Binding of anhydrotrypsin to alpha 2-macroglobulin

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Alpha 2-macroglobulin (alpha 2M) is a key plasma proteinase inhibitor.
• Understanding proteinase-alpha 2M interactions is crucial for biological and medical research.

Purpose of the Study:

• To investigate the binding characteristics of anhydrotrypsin, an inactive trypsin derivative, with alpha 2M.
• To compare the interaction of anhydrotrypsin with alpha 2M to that of active proteinases.

Main Methods:

• Radiolabelling of anhydrotrypsin for detection.
• Gel electrophoresis to assess binding affinity and stability.
• Displacement assays using trypsin inhibitors (aprotinin, soya-bean trypsin inhibitor, benzamidine).
• Competitive binding experiments with active trypsin.

Main Results:

• Anhydrotrypsin binds to alpha 2M, resisting gel electrophoresis separation.
• Approximately 2 moles of anhydrotrypsin bind per mole of alpha 2M.
• Binding affinity differs between 'fast' and 'slow' electrophoretic forms of alpha 2M.
• Anhydrotrypsin is displaced by specific trypsin inhibitors.
• Alpha 2M saturated with anhydrotrypsin can still bind and inhibit active trypsin.
• Bound anhydrotrypsin retains immunoreactivity with anti-trypsin antibodies, unlike bound active trypsin.

Conclusions:

• Alpha 2M exhibits distinct binding behavior towards inactive anhydrotrypsin compared to active proteinases.
• Anhydrotrypsin binding does not abolish alpha 2M's capacity to inhibit active trypsin.
• The retained immunoreactivity of bound anhydrotrypsin suggests conformational differences in the binding interaction.