[Preparation of thrombin affinity chromatography media]

Area of Science:

• Biochemistry
• Biotechnology
• Affinity Chromatography

Background:

• Thrombin plays a critical role in hemostasis and thrombosis.
• Developing efficient methods for thrombin purification and inhibitor screening is essential.

Purpose of the Study:

• To prepare and characterize thrombin affinity chromatography media.
• To optimize the coupling and reaction conditions for media preparation.
• To evaluate the stability and application potential of the prepared media.

Main Methods:

• Thrombin was coupled to cyanogen bromide-activated agarose.
• Enzyme activity was measured using a chromogenic substrate assay.
• Optimization of reaction buffer (pH 8.3), thrombin concentration (200 U/g agarose), and reaction time (10 h) was performed.

Main Results:

• Optimal conditions for thrombin coupling were established.
• The prepared affinity media demonstrated good stability, retaining 70.6% of thrombin activity after 40 days.
• The media is suitable for screening and purifying thrombin inhibitors.

Conclusions:

• A stable and effective thrombin affinity chromatography medium was successfully prepared.
• The optimized protocol provides a reliable method for media preparation.
• This affinity chromatography media has broad applications in biochemical research and drug discovery, particularly for identifying natural product-derived thrombin inhibitors.