Glycolytic pH oscillations in a flow reactor

Area of Science:

• Biochemistry
• Chemical Engineering
• Enzyme Kinetics

Background:

• Studying nonlinear enzyme-catalyzed reactions requires specialized reactor designs.
• Existing methods may limit analysis of enzymes with higher molecular weights or protein concentrations.
• Continuous flow reactors offer advantages for dynamic biochemical studies.

Purpose of the Study:

• To develop and validate a new ultrafiltration continuous flow stirred tank reactor (UCSTR) for studying nonlinear enzyme kinetics.
• To enable continuous monitoring of enzyme-catalyzed reactions under flow conditions.
• To investigate oscillatory behavior in biochemical systems.

Main Methods:

• Development of a UCSTR utilizing anisotropic ultrafiltration membranes.
• Characterization of reactor performance using the ferrocyanide-hydrogen peroxide reaction to reproduce pH oscillations.
• Experimental verification of oscillatory glycolysis using rat skeletal muscle extract with fructose-6-phosphate and ATP as input feeds.

Main Results:

• The UCSTR successfully accommodates enzymes with subunit molecular weights >= 9000 dalton and protein concentrations up to 2 mg/ml.
• Continuous potentiometric or spectrophotometric measurements were feasible without reactor redesign.
• Sustained pH oscillations in glycolysis were observed for over eight hours in the UCSTR.
• A six-step reaction mechanism accurately simulated the observed pH oscillations.

Conclusions:

• The UCSTR is a viable tool for studying complex, nonlinear enzyme-catalyzed reactions, including oscillatory phenomena.
• The reactor design facilitates the analysis of a wide range of enzymes under controlled flow conditions.
• The findings provide a new platform for investigating dynamic biochemical processes and enzyme mechanisms.