OR WAIT null SECS
Wyatt Application Note
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 Ã103 Mâ1 sâ1. Raw data (open symbols) and best fit curves are shown for varying Thr concentrations and constant AT concentration of 60 Âµg/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 kon = 6.09 × 103 M–1 s–1 , similar to results measured by other methods (e.g., ka = 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.
1. G. Izaguirre et al., Proc. Natl. Acad. Sci., 282, 33609–33622 (2007).
DAWN®, miniDAWN®, ASTRA®, Optilab® and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation.
Wyatt Technology Corp.
6300 Hollister Ave., Santa Barbara, California, USA
tel: +1 805 681 9009 fax: +1 805 681 0123