Antibody-antigen interactions measured by surface plasmon resonance: global fitting of numerical integration algorithms

The interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sensograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BIAcore measurements. The difference in results obtained with solid-phase BIAcore and competition BIAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BIAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BIAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.     

Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding

A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates.     

Preparation and characterization of monoclonal antibodies against adenylate kinase

Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAMD8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 × 108 M-1 and 9.6 × 108 M-1, while their interactions to AK in solution are much weaker, with affinity constants of 7.0 × 104 M-1 and 3.9×106M-1, respectively. Thus, McAb3D3 and McAMD8 react preferentially to the immobilized AKs. Since pro-teins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAMD8 are directed against non-native AK.     

A generalized kinetic model for the study of microbial growth

The following general equation is proposed to represent the kinetics of microbial growth \documentclass{article}\pagestyle{empty}\begin{document}$$\phi (dR/dt) + \psi R + X = 0$$\end{document}, where phi and psi depend on several parameters of the fermenting system. The values of phi and psi were calculated based on results obtained in a batch lactic acid fermentation, a batch cultivation of yeast on diesel oil, and a continuous cultivation of yeast on sugarcane molasses.     

Pathways of cadmium influx in mammalian neurons

The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2(+)-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 microM Cd2(+). This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.     

Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 2. Stopped-flow studies with agonists and antagonists.

The kinetics of cholinergic ligand binding to membrane-bound acetylcholine receptor from Torpedo californica have been followed in a stopped-flow photometer, by using the fluorescent probe ethidium. The overall reaction amplitude, as a function of ligand concentration, can be fit to the law of mass action for both agonist and antagonists. All agonists show at least biphasic kinetics, and the concentration dependence of the kinetic parameters is fit by a common mechanism involving sequential binding of ligands with increasingly lower affinity. The receptor-ligand precomplexes isomerize to different noninterconvertible final complexes depending on the number of ligands bound. In contrast, the kinetics observed with antagonists cannot be fit to a common model. These kinetics are always much slower than those observed with agonists, and the relaxation rates depend only weakly on antagonist concentration.     

A new monoclonal anti-idiotypic catalytic antibody with a CPA-like activity.

IIF9D8, a new monoclonal anti-idiotypic catalytic antibody with a CPA esterase-like activity was elicited by ID11D7, the monoclonal competitive inhibitory antibody to CPA. The hydrolysis of hippuryl-DL-phenyllactic acid by McAb IIF9D8 follows the Michaelis-Menten kinetics. The Km value and kcat are 0.036 M and 0.598 min(-1), respectively, and the rate acceleration (kcat/kuncat) is 30500. Compared with the previous McAb 32C3 induced by polyclonal antibodies to CPA, McAb IIF9D8 shows higher catalytic efficiency The catalytic antibodies with the catalytic properties similar to natural enzymes could be obtained by this approach.     

Enhancement of Epstein-Barr virus/C3d receptor (EBV/C3dR or CR2) and nuclear p120 ribonucleoprotein phosphorylation by specific EBV/C3dR ligands in subcellular fractions of the human B lymphoma cell line, Raji

We present herein the first evidence that interaction of specific EBV/C3dR ligands, as human C3bi/C3d and anti-EBV/C3dR MoAb, with EBV/C3dR enhanced significantly, in a dose dependent process, phosphorylation of EBV/C3dR and p120 RNP present in subcellular fractions, as purified plasma membranes and nuclei, of the human B lymphoma cell line, Raji. The use of kinase effectors allowed to detect some of the kinases involved in these phosphorylations. Pp60src-like phosphotyrosine kinase and protein kinase C were involved in the phosphorylation of plasma membrane or nuclear EBV/C3dR. An additional calcium/calmodulin-dependent kinase was also involved in nuclear EBV/C3dR phosphorylation. P120 RNP phosphorylation was under the control of protein kinase C and of CaCl2/Calmodulin-dependent kinase but also of casein kinase II.     

Kinetic characterization of the pH-dependent oligomerization of R67 dihydrofolate reductase.

Protein-protein recognition results from the assembly of complementary surfaces on two molecules that form a stable, noncovalent, specific complex. Our interest was to describe kinetic aspects of the recognition in order to understand the subtle molecular mechanism of association. R67 dihydrofolate reductase (DHFR) provides an ideal model to investigate kinetic parameters of protein-protein association since it is a homotetramer resulting from the pH-dependent dimerization of homodimers. We took advantage of the presence of a tryptophan residue at the dimer-dimer interface to monitor pH-dependent oligomerization of R67 DHFR using stopped-flow fluorescence techniques. Except for pH near neutrality where dissociation exhibited biphasic kinetics, association and dissociation followed monophasic kinetics fitted on a two-state model. Apparent rate constants of association k(on) and dissociation k(off) were determined at various pHs and pointed to the key role of a histidine located at the dimer-dimer interface in the pH control of tetramerization. The values of the tetramer-dimer equilibrium dissociation constant were calculated from the ratio k(off) /k(on) and correlated well with those previously measured at equilibrium. The thermodynamic parameters and the activation energies of both the association and dissociation were determined and indicated that the association is enthalpy driven and suggested that the formation of four hydrogen bonds (one per monomer) is responsible for the thermodynamic stability of the tetramer. Detailed analysis of the biphasic kinetics led to an original model, in which protonation of the tetramer is the triggering event for the dissociation process while the association involves primarily the unprotonated dimers.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

Immobilization of Lipases on Clay, Celite 545, Diethylaminoethyl-, and Carboxymethyl-Sephadex and their Interesterification Activity

Lipases AK, PS (both from Pseudomonas sp), and AY (from Candida rugosa) were immobilized on clay, Celite 545, DEAE-Sephadex, or CM-Sephadex and used to synthesize structured lipids from tricaprylin and trilinolein. Lipases AK and AY immobilized on Celite 545 had the highest interesterification activity (0.11 and 0.35 units, respectively) while lipase PS had the highest activity (0.18 units) when immobilized on CM-Sephadex. During 72 h reaction, the highest product yields were obtained in Celite AK (224 µmol), and CM PS (290 µmol) after 48 h and Celite AY (501 µmol) after 24 h. Lipase AK immobilized on DEAE-Sephadex was the most stable for repeated (5 times) operation.     

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)相似文献   

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Rate of allosteric change in hemoglobin measured by modulated excitation using fluorescence detection.

We have measured the forward and reverse rates of the allosteric transition of hemoglobin A with three CO molecules bound by using modulated excitation coupled with fluorescence quenching of the DPG analogue, PTS (8-hydroxy-1,3,6 pyrene trisulfonic acid). This dye is observed to bind to the T state with significantly larger affinity than to the R state, and thus provides an unequivocal marker for the molecule's conformational change. The allosteric rates obtained with the fluorescent dye (pH 7.0, bis-Tris buffer) are (3.4 +/- 1.0) x 10(3)s-1 for the R to T transition and (2.1 +/- 0.5) x 10(4)s-1 for the T to R transition. This gives an equilibrium constant L3 of 0.16 +/- 0.06. These results provide good agreement with modulated difference spectra calibrated from model compounds, arguing that there is little if any difference in the kinetics observed by the heme spectra and the kinetics of the full subunit motion. The equilibrium constant between structures (L3) is smaller in the absence of phosphates than observed in phosphate buffer (0.33). However, the rates of the allosteric transition increase in the absence of phosphates as compared with the corresponding rates in phosphate buffer of 1.0 x 10(3)s-1 and 3.0 x 10(3)s-1. The effects of inorganic phosphates on the equilibrium can be separated from the effects on kinetics. We find that phosphates also affect the dynamic behavior of hemoglobin, and the presence of 0.15 M phosphate can be viewed as raising the transition state energy between R and T conformations by approximately 0.5 kcal/mol exclusive of the T state stabilization. Dissociation constants for the dye were measured to be 104 +/- 25 microM for unligated T state and 930 +/- 300 microM for the fully ligated R state. The best fit equilibrium constant (125 +/- 40 microM) for three ligands bound does not differ significantly from that measured without ligands bound. Incidental to the measurement technique is the determination of the rates of binding and release of the dye. The association rate for binding to the T state is large, (at least 4 x 10(9) M-1 s-1) and may be diffusion limited, while the association and dissociation rates for R state binding, while not determined with precision, are clearly much smaller, of the scale of 10(5) M-1 s-1 for association.     

Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids

As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.