28 chromatography • spectroscopy
solution. After each injection, the flow stops to permit the reaction to reach equilibrium. Te Calypso software analyzes MALS and concentration data to compute the Mw,app for each step in the gradient and then quantifies the equilibrium association constant and stoichiometry of all complexes formed in solution.
Te Calypso is compatible with Wyatt DAWN HELEOS and miniDAWN TREOS multi-angle light scattering detectors. Te concentration of each binding partner can be measured using in-line UV/Vis absorption or a differential refractometer such as the Wyatt Optilab T-rEX.
Alternatively, the nominal concentrations based on calculated stock solution dilutions are automatically computed and saved with the data and can be used for analysis for experiments where no concentration detector is appropriate.
Experimental set-up Human thrombin a (Tr) and mouse monoclonal anti-human thrombin antibody (Ab) were purchased from Haematologic Technologies Inc. All experiments were performed in phosphate buffered saline (PBS;
137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2
HPO4 200ppm NaN3
, 1.76 mM KH2 , pH 7.4). Prior to
PO4
analysis, all protein solutions were filtered to 0.02µm by means of Anotop syringe filters (Whatman).
CG-MALS experiments were performed with a Calypso II composition gradient system (Wyatt Technology Corp) to prepare different compositions of protein and buffer and deliver to an online UV/Vis absorption detector (Waters Corp) and HELEOS MALS detector (Wyatt).
Anodisc filter membranes of 0.1-µm pore size were installed in the Calypso for sample and buffer filtration (Whatman). Tr and Ab were each diluted to stock concentrations of 20µg/mL in PBS, filtered to 0.02µm, and loaded on the Calypso II.
An automated Calypso method was run consisting of three distinct segments: two single component concentration gradients to quantify any self association and a dual- component ‘crossover’ composition gradient to assess the hetero- association behaviour (Fig. 1). For each composition, 0.8mL of protein solution at the appropriate concentration was injected into the
,
UV and MALS detectors. Te flow was then stopped for 180s (single protein gradients) or 300s (crossover gradient) to allow the solution to come to equilibrium within the MALS flow cells. Data collection and analysis of equilibrium association constants were performed using Calypso software.
Results and discussion Te automated Calypso method measured twenty-two different protein compositions to probe the potential interactions present in a solution of thrombin (Tr) and a monoclonal anti-thrombin antibody (Ab). A single-species concentration gradient for each protein was used to determine whether Tr or Ab homo-oligomerised. Tis data, combined with light scattering and concentration measurements at 12 different ratios of Tr and Ab, is all that is needed to determine the affinity and stoichiometry of the Tr-Ab binding.
Under these conditions, no self- interactions are observed for Tr or Ab. Te weight-average molar mass at each step in the self-association gradient for Ab corresponds to the expected monomer molecular weight. Likewise, Mw,app
for Tr is
calculated as 37kDa at all steps in the Tr self-association gradient.
Heterocomplex formation is evident in the crossover gradient light scattering profile. In the absence of a Tr-Ab interaction, this would consist of a simple linear ‘staircase’ of values (dashed purple plot, Fig. 2A). In fact, we observed an increase in scattering intensity indicating complex formation, reaching a maximum at the step where the Ab concentration becomes limiting (solid blue plot, Fig. 2A). Te position of the light scattering peak corresponds to the Ab:Tr stoichiometry, and the height of the peak gives a measure of the affinity. Binding kinetics, though clearly visible in the data, are not utilised to evaluate equilibrium constants. Analysis of binding kinetics via CG-MALS will be addressed in a separate tutorial.
As expected, the light scattering data in Fig. 2 is best fit in the Calypso software by an association model which describes two equivalent thrombin binding sites per antibody molecule. As the ratio of Ab:Tr concentration changes, so also does the fraction of bound species present. At high Ab:Tr ratios, the (1 Ab): (1 Tr) complex is most abundant; as the thrombin concentration increases and antibody concentration decreases, the (1 Ab):(2 Tr) complex dominates (Fig. 3). Te
Fig. 2. Light scattering signal resulting from protein compositions (see Fig. 1.) Fig 2A) Raw light scattering and composition data for self-association and hetero-association gradients between human thrombin α and an anti-thrombin antibody. Fig. 2B. Light scattering data for Ab self-association (plateaus 0-4), hetero-association (plateaus 5-18), and Thr self-association (plateaus 19-23). The best fit (red line) to the raw data (blue dots) is the sum of the LS contributions of each species present: free Thr, free Ab, (1Ab):(1 Thr) complex, and (1 Ab):(2 Thr) complex.
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