Affinity of antithrombin III for insoluble modified polystyrene

Area of Science:

• Biochemistry
• Polymer Science
• Chromatography

Background:

• Insoluble polystyrenes grafted with L-arginyl methyl ester (PAOM) demonstrate high affinity for thrombin.
• PAOM resins are utilized as stationary phases in affinity chromatography (AC) and high-performance liquid affinity chromatography (HPLAC) for thrombin purification.
• Previous AC studies indicated slight adsorption of antithrombin III (AT III) onto PAOM.

Purpose of the Study:

• To investigate the interaction of antithrombin III (AT III) with PAOM resin using a batch procedure.
• To quantify the adsorption characteristics and affinity constant of AT III on PAOM.
• To compare the behavior of PAOM with heparin-like resins regarding AT III interaction and thrombin-AT III complex generation.

Main Methods:

• Batch adsorption experiments using purified antithrombin III (AT III) and PAOM resin.
• Measurement of AT III adsorption to determine monolayer formation.
• Evaluation of the affinity constant (kAT) for AT III binding to PAOM.
• Comparative analysis with previously studied heparin-like resins.

Main Results:

• AT III adsorption on PAOM resin follows a monolayer model.
• The affinity constant for AT III on PAOM was determined to be kAT = 3.10(5) M⁻¹.
• PAOM resin does not exhibit heparin-like behavior in catalyzing thrombin-AT III complex formation.
• When thrombin is bound to the PAOM surface, AT III is unable to interact with the enzyme's active site.

Conclusions:

• PAOM resin exhibits specific binding characteristics for AT III, quantifiable by an affinity constant.
• PAOM does not mimic heparin's catalytic activity in the context of thrombin-AT III complex formation.
• The presence of bound thrombin on PAOM sterically hinders AT III interaction with the enzyme, preventing complex formation.