Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy

Abstract

We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated.

Biochemical Characterization of a Thrombin Inhibitor from the Bloodsucking Bug <i>Dipetalogaster maximus</i>

“…One of these, the fibrinogen recognition or anion-binding exosite, plays an important role in the selection of thrombin substrates and inhibitors [27]. The thrombin inhibitors isolated from bugs described so far are known to interact either with both the active site and the anion-binding exosite of thrombin simultaneously, like rhodniin, or only with the anion-binding exosite, like triabin [28][29][30]. The potent inhibition of thrombin by dipetalogastin also in amidolytic assays using small substrates suggests that unlike triabin, which prolongs TT and aPTT but only minimally suppresses the amidolytic activity of thrombin, dipetalogastin binds to the active site of thrombin.…”

Applications of Fluorescence Correlation Spectroscopy: Polydispersity Measurements

“…[7] we set the first derivative with respect to c j equal to zero: Different methods for solving Eq. [9] could be explored. The Newton-Raphson method (19) is an iterative procedure, in which the solution is searched by starting from an initial approximation.…”

“…As a result, the sensitivity has been substantially increased and it has become possible to perform single-molecule detection measurements (3,4). Until now the FCS method has been applied mainly in biochemical and biophysical investigations such as measurement of lateral transport on cell surfaces (5), nucleic acid hybridization (6,7), and receptor/ligand interaction (8,9). The method, however, can also be very helpful in environmental studies, both for analytical determination and interaction studies of small particles and trace components in natural water and for investigation of the particle dynamics of model systems.…”

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

“…For all these purposes, fluorescence correlation spectroscopy (FCS) (5)(6)(7)(8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled biomolecules that undergo temporal changes in their diffusion properties (10)(11)(12)(13)(14)(15)(16)(17). The dual-color cross-correlation method, as proposed by Rigler and Eigen (2,18), circumvents the necessity of evaluating the diffusion characteristics of the product fractions; therefore, simple mathematical evaluation (18,19) and much shorter readout times for screening applications can be achieved as shown in the subsequent article (20).…”