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Preparation and activity of nitrated insulin dimer
International Journal of Peptide and Protein Research
|April 1, 1986
Summary
Researchers created a covalent insulin dimer by nitrating insulin crystals. This cross-linked dimer, involving specific tyrosyl residues, retained significant biological activity, indicating key functional sites are surface-exposed.
Area of Science:
- Biochemistry
- Protein Chemistry
- Crystallography
Background:
- Insulin's biological activity is modulated by its oligomeric state.
- Tyrosyl residues are crucial for insulin structure and function.
- Chemical modification can alter protein structure and interactions.
Purpose of the Study:
- To create a covalently linked insulin dimer using nitration.
- To investigate the structural and functional consequences of cross-linking specific tyrosyl residues in insulin.
- To determine if the functional sites of insulin are accessible in a dimeric form.
Main Methods:
- Nitration of insulin crystals using tetranitromethane.
- Selective blocking of reactive tyrosyl residues (A14).
- Isolation and purification of the covalent dimer using oxidative sulphitolysis and High-Performance Liquid Chromatography (HPLC).
- Biological activity assay (lipogenesis).
Main Results:
- A covalent insulin dimer was successfully isolated by cross-linking B16 and B26 tyrosyl residues.
- Cubic pig insulin crystals yielded a pure dimeric product.
- The nitrated insulin dimer exhibited approximately 10% of the potency of monomeric insulin in a lipogenesis assay.
Conclusions:
- Specific tyrosyl residues (B16-B26) can be covalently cross-linked in insulin crystals.
- The resulting covalent dimer retains significant biological activity.
- This suggests that the active sites of insulin are located on the surface of the dimer, not buried within the interface.