共查询到20条相似文献,搜索用时 15 毫秒
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K.S. Joseph David S. Hage 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(19):1590-1598
Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (±0.2) × 105 and 3.5 (±3.0) × 102 M−1 for acetohexamide and values of 8.7 (±0.6) × 104 and 8.1 (±1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (±0.1) × 105 and 4.3 (±0.3) × 104 M−1, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (±0.2) × 104 and 5.3 (±0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions. 相似文献
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Meina Li Lei Lei Linghan Jia Xiaomei Ling Jianmei Zhang Yiran Zhao KeWei Wang 《Analytical biochemistry》2014
The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 106 L mol–1 and (5.26 ± 0.71) × 106 L mol–1, respectively. In addition, in the presence of calcium (10 μmol L–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 107 L mol–1. In addition, the binding constant of KChIP4a with Ca2+ was (7.1 ± 1.5) × 107 L mol–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca2+, and the integrity of the molecular structure of KChIP4a was important for Ca2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming. 相似文献
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Abdol-Khalegh Bordbar Fakhrossadat Mohammadi Chris Orvig 《Journal of inorganic biochemistry》2009,103(4):643-647
Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO4), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO4 to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO4 were used to determine binding constants; at 25 °C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (±1) × 105 and 1.8 (±0.7) × 105 M−1, respectively. The corresponding values for VOSO4 were 1.7 (±0.3) × 105 and 7 (±2) × 104 M−1. The results show that the vanadium species initially presented as either BMOV or VOSO4 had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate () was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO3) and hTf. 相似文献
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Irina V. Nesmelova Elena Ermakova Mabel Pang Laura Lagartera Dolores Solís Linda G. Baum 《Journal of molecular biology》2010,397(5):1209-5276
Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K1 = 21 ± 6 × 103 M− 1) than the second (K2 = 4 ± 2 × 103 M− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K1 = 20 ± 10 × 103 M− 1 and K2 = 1.67 ± 0.07 × 103 M− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general. 相似文献
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Cannon MJ Papalia GA Navratilova I Fisher RJ Roberts LR Worthy KM Stephen AG Marchesini GR Collins EJ Casper D Qiu H Satpaev D Liparoto SF Rice DA Gorshkova II Darling RJ Bennett DB Sekar M Hommema E Liang AM Day ES Inman J Karlicek SM Ullrich SJ Hodges D Chu T Sullivan E Simpson J Rafique A Luginbühl B Westin SN Bynum M Cachia P Li YJ Kao D Neurauter A Wong M Swanson M Myszka DG 《Analytical biochemistry》2004,330(1):98-113
To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 106 M−1 s−1 and 6.7 ± 2.5 × 10−2 s−1, respectively, which corresponded to an average equilibrium dissociation constant (KD) of 2.6 ± 1.4 × 10−8 M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions. 相似文献
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Paolo Ascenzi Alessandra di Masi Massimo Coletta 《Biochemical and biophysical research communications》2009,387(1):83-189
Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (koff) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The koff value increases from (1.4 ± 0.2) × 10−4 s−1, in the absence of the drug, to (9.5 ± 1.2) × 10−3 s−1, in the presence of 1.0 × 10−2 M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of koff on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K1 = (3.1 ± 0.4) × 10−7 M, K2 = (1.7 ± 0.2) × 10−4 M, and K3 = (2.2 ± 0.2) × 10−3 M) were determined. The K3 value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10−3 M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics. 相似文献
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Ilona Fitos Júlia VisyFerenc Zsila György MádyMiklós Simonyi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α1-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of Ka = 1.7(± 0.2) × 106 M− 1 and n = 0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nKa ≈ 3 × 104 M− 1) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP. 相似文献
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Shmykov AY Filippov VN Foteeva LS Keppler BK Timerbaev AR 《Analytical biochemistry》2008,379(2):216-218
The performance of capillary electrophoresis (CE) operating with a sulfonated capillary for the separation of protein adducts of anticancer ruthenium(III)-based drugs was evaluated. The coated capillary was shown to yield improved resolution of albumin- and transferrin-bound species of ruthenium compared with that attained with the bare fused-silica capillary. The coating also showed an increased reproducibility of migration times and peak areas and allowed reasonably high efficiency separation of analytes (up to 1300 theoretical plates per meter), which display high affinity toward a fused-silica surface. In addition, due to rather high electroosmotic flow (EOF, > 45 × 10−5 cm2 V−1 s−1) in the coated capillary, it enabled fast counter-EOF monitoring of albumin and transferrin adducts. This benefit, together with requiring only a short flush with the background electrolyte to have migration times reproducible (at < 1.5% relative standard deviation), makes this wall-modified capillary holding promise for CE examination of fast reactions such as those accompanying protein-drug interactions and biotransformations associated with drug delivery via protein binding. 相似文献
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Essemine J Hasni I Carpentier R Thomas TJ Tajmir-Riahi HA 《International journal of biological macromolecules》2011,49(2):201-209
The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of Kspm-β-LG = 3.2(±0.6) × 104 M−1, Kspmd-β-LG = 1.8(±0.5) × 104 M−1, KBE-333-β-LG = 5.8(±0.3) × 104 M−1 and KBE-3333-β-LG = 6.2(±0.05) × 104 M−1. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding. 相似文献
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Xinfeng Zhao Yefei Nan Chaoni Xiao Jianbin Zheng Xiaohui Zheng Yinmao Wei Youyi Zhang 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(22):2029-2034
A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β2-adrenoceptor (β2-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β2-AR affinity column. The retention compounds eluted from the β2-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β2-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β2-AR. Their association constants were (2.28 ± 0.11) × 104/M and (3.00 ± 0.10) × 104/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β2-AR, and the corresponding association constants were (2.20 ± 0.09) × 10−4/M and (6.78 ± 0.001) × 105/M. 相似文献
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The present study reports a detailed investigation with the interaction of [Cr(phen)2(dppz)]3+ with serum albumins, the key protein for the transport of drugs in the blood plasma, which allows us to understand further the role of [Cr(phen)2(dppz)]3+ as sensitizer in Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)2(dppz)]3+, (dppz = dipyridophenazine and phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly with bovine and human serum albumins (BSA and HSA) with an intrinsic binding constants, Kb, of (1.7 ± 0.3) × 105 M− 1 and (2.2 ± 0.3) × 105 M− 1 at 295 K, respectively. The interactions of serum albumins with [Cr(phen)2(dppz)]3+ were assessed employing fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy. The serum albumins-[Cr(phen)2(dppz)]3+ interactions caused conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the albumin (BSA or HSA) bound to the Cr(III) complex decreased, suggesting that perturbation around the Trp 214 residue took place. The analysis of the thermodynamic parameters ΔG, ΔH, ΔS indicated that the hydrophobic interactions played a major role in both BSA-Cr(III) and HSA-Cr(III) association processes. The binding distances and transfer efficiencies for BSA-Cr(III) and HSA-Cr(III) binding reactions were calculated according to the Föster theory of non-radiation energy transfer. All these experimental results suggests that [Cr(phen)2(dppz)]3+ binds to serum albumins, by which these proteins could act as carriers of this complex for further applications in PDT. 相似文献
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Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate protoporphyrinogen IX (Km of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min-1 μg−1, respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μМ. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study. 相似文献
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Magdalena Soko?owska Jaros?aw Poznański Wojciech Bal 《Journal of inorganic biochemistry》2009,103(7):1005-1013
Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 104 M−1, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed. 相似文献
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Escherichia coli HUαβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 106 M− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 105 M− 1 for the 10 bp mode, 3.5 ± 1.4 × 104 M− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. 相似文献
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Calmodulin (CaM) is a Ca2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the Kd for the interaction of CaM with the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca2+ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant Kd in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a Kd value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (Kd = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca2+ to the binding sites in CaM. 相似文献
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McKnight RE Reisenauer E Pintado MV Polasani SR Dixon DW 《Bioorganic & medicinal chemistry letters》2011,21(14):4288-4291
A combination of isothermal titration calorimetry (ITC), topoisomerase I DNA unwinding assays, and ethidium bromide displacement studies were employed to investigate the binding of a homologous series of naphthalene diimides (NDI) to DNA. Our results suggest that the nature of the substituent plays a significant role in both the preferred binding mode and relative binding affinity of the compounds of this study. Only intercalative-type binding (K = 15 ± 3 × 106 M−1) was observed for the NDI with the smallest substituent (trimethyl-ethylamino), while larger members of the series (diethylmethyl-, dipropylmethyl- and dibutylmethyl-ethylamino substituents) adopted an additional binding mode of higher affinity (K1 = 31 − 78 × 106 M−1). 相似文献