Kinetic Binding Characteristics Research Articles

Measuring Antibody-Antigen Binding Kinetics Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species, and the binding event is visualized in real time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns not only concentration and affinity data but when used correctly can resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics

BackgroundResolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A3R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the Ki of eight different A3R antagonists, using CHO-K1 cells stably expressing the hA3R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants kon and koff, as well as the dedicated Kd of the A3R agonist [125I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds.ResultsTwo novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A3R antagonists, no influences on the experimental performance and the resulting affinity were investigated.ConclusionsBoth kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays.

The Heliothis virescens 170 kDa aminopeptidase functions as “Receptor A” by mediating specific Bacillus thuringiensis Cry1A δ-endotoxin binding and pore formation

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac δ-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with K D ranging from 41 to 95 nM and a lower affinity site with K D in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb + release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb + release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb + release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

Interaction analyses of Bacillus thuringiensis Cry1A toxins with two aminopeptidases from gypsy moth midgut brush border membranes

A 100 kDa aminopeptidase N isolated from Lymantria dispar (gypsy moth) larval midgut brush border membrane vesicles (BBMVs) has previously been reported to function as a surface binding protein for the entomocidal protein toxin CrylAc from Bacillus thuringiensis (Valaitis et al., 1995; Lee et al., 1996). Fractionation of detergent-solubilized, phosphatidylinositol-specific phospholipase C-digested BBMV membrane proteins by ion-exchange chromatography revealed two distinct peaks of aminopeptidase activity from which two proteins, APN-1 and APN-2, were purified. Western blot immunoanalysis revealed that the previously reported 100 kDa APN (APN-1 in this study) was antigenically distinct from the newly identified 105 kDa APN-2. Both ligand blots and CrylAc-Sepharose affinity chromatography revealed that only APN-1 was able to bind CrylAc. The narrow specificity and kinetic binding characteristics of APN-1 for CrylAc were determined using a surface plasmon resonance-based optical biosensor. APN-1 from the gypsy moth possessed a single CrylAc toxin-binding site and did not interact with either CrylAa or CrylAb. The association and dissociation rate constants of CrylAc and APN-1 were determined to be 7.2 × 10 4 Ms −1 and 2.3 × 10 −3s −1, respectively, with an apparent affinity constant of 3.2 × 10 −8M. Toxin binding to APN-1 was directly inhibited with N-acetylgalactosamine, suggesting that this aminosugar forms an integral part of the binding site. The absence of recognition of all Cry toxins by APN-2 suggests that either APN-2 recognizes an untested subclass of Cry toxins, or alternatively, not all APN molecules in larval midguts serve to function as toxin-binding proteins.

The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin.

Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.

Characteristics of inositol 1,4,5-trisphosphate binding to rat cerebellar and bovine adrenal cortical membranes: evidence for the heterogeneity of binding sites

The equilibrium and kinetic binding characteristics of D-inositol 1,4,5-trisphosphate (Ins(1.4.5)P 3) have been studied in membrane preparations of rat cerebellum and bovine adrenal cortex. Saturation analysis of isotopic dilution binding data demonstrated apparent K D values for Ins(1,4,5)P 3 binding of 23 ± 5 nM and 3.0 ± 1.3 nM for cerebellar and adrenal cortical preparations, respectively, with approximately 20-fold greater receptor density present in the cerebellar preparation (B max: 10.2 ± 2.5 pmol/mg protein). Kinetic analysis confirmed the equilibrium binding-derived K D value for cerebellum (K D: 39.9 nM), but revealed a second, very high affinity site (K D: 0.06 nM) to be present in adrenal cortex. The affinity differences between the investigated preparations was also observed with respect to the IC 50 values obtained for inhibition of specific [ 3H]Ins(1,4,5)P 3 binding by a number of inositol polyphosphate analogues including D-inositol 2,4,5-trisphosphate, DL-inositol 1,4,5-triphosphorothioate and L-Ins(1,4,5)P 3. In contrast, the Ins(1,4,5)P 3-receptor antagonist heparin displayed greater potency for the cerebellar (IC 50: 16.5 ± 6.2 μg · ml −1) compared to the adrenal cortical preparation (IC 50: 51.0 ± 6.1 μg · ml −1). The apparent differences between the Ins(1,4,5)P 3 receptors characterized in the two tissue preparations are discussed.

Interleukin-2 monoclonal antibody affinity adsorption The critical role of binding kinetics for optimal immunoadsorption

With the ready availability of monoclonal antibodies reactive with an extensive spectrum of antigens, immunoaffinity adsorption has become more widely applicable for protein purification. However, given several monoclonal antibodies reactive with the same antigen, most investigators have found that only a few antibodies are useful for solid-phase immunoaffinity antigen purification. Accordingly, in order to determine the parameters of monoclonal antibody-antigen binding most important for effective immunoaffinity adsorption, equilibrium and kinetic binding experiments were performed using radiolabeled interleukin-2 (IL-2) as antigen and four different IL-2-reactive monoclonal antibodies. The antibodies were found to differ primarily with respect to their kinetic binding characteristics; at 37°C IL-2 bound to two of these antibodies very rapidly, while it bound to the other two more slowly. When binding was performed at 4°C, the equilibrium dissociation constants for all of the antibodies decreased due to a more marked prolongation of the dissociation rate than the association rate. However, at 4°C the association rates of the two slow-reactive antibodies became retarded so markedly that efficient affinity adsorption did not occur. By comparison, for both of these antibodies, efficient removal of IL-2 could be obtained if adsorption was performed at 37°C, provided the column flow rate was adjusted according to the IL-2 association rate. Kinetic considerations also dictated IL-2 adsorption to mixtures of two or more monoclonal antibodies: IL-2 immunoadsorption correlated with the association rates of the individual antibodies, rather than the equilibrium binding constants. These results indicate that the most important parameter for efficient affinity adsorption is the association rate constant. In addition, the results obtained indicate that monoclonal antibodies may differ markedly as regards their kinetic binding characteristics, and that all antibodies can serve as effective immunoadsorbents, provided their antigen binding characteristics are known.

Binding characteristics of (+)-, (+/-)- and (-)-[125iodo] cyanopindolol to guinea-pig left ventricle membranes.

[125Iodo]cyanopindolol [(±)-ICYP], a potent and selective ligand for β-adrenoceptors, exhibited complex biphasic dissociation kinetics. Consequently, in receptor binding studies, the pure (+)- and (-)-enantiomers of ICYP were synthesised and their equilibrium and kinetic binding characteristics were investigated on a membrane preparation of guinea pig left ventricle containing almost only β1-adrenoceptors. All three ligands, i. e. (+)-, (-)- and (±)-ICYP, bind to β-adrenoceptors as assessed by competition experiments with different β-blocking agents; irrespective of the ligand used, the same dissociation constant was found for the competitor. In a first series of saturation binding experiments performed in a low concentration range of free ligand (0–250 pM), ICYP showed the following dissociation constants: K D=93, 9 and 23 pM, and number of binding sites: B max=40,128 and 124 fmoles/mg protein for (+)-, (-)-, and (±)-ICYP, respectively. Asexpected, (±)-ICYP showed the same B max as (-)-ICYP, whereas its K D was approximately two times higher than that of (-)-ICYP. Surprisingly, the B max of (+)-ICYP represented only ∼ 30% of the B max of (-)-ICYP. All three ligands bound apparently to a single class of binding sites. In dissociation experiments, the enantiomers of ICYP showed biphasic dissociation curves as observed for the racemic ligand. (+)-, (-)- and (±)-ICYP showed a rapidly dissociating (k -1=0.488, 0.047 and 0.049 min−1) and a slowly dissociating component (k -2=0.0205, 0.0033 and 0.0025 min−1). The ratio slow dissociating/fast dissociating component represented respectively for (+)-, (-)- and (±)-ICYP 40/60, 90/10 and 90/10. For all three ligands, the association rate constants were of the same order of magnitude (ca. 109 M−1 min−1), typical for a diffusion controlled reaction. In contrast to equilibrium binding studies, the existence of multiple receptor affinity sites was evident from the biphasic dissociation behaviour observed especially with the nonracemic ligands (+)-ICYP and (-)-ICYP. Simulation of theoretical saturation curves performed with the ratios of high versus low affinity sites and the K D-values suggested by kinetic analysis, indicated that the delineation into two affinity states might be visible in saturation experiments, under certain conditions. Therefore, equilibrium binding studies were repeated with an increased number of ligand concentrations covering a large concentration range of 0–800 pM. Simultaneous analysis of saturation curves from the same experiment using three different ligands, provided more accurate estimates of the ratio of high and low affinity sites, as well as the affinity constants of the ligand for each receptor affinity state, in good agreement with the results from kinetic analysis. The contribution of the (+)enantiomer in the binding of the racemic ligand under low receptor concentrations could be neglected since dissociation characteristics of (±)- and (-)-ICYP were identical. A model that explains the biphasic dissociation of (±)-ICYP by differential binding of both enantiomers could be rejected. Kinetic and equilibrium binding characteristics of the three radioligands were not influenced by the guanylnucleotide Gpp(NH)p (10−4 M). The antagonist ICYP binds to β-adrenoceptors in a high and low affinity state which are probably interconvertible.