Preparation and characterization of anhydrothrombin

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Thrombin is a key enzyme in coagulation.
• Catalytically inactive thrombin derivatives are valuable research tools.
• Understanding thrombin's active site is crucial for inhibitor development.

Purpose of the Study:

• To develop a novel method for preparing anhydrothrombin.
• To characterize the structure and properties of anhydrothrombin.
• To assess the ligand-binding capabilities of anhydrothrombin.

Main Methods:

• Beta-elimination reaction on phenylmethylsulfonyl fluoride-inactivated thrombin.
• Denaturant unfolding and refolding for anhydrothrombin preparation.
• Purification using PAB affinity chromatography.
• Peptide analysis via cyanogen bromide digestion, HPLC, amino acid analysis, and electrospray mass spectrometry.
• Sedimentation equilibrium for monomeric assessment.
• Competitive titration with hirudin to assess binding.

Main Results:

• Successfully synthesized anhydrothrombin by replacing active-site serine with dehydroalanine.
• Confirmed the absence of serine and a mass reduction consistent with water elimination in the anhydrothrombin active-site peptide.
• Demonstrated that anhydrothrombin is monomeric.
• Showed that anhydrothrombin binds to hirudin with native-like interactions.

Conclusions:

• A novel and effective method for anhydrothrombin preparation was established.
• Anhydrothrombin serves as a catalytically inactive but structurally intact thrombin analog.
• Anhydrothrombin retains the ability to interact with macromolecular ligands, validating its utility in studying thrombin-binding interactions.