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Probing the Interactions Among Three Proteins to Evaluate Whether an Antibody and Inhibitor Compete for the Same Binding Site on an Enzyme


Sophia Kenrick Multi-Angle static Light Scattering (MALS) is a powerful tool for quantifying multiple types of protein-protein interactions. This article provides an


account of research conducted to study the interactions between three proteins: human thrombin–α (Thr), antithrombin III (AT), and an anti- thrombin monoclonal antibody (Ab) to evaluate whether the antibody can recognise AT-inactivated Thr.


Introduction


Composition-gradient multi-angle light scattering (CG-MALS) and dynamic light scattering (CG-DLS) quantify the affinity and stoichiometry of equilibrium protein-protein interactions in solution, as well as nonspecific interactions leading to thermodynamic nonideality. In previous studies, CG-MALS was applied to characterise the reversible equilibrium between human


thrombin–α and an anti-thrombin antibody, revealing the expected 2:1 stoichiometry and equilibrium dissociation constant KD = 8.8 nM [1]. In addition, the second order association rate constant for the covalent association of human thrombin–α to antithrombin was calculated using time-dependent MALS TD-MALS [2].. In this article, the CG-MALS is extended to probe the recognition of the bound thrombin-antithrombin complex by the same antibody.


Experimental Reagents and Instrumentation


For each experiment, human antithrombin III (AT), human thrombin–α (Thr) and mouse monoclonal anti-human antibody (Ab) were diluted to the appropriate concentrations in


phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4). The CG-MALS experiments were performed with a Calypso II composition gradient system (Wyatt Technology Corporation, Santa Barbara, CA), which prepared different


compositions of protein and buffer and delivered them to an online UV/Vis detector (Waters Corporation, Milford, MA) and DAWN HELEOS MALS detector (Wyatt). Inline filter membranes with 0.1µm pore size were installed using a Calypso for sample and buffer filtration. Control of the Calypso pumps, data acquisition, and analysis were performed with the Calypso software.


Size exclusion chromatography (SEC) was performed using a column with 300 Å pore size (030S5 column, Wyatt), with injections performed by an HPLC pump and autosampler (Agilent Technologies, Santa Clara, CA). The separation between the covalent Thr-AT complex and Thr and AT monomers was confirmed by SEC coupled with multi-angle light scattering (SEC-MALS) using a UV detector, DAWN HELEOS, and Optilab rEX refractive index detector (Wyatt).


Determination of Three-Component Binding


For initial determination of Ab binding to bound Thr-AT complex, CG-MALS experiments were performed such that Ab solution was exposed to an unfractionated mixture of Thr, AT and Thr-AT complex, as follows. Thr and AT were diluted to stock concentrations of 24µg/ml and 80 µg/ml, respectively, in PBS, and each solution was filtered to 0.02µm. Equal volumes of filtered stock solution were combined and allowed to incubate at room temperature for ~2 hours with gentle shaking. Ab was diluted to 22µg/mL in PBS and filtered to 0.02µm. A composition gradient was performed, consisting of six compositions of premixed AT + Thr solution at constant concentration of 25µg/mL and Ab concentrations from 0 to 8 µg/mL. For each composition, the Calypso mixed the appropriate volumes of AT-Thr solution and Ab and delivered the solution to the downstream detectors. After each injection, the flow was stopped to allow any binding interactions to come to equilibrium. The measured weight-average molar mass of each composition at equilibrium was used to determine qualitatively whether the antibody would recognise the bound Thr-AT complex.


To quantify the affinity of the Ab-complex binding, a second composition gradient was performed with multiple compositions of Ab and purified Thr-AT complex (Figure 1). Thr and AT were diluted to 2mg/mL each in PBS, mixed at a 1:1 ratio, and allowed to incubate at room temperature 0.5-1 h. Pre-mixed aliquots of 0.1mL were injected onto an SEC column, and fractions of bound Thr-AT complex were collected at the outlet of the UV detector. Multiple injections were performed and the fractions pooled and filtered to 0.1µm, with a final protein concentration ~70µg/mL. The purified Thr-AT complex was then used in further CG-MALS experiments. Ab was diluted to 15µg/ml in PBS and filtered to 0.02µm. The interaction between purified Thr-AT complex and Ab was measured using a single “crossover” gradient consisting of eight compositions of Ab and Thr-AT complex. For each composition, the Calypso mixed purified Thr-AT complex with Ab, injected the solution into the UV and MALS detectors, and stopped the flow to allow all binding reactions to come to equilibrium. The light scattering data as a function of composition was fit to the appropriate binding model to quantify the interaction stoichiometry and equilibrium dissociation constant, KD, at each binding site [3].


Figure 1. Strategy for quantifying the binding between the covalent thrombin-antithrombin complex and an anti-thrombin antibody


Results


Interaction of Ab with Unfractionated Thr-AT Complex


Once Thr was bound irreversibly to AT, the ability of Ab to retain the Thr-binding activity was measured with CG-MALS.


In a preliminary experiment, AT and Thr were pre-mixed and the reaction allowed to come to completion before loading the unfractionated solution on the Calypso and performing a crossover gradient with this complex solution and Ab. Although binding was evident.


The weight-average molar mass measured by light scattering was significantly less than expected for an interaction between Ab and Thr-AT complex with 2:1 stoichiometry and


KD ~9 nM per binding site, the affinity previously measured for Ab binding pure Thr [1] (Figure 2).


Figure 2. The equilibrium Mw for a composition gradient with constant concentration of unfractionated Thr, AT, and Thr-AT complex (25 µg/mL total protein) and varying concentrations of Ab indicated binding was occurring


but could not differentiate between binding with decreased affinity and Ab binding only free Thr in solution.


The decreased Ab-binding could be explained by two possible mechanisms: 1) the Ab bound the Thr-AT complex with decreased affinity compared to free Thr or 2) the Ab did not bind Thr-AT complex, and the observed increase in Mw resulted from Ab binding free Thr in solution.


INTERNATIONAL LABMATE - APRIL 2013


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