Desorption of bound thrombin

In the present paper, we report high-performance affinity chromatography of thrombin in presence of AT III and Hep, using two types of resins as stationary phases either heparin-like PSSO or AT Ill-like PAOM. In order to differentiate their mechanisms of interaction with thrombin, we examined the chromatographic behavior of thrombin in the presence, or in the absence of AT III and/or heparin. Finally, thrombin was injected on the columns at low ionic strength. The desorption of bound thrombin from the two solid surfaces was then carried out using AT III, heparin and the AT Ill-Hep complex, to elucidate the specificity of the interactions involved. 

Desorption of Bound Thrombin by Antithrombin III, Heparin or Hep-AT III Complex. Thrombin was injected onto PAOM or PSSO chromatographic supports in 0.1 M NaCl solution. To minimize possible kinetic effects, the flow-rate was reduced to 0.2 ml/min. After washing with 0.1 M NaCl buffer, an excess of either AT III, Hep, or AT Ill-Hep complex was injected. The flow was then stopped for five minutes in order to obtain a better exchange of macromolecules between the mobile and the stationary phases. The objective was to determine whether AT III, Hep or AT Ill-Hep complex could act as eluents for the "biospecific desorption" of the bound enzyme at 0.1 M NaCl. 

The comparison between the chromatogram of Th-AT complex (Figure 4A) and the desorption of bound thrombin by antithrombin III from the PAOM resins (Figure 5A) indicates that the active... 

Figure 5. Desorption of bound thrombin from PAOM (A) by AT III, Hep and AT Ill-Hep and from PSSO resins by antithrombin III (B), AT Ill-Hep complex (C) and heparin (D), at 25 °C. Eluent 0.05 M phosphate buffer (pH = 7.4), NaCl 0.1 M and 2 M, flow rate 0.2 ml/min.

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See also in sourсe #XX -- [ , , ]

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