The binding of pefloxacin mesylate （PFLX） to bovine lactoferrin （BLf） and human serum albumin （HSA） in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant ...The binding of pefloxacin mesylate （PFLX） to bovine lactoferrin （BLf） and human serum albumin （HSA） in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Forster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.展开更多
Characterizing how chemical compounds binding to serum albumin is essential in evaluating drug candidates and is the focus of this study. A surface plasmon resonance biosenser developed in this laboratory was used to ...Characterizing how chemical compounds binding to serum albumin is essential in evaluating drug candidates and is the focus of this study. A surface plasmon resonance biosenser developed in this laboratory was used to determine the binding constants of antibiotics with serum albumin. The binding constants of five antibiotics（asithromycin, spectinomycin, gentamycin, metacycline and kanamycin） with serum albumins were obtained.展开更多
A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include...A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include drug candidates, performance enhancing drugs and toxic nerve agents that also interact quite strongly with HSA. Endogenous ligands are natural circulating compounds whose abundance corresponds to normal hemostasis or elevated levels that could be disease-specific molecular biomarkers. Melting curves of plasma solutions measured by differential scanning calorimetry produce “so-called” plasma thermograms that are physical signatures of the plasma solution. Patterns displayed by thermograms can be sensitive indicators of the presence of abnormal levels of exogenous and endogenous ligand components. Effects of ligand interactions on thermodynamic stability of proteins in plasma that they bind, primarily HSA, manifest on the plasma thermogram. The capture strategy is demonstrated for HSA binding in plasma of four “ideal” ligands of different types. The particular ligands were naproxen, bromocresol green, short double stranded and single strand DNA. Thermogram shapes and features were sensitive to the presence of ligands as thermograms of mixtures of plasma and HSA with these ligands were significantly different than thermograms of plasma or HSA alone. These results demonstrated directly that significant perturbations of plasma thermograms corresponded to ligand interactions with HSA in plasma.展开更多
Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). ...Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.展开更多
基金Project (No. 20173050) supported by the National Natural ScienceFoundation of China
文摘The binding of pefloxacin mesylate （PFLX） to bovine lactoferrin （BLf） and human serum albumin （HSA） in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Forster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.
文摘Characterizing how chemical compounds binding to serum albumin is essential in evaluating drug candidates and is the focus of this study. A surface plasmon resonance biosenser developed in this laboratory was used to determine the binding constants of antibiotics with serum albumin. The binding constants of five antibiotics（asithromycin, spectinomycin, gentamycin, metacycline and kanamycin） with serum albumins were obtained.
文摘A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include drug candidates, performance enhancing drugs and toxic nerve agents that also interact quite strongly with HSA. Endogenous ligands are natural circulating compounds whose abundance corresponds to normal hemostasis or elevated levels that could be disease-specific molecular biomarkers. Melting curves of plasma solutions measured by differential scanning calorimetry produce “so-called” plasma thermograms that are physical signatures of the plasma solution. Patterns displayed by thermograms can be sensitive indicators of the presence of abnormal levels of exogenous and endogenous ligand components. Effects of ligand interactions on thermodynamic stability of proteins in plasma that they bind, primarily HSA, manifest on the plasma thermogram. The capture strategy is demonstrated for HSA binding in plasma of four “ideal” ligands of different types. The particular ligands were naproxen, bromocresol green, short double stranded and single strand DNA. Thermogram shapes and features were sensitive to the presence of ligands as thermograms of mixtures of plasma and HSA with these ligands were significantly different than thermograms of plasma or HSA alone. These results demonstrated directly that significant perturbations of plasma thermograms corresponded to ligand interactions with HSA in plasma.
文摘Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.
