A Thermodynamic Characterization of the Binding of Thrombin Inhibitors to Human Thrombin, Combining Biosensor Technology, Stopped-Flow Spectrophotometry, and Microcalorimetry
✍ Scribed by Johanna Deinum; Lars Gustavsson; Erika Gyzander; Mari Kullman-Magnusson; Åsa Edström; Robert Karlsson
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 152 KB
- Volume
- 300
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399 -575 Da) to human ␣-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K D (nM to M range) at 25°C obtained from the BIACORE analysis correlated well with the inhibition constants K i in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K i and K D values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, ⌬H, and entropy, ⌬S, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in ⌬S for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the ⌬H by 20 kJ mol ؊1 . ⌬H values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37°C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.