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PubMed1982

Carbon monoxide binding kinetics in "capped" porphyrin compounds.

The rate constants for CO binding to the five-coordinate ferrous iron complexes of 5,10,15,20-[pyromellitoyl(tetrakis-o-oxyoxyphenyl)]porphyrin and 5,10,15,20-[pyromellitoyl(tetrakis-o-oxypropoxyphenyl)]porphyrin have been measured and compared with the corresponding rate constants for other hemes and hemoproteins. The second-order rate constant is independent of cap size and is comparable to that of high-affinity state hemoglobin (k5 approximately 4 X 10(6) M-1s-1). Therefore, these capped porphyrins provide no steric hindrance to CO binding. In addition, a kinetic scheme involving an unusual seven-coordinate porphyrin species is described.

Carbon Monoxide↗
PubMed1989

Binding kinetics of tetrachloro-1,2-diaminocyclohexaneplatinum (IV) (tetraplatin) and cis-diamminedichloroplatinum (II) at 37 degrees C with human plasma proteins and with bovine serum albumin. Does aquation precede protein binding?

Experiments were conducted at 37 degrees C to study the kinetics of (a) binding of cis-diamminedichloroplatinum (II) (CDDP) and of a racemic mixture of d- and l-isomers of trans-tetrachloro-1,2-diaminocyclohexaneplatinum (IV) [or tetraplatin (TP)] to protein [human plasma proteins or bovine serum albumin (BSA)]; (b) aquation (acid hydrolysis) of CDDP and of TP; and (c) binding of charged (aquated) CDDP species to BSA. The experiments were performed at clinically relevant concentrations for CDDP, so that the proportional concentrations of platinum complexes relative to the concentrations of other chemical species in blood plasma were similar to those obtaining in the clinical use of the drug. "Free" (unbound) platinum complexes were separated from the protein-bound complexes were separated from the protein-bound complexes by gel filtration chromatography. By use of ion-exchange chromatography, charged platinum species were separated from the uncharged species and free charged platinum species of CDDP were separated from those bound to BSA. Platinum in various fractions was quantitated by atomic absorption spectrophotometry with electrothermal atomization; proteins were quantitated by te Bradford method with Coomassie blue dye. The kinetic data obtained by the application of these methods for CDDP are in good agreement with those obtained by other methods, e.g., binding rates based on separations by centrigugal ultrafiltration. The overall protein-binding reaction of CDDP was consistent with a binding process comprising two consecutive first-order reaction steps: the rate-controlling aquation reaction [half-life (t 1/2), approximately 2 hr] followed by a more rapid binding reaction of the charged (aquated) CDDP species to the protein (t 1/2, approximately 23 min). However, the results for TP indicated that prior aquation was not required for protein binding, and we could surmise that binding of TP to protein proceeds via a direct nucleophilic attack. An unexpected finding was the marked, reproducible difference in rates of aquation between the two lots of TP that we used; this finding suggests the need for cautions evaluation of pharmacokinetic data describing the behavior of TP.

Antineoplastic Agents↗
PubMed1994

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes.

Ankyrins↗
PubMed1996

An analysis of antigen-antibody binding kinetics for biosensor applications utilized as a model system: influence of non-specific binding.

The influence of non-specific binding on the specific binding of antigen in solution to antibody immobilized on a biosensor surface is presented for first-, one and a half-, second-, and other order reactions occurring under external diffusion-limited conditions. Both single-step and dual-step binding of antigen to antibody is considered. For a half-order reaction the value of the ratio of non-specific binding to specific binding (alpha) does not affect the rate of specific binding since a single curve represents the binding curve for alpha = 0 to 0.5. An increase in the alpha value leads to a decrease in the rate of binding and in the amount of antigen bound specifically to the antibody on the surface for first-, one and a half-, and second-order reactions. Also, an increase in the reaction order increases the sensitivity of the specific binding to the alpha value. An increase in the antigen concentration in solution increases the amount and the rate of specific binding for first-, one and a half-, and second-order reactions. The introduction of non-specific binding leads to complexities in the specific binding of the antigen for the one and half- and second-order reactions as the antibody concentration on the surface is varied. When non-specific binding is present there is an optimum value of the antibody concentration on the surface that yields the maximum rate and amount of antigen specifically bound for one and a half- and second-order reactions. Though this optimum amount of antibody immobilized on the surface is the same for the one and a half- and second-order reactions, it is different for different alpha values. No such complexities are observed for first-order reactions. The inclusion of non-specific binding in the analysis provides a more realistic picture of the binding of the antigen in solution to the antibody immobilized on the surface. The figures that show the numerically calculated binding rates for different orders when non-specific binding is present, represent the most useful part of the analysis for readers who are interested in constructing biosensors, and should assist in the control and manipulation of these interactions at the surface. These curves can be used to interpret a far from ideal binding of antigen to an immobilized antibody surface or vice versa. More-or-less all of the analysis should also be applicable to analytical systems that would not be classified as biosensors (that is, immuno and receptor assays).

Antibodies↗
PubMed1989

Effect of the distal histidine modification (Cyanation) of myoglobin on the ligand binding kinetics and the heme environmental structures.

The kinetics of carbon monoxide (CO) binding to myoglobin (Mb) modified at the distal histidine (His) by cyanogen bromide (BrCN) has been studied. The CO association and dissociation rates of BrCN-modified Mb were obtained as 1.8 x 10(3) M-1 s-1 and 0.13 s-1, respectively (20 degrees C and pH 7.0). Thermodynamic parameters were obtained as well. These values are notable, compared with those for other hemoproteins, the slowest association and the fastest dissociation rates among various hemoproteins examined so far. On the basis of the available structural data obtained from the absorption, 1H NMR, and IR spectral measurements, these unique kinetic and thermodynamic properties were reasonably explained in terms of the steric restriction at the modified distal side.

Animals↗
PubMed1998

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.

Amino Acid Sequence↗
PubMed1998

Construction and binding kinetics of a soluble granulocyte-macrophage colony-stimulating factor receptor alpha-chain-Fc fusion protein.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha). We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha. The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 x 10(-2)/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.

Animals↗
PubMed1995

Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor.

We have used a resonant mirror biosensor to determine the kinetics of binding of four antibodies, and their Fab' fragments, to their antigen, the plant-derived ribosome-inactivating protein (RIP) saporin. The analysis of the affinity of the antibodies was in reasonable agreement with values obtained by conventional techniques. However, the kinetic data showed that all four antibodies have a high dissociation rate constant (kdiss). These antibodies have been used in the construction of bispecific antibodies used to deliver saporin to tumour cells, and it is highly probable that the in vivo efficacy of the bispecific antibodies is limited by the high rate of dissociation of antibody-toxin complexes.

Allergy and Immunology↗
PubMed1989

Statistical comparison of ligand-binding kinetics.

We describe the application of generalized linear model methodology to the problem of testing differences among ligand-receptor interactions, and show that the method is analogous to weighted least squares regression methodology and F tests familiar to many investigators. The method accommodates incomplete block designs so that one can obtain kinetic parameter estimates directly comparable among samples analysed on incompletely overlapping sets of experimental runs. We demonstrate the method with data that compare saturation kinetics for a single penicillin-binding protein in isogenic ampicillin susceptible and resistant bacteria.

Ampicillin Resistance↗
PubMed2000

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen.

The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor. Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K(i)), and viral replication (EC(50)) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K(i) and EC(50) values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.

Biosensing Techniques↗
PubMed1995

Kinetic, binding, and NMR studies of perdeuterated yeast phosphoglycerate kinase and its interactions with substrates.

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 A apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme-bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme-bound dATP is anti with O4'/C2' endo ribose pucker and trans, gauche about the C4'-C5' bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

Binding Sites↗
PubMed1980

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model.

Saturation analysis of equilibrium binding of iodinated thyrotropin (125I-TSH) to normal human thyroid preparations yielded linear Scatchard plots under non-physiological conditions of pH 6.0 or 20 mM Tris/acetate buffer, pH 7.4. The apparent equilibrium dissociation constant of this binding was approximately 10(-8) M. By contrast, nonlinear plots were obtained under standard conditions of pH 7.4 and 40 mM Tris/acetate buffer. Resolution of the components of these curves by computer analysis revealed the presence of at least two classes of binding sites, one of which is of a low capacity and high affinity (approximately 10(-10) M) consistent with receptor binding. The other component is of a high capacity and lower affinity. Binding to non-target tissues of muscle, parathyroid, mammary carcinoma, and placenta was only demonstrable at pH 6.0 or in 20 mM Tris/acetate buffer, pH 7.4, yielding linear Scatchard plots with similar binding affinity (approximately 10(-8)M) to normal thyroid but much reduced capacity. Preincubation of thyroid tissue at 50 degrees C resulted in an apparent selective loss of the high affinity component of binding measured under standard conditions. Kinetic experiments on the dissociation of bound 125I-TSH were undertaken to determine whether the non-linearity of Scatchard plots was due to two or more classes of binding sites or negative cooperativity. It was found that the experimental determinant that is presently ascribed to a negative cooperativity phenomenon regulating receptor affinity (i.e. an enhanced dilution-induced dissociation rate in the presence of excess native hormone), although apparently hormone-specific, was demonstrated under nonphysiological binding conditions and in non-target tissue. Significantly, the phenomenon was found under conditions of pH 6.0 or 20 mM Tris where a linear Scatchard plot was obtained. The evidence thus suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site (maybe gangliosides).

Breast Neoplasms↗
PubMed1991

In vivo binding of nimodipine in the brain: II. Binding kinetics in focal cerebral ischemia.

We report the binding characteristics of [3H]nimodipine to normal and ischemic brain in vivo. We used the 1,4-dihydropyridine, nimodipine, to label the L-type voltage-sensitive calcium channel in focal cerebral ischemia after occlusion of both the middle cerebral and ipsilateral common carotid arteries in rats. Varying concentrations of [3H]nimodipine were infused 3.5 h after the onset of ischemia and circulated for 30 min before the brain was obtained for autoradiography and determination of regional nimodipine content. In separate sets of experiments, the metabolites of nimodipine were determined and the conditions for equilibrium of nimodipine distribution were established. Increased nimodipine uptake was observed in ischemic regions. This increased binding was saturable and specific with an affinity constant, KD, of 0.45 nM and a maximal regional binding capacity, Bmax, ranging from 3.1 to 10.9 pmol/g. Only binding to ischemic tissue was specific and saturable whereas that in nonischemic tissue was nonspecific. In vivo binding of nimodipine may be used to identify cell membrane depolarization and calcium channel activation in focal cerebral ischemia.

Animals↗
PubMed2006

Multiple receptor states are required to describe both kinetic binding and activation of neutrophils via N-formyl peptide receptor ligands.

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).

Actins↗
PubMed1994

Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation.

Nitric oxide (NO) is synthesized from L-arginine by different NO synthase isozymes, which are inhibited by the substrate analogs NG-methyl- and NG-nitro-L-arginine. We studied binding of 3H-labeled NG-nitro-L-arginine to purified brain NO synthase and compared the data with results obtained in enzyme kinetic experiments. Binding data revealed a single binding site for NG-nitro-L-[3H]arginine (KD = 0.17 microM). Binding was competitively antagonized by L-arginine (KI = 2.9 microM). The half-time of dissociation was remarkably slow (9.4 min) and closely correlated with the time necessary for surmounting NO synthase inhibition by dilution. Although an apparently less potent inhibitor, NG-methyl-L-arginine exhibited the same affinity for brain NO synthase as the nitro derivative (KI = 0.17 microM), and in initial rate experiments, almost equal KI values were obtained for NG-methyl-L-arginine (0.61 microM) and NG-nitro-L-arginine (0.53 microM). However, after prolonged incubation periods, NG-nitro-L-arginine induced a rapid inactivation of the enzyme, whereas the methyl derivative turned out to be a substrate of NO synthase, which was slowly converted into stoichiometric amounts of NO and L-citrulline.

Amino Acid Oxidoreductases↗
PubMed1999

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein.

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.

2-Naphthylamine↗