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Characterization of thrombin binding to alpha 2-macroglobulin.

D L Straight, P A McKee

The Journal of Biological Chemistry

|January 25, 1984

Summary

Human alpha 2-macroglobulin (alpha 2M) binds thrombin and trypsin, forming covalent bonds with uncleaved subunits. Thrombin rapidly binds one site on alpha 2M, with difficulty occupying a second site.

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Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Alpha 2-macroglobulin (alpha 2M) is a crucial human plasma proteinase inhibitor.
Understanding alpha 2M's interaction with proteinases like thrombin is vital for biological and medical research.

Purpose of the Study:

To elucidate the formation and structural characteristics of the human alpha 2-macroglobulin-thrombin complex.
To investigate the binding mechanism and stoichiometry of thrombin and trypsin to alpha 2M.

Main Methods:

Intrinsic protein fluorescence
Sulfhydryl group titration
Polyacrylamide gel electrophoresis (SDS-PAGE)
Macromolecular inhibitor assays
Staphylococcus aureus V-8 proteinase digests

Main Results:

Thrombin maximally affects alpha 2M at a 1.3:1 molar ratio, with covalent binding peaking at 1.5:1, resulting in one mole of thrombin covalently bound per mole of alpha 2M.
Thrombin binds rapidly to one site on alpha 2M, but occupies a second site with difficulty.
Proteinases form covalent bonds with uncleaved alpha 2M subunits, suggesting initial reversible interaction determines binding ratio.

Conclusions:

The study provides insights into the mechanism of proteinase binding to alpha 2M.
Findings highlight similarities and differences in thrombin and trypsin interactions with alpha 2M across various pH levels.
Results support a model where proteinase affinity dictates binding stoichiometry with alpha 2M.

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