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A simple activity assay for thrombin and hirudin

Q D Dang¹, E Di Cera

¹Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis 63110.

Journal of Protein Chemistry

|May 1, 1994

Summary

Researchers developed a new assay for measuring thrombin activity. This method accurately quantifies thrombin and hirudin at picomolar concentrations, overcoming limitations of standard enzyme assays.

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Cloning thrombin cDNA enables site-directed mutagenesis for functional studies.
Standard enzyme activity assays require high protein concentrations, limiting studies on low-yield thrombin mutants.
Accurate quantification of enzyme activity is crucial for interpreting mutagenesis data.

Purpose of the Study:

To develop a novel, sensitive method for active-site titration of thrombin.
To enable quantitative studies of thrombin mutants produced in limited quantities.
To establish a method for determining hirudin activity.

Main Methods:

Utilized the synthetic substrate S-2238.
Employed hirudin, a potent thrombin inhibitor, for active-site titration.
Developed a novel assay applicable to very low enzyme concentrations.

Main Results:

Successfully performed active-site titration of thrombin at concentrations as low as 20 pM with <5% error.
Determined hirudin activity at concentrations as low as 40 pM with <5% error.
The novel method overcomes limitations of standard assays for low-concentration enzyme studies.

Conclusions:

The developed assay provides a sensitive and accurate method for quantifying thrombin and hirudin activity.
This technique facilitates the study of thrombin mutants and inhibitors produced in small quantities.
The method significantly advances the ability to perform quantitative biochemical analyses in molecular biology research.

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