Homotropic cooperative binding of organic solvent vapors by solid trypsin

Area of Science:

• Biochemistry
• Physical Chemistry
• Enzyme Science

Background:

• Enzyme activity and stability are significantly influenced by solvent environment.
• Understanding protein-solvent interactions is crucial for biocatalysis and biotechnology.
• Dried enzymes offer unique opportunities for studying solvent interactions without bulk liquid phases.

Purpose of the Study:

• To investigate the vapor sorption behavior of organic solvents by dried solid trypsin.
• To characterize the binding mechanism, stoichiometry, and thermodynamics of solvent inclusion.
• To explore the implications of these interactions for enzyme activity and stability in low-water media.

Main Methods:

• Vapor sorption isotherms were measured using a static method with gas chromatographic headspace analysis at 298 K.
• The 'vapor solvent+solid trypsin' systems were studied in the absence of a liquid phase.
• Isotherm data were approximated using the Hill equation to determine binding parameters.

Main Results:

• Homotropic cooperative binding of organic solvents (acetonitrile, alcohols, nitroethane) by dried trypsin was observed.
• Sorption isotherms showed a sigmoidal shape, with significant uptake above a threshold thermodynamic activity.
• Formation of inclusion compounds with phase transitions and stoichiometry (S) dependent on sorbate size/shape was proposed, with Hill constants (N) indicating strong cooperativity.

Conclusions:

• Dried trypsin forms inclusion compounds with organic solvent vapors, demonstrating cooperative binding.
• The binding stoichiometry and thermodynamics are influenced by the sorbate's molecular characteristics.
• These findings provide insights into solvent effects on enzyme function in non-aqueous environments.