High-performance affinity chromatography of human thrombin on modified polystyrene resins

Area of Science:

• Biochemistry
• Chromatography
• Materials Science

Background:

• Antithrombin III and thrombin substrates share binding sites.
• Cross-linked polystyrenes can be functionalized to mimic these binding sites.

Purpose of the Study:

• To develop and evaluate cross-linked polystyrenes modified with L-arginyl methyl ester as stationary phases for high-performance affinity chromatography (HPAC) of thrombin.
• To investigate the adsorption and desorption characteristics of thrombin on these novel stationary phases.

Main Methods:

• Synthesis of cross-linked polystyrenes modified with L-arginyl methyl ester.
• High-performance affinity chromatography (HPAC) for thrombin separation.
• Isocratic and gradient elution with varying sodium chloride concentrations.
• Analysis of thrombin recovery and enzymatic activity.
• Investigation of kinetic parameters including flow-rate and salt gradient slope.

Main Results:

• Thrombin strongly adsorbed on the modified resins at sodium chloride concentrations below 0.5 M.
• Selective desorption of thrombin achieved at approximately 1.2 M sodium chloride.
• High recovery of thrombin enzymatic activity demonstrated with linear salt gradient elution.
• Thrombin decomposition products (e.g., from sodium dodecyl sulphate treatment) were not retained.
• Adsorption and desorption were significantly influenced by kinetic parameters like flow-rate and salt gradient slope.

Conclusions:

• L-arginyl methyl ester-modified polystyrenes serve as effective stationary phases for HPAC of thrombin.
• The developed method allows for selective and efficient separation of thrombin while preserving its activity.
• Kinetic factors play a crucial role in optimizing the adsorption and desorption processes for thrombin purification.