Solid surface-catalysed inactivation of bovine alpha-chymotrypsin in dilute solution

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Chymotrypsin is a key digestive enzyme.
• Understanding enzyme stability in solution is crucial for its applications.
• Previous studies have not fully elucidated the mechanisms of chymotrypsin activity loss.

Purpose of the Study:

• To investigate the kinetics and mechanisms of chymotrypsin activity loss in dilute solutions.
• To determine the role of solid surfaces in enzyme inactivation.
• To identify the molecular changes responsible for loss of chymotrypsin activity.

Main Methods:

• Kinetic analysis of chymotrypsin activity loss at varying concentrations.
• Investigating the effect of surface area-to-volume ratio and vessel material on reaction rates.
• Monitoring intrinsic fluorescence changes of chymotrypsin over time.
• Assessing autolysis as a contributing factor to activity loss.

Main Results:

• Chymotrypsin activity loss followed first-order kinetics above 4 nM enzyme concentration.
• Rate constants for activity loss were dependent on surface area-to-volume ratio, vessel type, and surface coatings (DS, polybrene, lecithin, BSA).
• Intrinsic fluorescence decreased over time, indicating conformational changes, with no significant autolysis observed.

Conclusions:

• Loss of chymotrypsin activity in dilute solution is significantly influenced by solid surface-catalyzed reactions.
• The primary mechanism for activity loss is an irreversible conformational change, not autolysis.
• Enzyme stability is modulated by interactions with various surfaces, impacting its practical use.