Light Scattering for the Masses Kinetics of Enzyme-Inhibitor Associations

The kinetics of association or dissociation is of paramount importance in many biological systems. For example, antithrombin III forms a covalent bond with the catalytic serine of several proteases involved in the coagulation cascade, and the rate of this reaction can increase up to 1000× in the presence of certain macromolecules. Here, we determine the second-order rate constant for the covalent association of thrombin α (Thr) and antithrombin III (AT) by Composition Gradient Multi-Angle Light Scattering (CG-MALS).

Figure 1: Light scattering and concentration data for thrombin titration at constant antithrombin concentration.

Human thrombin α and antithrombin III (Haematologic Technologies, Inc.) were diluted to 60 µg/mL and 120 µg/mL, respectively, in phosphate buffered saline (PBS, pH 7.4) and filtered to 0.02 µm. Composition gradients were created using a Calypso II and delivered to an online UV/vis concentration detector and DAWN HELEOS. The method consisted of six injections at constant AT concentration of 60 µg/mL and Thr concentrations from 0 to 30 µg/mL. After each injection into the UV and MALS detectors, the flow was stopped for 2000 s to allow the reaction to come to completion. Calypso software was used to run the method, acquire MALS and UV signals, and calculate the apparent weight-averaged molar mass as a function of time

Figure 2: The increase in Mw as a function of time was fit to the appropriate association model to calculate the second order rate constant for the inhibition of Thr by AT, kon = 6.09 ×103 M–1 s–1. Raw data (open symbols) and best fit curves are shown for varying Thr concentrations and constant AT concentration of 60 µg/mL.

The light scattering data for all five measurements with nonzero thrombin concentrations were fit simultaneously to a model of 1:1 irreversible association between thrombin and antithrombin (Figure 2). Using this model, the second order rate constant was calculated as k_on = 6.09 × 10³ M^–1 s^–1 , similar to results measured by other methods (e.g., k_a = 5.8 × 10³ M^–1 s^–1 measured at 25 °C by fluorescence) (1). The analysis simultaneously determined the fraction of thrombin capable of binding antithrombin. This calculated fraction, 77%, compared favourably with the manufacturer's reported specific activity of the antithrombin (0.79 mol Thr/mol AT).

Thus, CG-MALS provided a complete picture of the association between thrombin and antithrombin in solution, including identifying a fraction of thrombin incapable of binding its inhibitor. This technique enabled observation of slow, irreversible binding and measurement of rates of 1:1 association consistent with other techniques.

References

1. G. Izaguirre et al., Proc. Natl. Acad. Sci., 282, 33609–33622 (2007).

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