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Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body 总被引:1,自引:0,他引:1
The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Motivated by the problem of microbial deposition, a dynamic model is developed for the attachment of a Brownian particle to a surface mediated by colloidal forces as well as macromolecular bridging. The model predicts the attachment probability of the particle to the surface based upon the free energy as a function of fluctuating bond number and separation distance from the surface. From this model, the mean first-passage time approach is used to predict the mean time required for the particle moving from the unattached state to the attached state based on the properties of the binding macromolecules. This approach provides an analytical approximation for mean transition time from the secondary energy minimum as well as the attachment rate constant for the general case where neither binding nor particle diffusion are necessarily rate-limiting. 相似文献
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Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation. 相似文献
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Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins’ respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field. 相似文献
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Birth of the macromolecule 总被引:1,自引:0,他引:1
Eisenberg H 《Biophysical chemistry》1996,59(3):247-257
The science of chemistry has made considerable advances over the last few hundred years in the characterization of "small" molecules which can be purified and studied by melting, distillation, crystallization and solubility in various liquids. When the study of "large" natural and biological molecules, limited in these properties, rose in significance at the turn of the century, it was first attempted to explain their properties by the concepts of colloid chemistry of aggregation and complex formation. The struggle for the acceptance of the concept of the natural or biological covalently bonded macromolecule, as recalled by Herman Mark, is one of the interesting chapters in recent science history. A specific phase in the establishment of the macromolecular concept centered around the development by The Svedberg of the analytical ultracentrifuge, a versatile tool of highly practical and profound thermodynamic significance. 相似文献
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《生物化学与生物物理学报:生物膜》2018,1860(4):878-886
Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. 相似文献
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The electron density distribution in deoxycytidine-5'-monophosphate (5'-dCMP) molecule and dianion has been studied by the method of CNDO/2. The comparison between the results of calculation for the neutral molecule and the data obtained by Pearlman and Kim shows that there is a linear correlation between the atomic charges calculated using quantum chemistry and those derived from X-ray results. However, partial charges for the deoxyribose fragment are correlated in a nonlinear manner. The influence of the protons added to the cytosine and phosphate residues on the atomic charges and bond orders of deoxy-cytidine-5'-monophosphate has been analyzed here. The conclusion has been drawn that the semiempirical quantum-chemical CNDO/2 technique is applicable to the mononucleotide studies. 相似文献
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We formulate the proper statistical mechanics to describe the stretching of a macromolecule under a force provided by the cantilever of an Atomic Force Microscope. In the limit of a soft cantilever, the generalized ensemble of the coupled molecule-cantilever system reduces to the Gibbs ensemble for an isolated molecule subject to a constant force in which the extension is fluctuating. For a stiff cantilever, one obtains the Helmholtz ensemble for an isolated molecule held at a fixed extension with the force fluctuating. Numerical examples and predictions for experiments with cantilevers of differing stiffness are given for short and long chains of poly (ethylene glycol), based on parameter-free ab initio calculations. 相似文献
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Amanda C. Crisostomo Loan Dang Jyothi L. Digambaranath Andrea C. Klaver David A. Loeffler Jeremiah J. Payne Lynnae M. Smith Adam L. Yokom John M. Finke 《Analytical biochemistry》2015
Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease. 相似文献
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We investigate the time-dependent solution of a set of equations used to approximate the binding of flexible polymers to the surface of suspended targets. The equations account, for the possibility that the first adsorption step can be a rate limiting step. Rate constants for other steps of adsorption and desorption are proportional to the number of free and occupied polymer binding sites, respectively. 相似文献
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The class C beta-lactamase of Enterobacter cloacae P99 is known to catalyze the hydrolysis of certain acyclic (thio)esters. Previous experiments have employed thioglycolate, m-hydroxybenzoate, and phenylphosphate leaving groups. The relative effectiveness of these leaving groups has now been quantitatively assessed by employment of a series of compounds with common acyl groups, and found to rank in the order phenylphosphate >m-hydroxybenzoate >thioglycolate. Structural models suggest that these leaving groups interact during acylation principally with Tyr 150, Lys 315, and Thr 316 of the beta-lactamase active site. The positions of the leaving group carboxylates in these models is compared with those in published crystal structures of complexes of class C beta-lactamases with beta-lactams. The particular effectiveness of the acyl phosphate indicates the positions of two oxyanions that strongly interact with the active site. This information should be useful in the design of inhibitors of class C beta-lactamases. 相似文献
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Diacovich L Peirú S Kurth D Rodríguez E Podestá F Khosla C Gramajo H 《The Journal of biological chemistry》2002,277(34):31228-31236
Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases. 相似文献
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Muñoz-López A Sotos-Lomas A Arribas E Masia-Perez J Garcia-Molina F García-Moreno M Varon R 《Journal of enzyme inhibition and medicinal chemistry》2007,22(2):147-155
Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. 相似文献
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A mathematical model of electron excitation energy transport between molecular probes sorbed on the polymeric chain in solution was proposed. The kinetics of the process was described in terms of the conception of stochastic changes in macromolecule conformation. The results of computer simulation and the analytical expressions obtained in the framework of the perturbation theory for the cases of low transfer rate and/or fast conformation motion of the macrochain are presented. A channel of nonlinear deactivation as a result of binary annihilation of closely-spaced excited centers was considered. Expressions for the effective rate of mutual quenching and delayed annihilation fluorescence of the probe were obtained. Time dependencies of typical luminescent signals and parameteric curves of relative fluorescence quantum yield are presented. 相似文献