Visualization of the reaction layer in the immediate membrane vicinity.

Coupled processes of diffusion and chemical reaction are quantitatively analyzed by means of proton concentration profile measurements in the immediate membrane vicinity. Because mass transfer across the unstirred layer (USL) is thought to proceed via diffusion, only the size of the USL is used as a scale for diffusion restrictions. Limitations associated with the finiteness of the reaction rate are characterized by the size of the reaction layer (RL). The latter is defined as the distance from the membrane where equilibrium is reached if transmembrane diffusion is followed by chemical reactions proceeding in the solution layers adjacent to the membrane. The well-known reaction of acetaldehyde oxidation catalyzed by alcohol dehydrogenase in the presence of NADH was selected because it is accompanied by an easily measurable alkalinization of the solution. The enzyme/coenzyme and the substrate were added to opposite sides of the membrane. A theoretical model describing the reaction and diffusion kinetics enabled us to calculate the size of the RL from the pH profile within the USL of a planar bilayer membrane showing a high proton permeability. As the enzyme concentration increased, the initially reaction-limited kinetics of the total turnover became diffusion limited under our experimental conditions due to a dramatic shortening of the RL.