR spectroscopic measurements.     

Stability of bovine serum albumin at different pH

Summary The effect of pH on the thermal denaturation of BSA containing fatty acids was studied by use of differential scanning calorimetry (DSC). Thermal scanning of BSA aqueous solutions gave various types of DSC curves depending on the protein concentration and on the pH. The broad bimodal endothermic transition was suggested to be connected with loose protein structure in contradistinction to single peak for compact molecule structure. The propensity toward precipitation at pH conditions ranging from 3.8 to 5 was observed. A scan-rate independent and partly reversible behavior of the thermal heating of BSA was found. Deconvolution of DSC traces in non-two-state model with assumption of two- or three-component transition allowed to study the effect of pH on different parts of BSA molecule.     

Susceptibility to hydrolysis of phenylboronic pinacol esters at physiological pH

Boronic acids and their esters are highly considered compounds for the design of new drugs and drug delivery devices, particularly as boron-carriers suitable for neutron capture therapy. However, these compounds are only marginally stable in water. Hydrolysis of some phenylboronic pinacol esters is described here. The kinetics is dependent on the substituents in the aromatic ring. Also the pH strongly influences the rate of the reaction, which is considerably accelerated at physiological pH. Therefore, care must be taken when considering these boronic pinacol esters for pharmacological purposes.      

光谱法研究喜树碱与牛血清白蛋白的相互作用

采用多种光谱技术对喜树碱和牛血清白蛋白的相互作用进行了研究.结果表明喜树碱和牛血清白蛋白可形成基态复合物,引起牛血清白蛋白内源荧光猝灭.通过计算获得了二者在不同温度下的结合常数及结合位点数.根据喜树碱和牛血清白蛋白结合的热力学参数,确定了二者之间主要为疏水作用力.根据F(o)rster非辐射能量转移理论确定了喜树碱和牛血清白蛋白的作用距离.同步荧光光谱显示喜树碱主要与蛋白中色氨酸残基发生相互作用,改变其周围的局部构象.红外光谱提示喜树碱可引起蛋白的构象发生改变,α-螺旋二级结构减少.     

A thermodynamic study on the binding of theophylline with human serum albumin

The thermodynamic parameters of interaction between theophylline and Human Serum Albumin (HSA) in buffer solution (30 mM) of pH = 7 at 27 °C was investigated by isothermal titration calorimetry (ITC). The thermodynamic quantities of the binding mechanism, the number of binding sites (g), the dissociation binding constant (K _d), the molar enthalpy of binding (ΔΗ) and other thermodynamic parameters can be obtained by the extended solvation theory.     

Polyoxometalate binding to human serum albumin: a thermodynamic and spectroscopic approach

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.     

Solvation effects in human serum albumin radiolysis in the presence of dimethyl sulfoxide

PMR and electrophoresis have been applied to examine hydration changes in protein molecules due to the presence of the electron donor dimethylsulfoxide DMSO, which influences the radiolysis of human serum albumin HSA. The reactions of aqueous HSA with added DMSO show that the DMSO on the one hand acts as a protector, which prevents the formation of low-molecular protein forms on reaction with hydroxyl radicals, and on the other alters the protein hydration, which facilitates thiol-di-sulfide exchange, which leads to oligomers.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 26, No. 1, pp. 107–111, January–February, 1990.     

Epimerization and racemization of some chiral drugs in the presence of human serum albumin

Epimerization and racemization of carbenicillin, ethiazide, etoposide and oxazepam acetate were examined kinetically in the presence of human serum albumin (HSA). The concentration of both optical isomers of each drug was determined by stereospecific high-performance liquid chromatography. The apparent rate constants of epimerization or racemization and hydrolysis were estimated from the concentration-time data. HSA retarded the racemization of ethiazide and the epimerization of etoposide. The binding of the drugs to HSA may inhibit the attack of hydroxy ion and/or water molecule and thus retard the epimerization and the racemization. HSA accelerated the epimerization of carbenicillin, which is charged at the pH studied. Ion-ion and ion-dipole interactions between carbenicillin and HSA activate the carbenicillin molecule favorable for the attack of hydroxy ion and/or water molecule. The hydrolysis rates of ethiazide, carbenicillin and oxazepam acetate were increased by the addition of HSA. The hydrolysis rate of d-oxazepam acetate enantiomer bound to HSA was twice that of the l-enantiomer, which suggests that the esterase-like activity of HSA is enantioselective. Differences in the binding affinities of the drug's enantiomers to HSA may account for the selectivity.     

Site-specific cleavage of glycated human serum albumin in the presence of iron.

Nonenzymatically glycated human serum albumin was incubated with ferric ion at 37 degrees C in 0.2 M phosphate buffer, pH 7.4, up to 30 days. In the incubation mixture, amino acids, tyrosine and phenylalanine, were detected, suggesting site specific cleavage of glycated human serum albumin.     

Kinetic and thermodynamic study of the behaviour of diphenylcarbazide in aqueous solution with pH

In order to find out about the chemical behaviour of the diphenylcarbazide (DPCI) in water, kinetic and thermodynamic studies were carried out using UV-Vis spectrophotometry within the 0-12 pH range. The kinetic parameters and acidity constant were then determined, and the results show that a pseudo first order reaction rate was observed during hydroxilation, while the reaction rate was also observed to increase with the pH. The acidity constant obtained with the aid of SQUAD was pK(a)=10.718+/-0.027.     

Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent

In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation.     

Study on the thermodynamic characteristics between fluoroquinolone and bovine serum albumin

The binding reactions of the fluoroquinolone with bovine serum albumin (BSA) were investigated by microcalorimetry. The thermodynamic parameters were measured with the help of spectroscopy in a Tris–HCl buffer solution (pH 7.0, made isotonic with sodium chloride) at T = 298 K. Microcalorimetric measurements show that the molar change of enthalpy Δ_rH_m is insignificant for the reaction, which may suggest that the interaction is governed mainly by entropy, and the interaction between the protein and the drugs is stronger. The results also reveal an entropy–enthalpy compensation relationship of the interaction.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Kinetic relationships of hexachlorocyclotriphosphazatriene hydrolysis in aqueous acetonitrile in the presence of triethylamine

The kinetic and thermodynamic relationships of hexachlorocyclophosphazatriene hydrolysis in aqueous acetonitrile in the presence of triethylamine were determined. The hydrolysis mechanism involving base catalysis with triethylamine is described. The halogen substitution in hexachlorocyclotriphosphazatriene occurs by the mechanism of bimolecular nucleophilic substitution.     

Kinetic study of Berthelot reaction steps in the absence and presence of coupling reagents

Several reaction steps in the Berthelot reaction for the determination of ammonia have been separately studied. A reaction order of two has been confirmed for the reaction between HOCl and NH(3). The rate constant for this reaction has been determined to be 3.2 x 10(6)l.mole(-1).sec(-1). The first evidence for the formation of benzoquinonechlorimine is presented. Pentacyanoferrate coupling reagents which accelerate the production of indophenol have been found to operate on the reaction between NH(2)Cl and phenol. The rate constant for the final step of the reaction sequence has been determined to be 5.3 x 10(-3)l.mole(-1).sec(-1). A reaction between chlorimine and pentacyanoferrate compounds has been found to be responsible for the formation of a green product in the presence of excess of coupling reagent.     

Adsorption and interaction of fibronectin and human serum albumin at the liquid-liquid interface

The goal of this work was to investigate the dynamics of human plasma fibronectin (HFN) at the oil-water interface and to characterize its interactions with human serum albumin (HSA) by total internal reflection fluorescence microscopy (TIRFM). Among key results, we observed that fibronectin adsorption at the oil-water interface is rapid and essentially irreversible, even over short time scales. This may be due to the highly flexible nature of the protein, which allows its various domains to quickly attain energetically favorable conformations. On the other hand, HSA adsorption at the oil-water interface is relatively reversible at short times, and the protein is readily displaced by fibronectin even after HSA has been adsorbed at the interface for as long as 2 h. At longer adsorption times, HSA is able to more effectively resist complete displacement by fibronectin, although we observed significant fibronectin adsorption even under those conditions. Displacement of adsorbed fibronectin by HSA was negligible under all conditions. Fibronectin also adsorbs preferentially from a mixture of HFN and HSA, even when the concentration of HSA is substantially higher. This study is relevant to such emerging research thrusts as the development of biomimetic interfaces for a variety of applications, where there is a clear need for better understanding of the effects of interfacial competition, adsorption time scales, and extent of adsorption irreversibility on interfacial dynamics.     

Binding of glycyrrhizin to human serum and human serum albumin

The binding of glycyrrhizin (GLZ) to human serum and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in both human serum and HSA. The association constants (K) for the specific bindings were very similar: 1.31 x 10(5) M-1 in human serum and 3.87 x 10(5) M-1 in HSA. The number of binding sites (n) and the linear binding coefficient (phi) in HSA were 1.95 and 3.09 x 10(3) M-1, respectively. When the human serum protein concentration was assumed to be 4.2% (equal to the measured serum albumin concentration), n in human serum was 3.09, which is similar to the n value in HSA, and phi in human serum was 0.71 x 10(3) M-1, which is reasonably close to that for HSA. The binding pattern of GLZ with human serum protein on Sephadex G-200 column chromatography showed that GLZ binds to only the albumin fraction. It was concluded that the GLZ-binding sites in human serum exist mainly on albumin and GLZ binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.     

CE determination of quinolones in the presence of bovine serum albumin

This article describes the influence of bovine serum albumin (BSA) as an additive on the capillary electrophoresis–potential gradient determination of five quinolones, enoxacin, norfloxacin, ofloxacin, fleroxacin, and pazufloxacin. With 10 mg/L of BSA present in the buffer of 30 mM Tris and 3 mM phosphoric acid at pH 9, the detection limits of the five quinolones were in the range of 0.24–0.68 mg/L, i. e. 5.8–16.5‐fold lower than those obtained with the buffer devoid of BSA, and the analysis time was shortened. We suggest that the inner wall‐adsorbed BSA suppresses the adsorption of quinolones and simultaneously enhances the electroosmotic flow rate. Our experiments indicated that adopting the potential gradient detection technique could eliminate the interference of the UV‐active proteins on the detection of quinolones that would occur with conventional optical detection, and therefore offer high detection sensitivity. As a demonstration, the method was applied to the determination of QNs in fortified chicken muscle sample with satisfactory results.