IMMOBILIZATION AND KINETICS OF LACTATE-DEHYDROGENASE AT A ROTATING NYLON DISK

Lactate dehydrogenase (LDH) was covalently attached to an impervious nylon surface by an improved technique. The procedure allowed the kinetics of the rotating enzyme disk reactor to be successfully explored. This enzyme-disk configuration has potential applications in assays for lactic acid or pyruvic acid in fluids of biological importance (e. g. , urine). In order to evaluate and understand the physics and chemistry underlying the kinetics of the heterogeneous biocatalyst, a mathematical model based on the I. von Karman-V. G. Levich theories of rotating electrodes, was developed. It applied well to LDH attached to a disk, under variable NADH concentrations and fixed pyruvic acid. The new theory, leads to the conclusion that the apparent Michaelis constant K//m(app), varies linearly with f** minus ** one-half , where f is the speed of rotation of the disk. Extrapolation of f** minus ** one-half to zero gives the Michaelis-Menten constant, K//m, corresponding to the diffusion-free behavior. With immobilized LDH, the diffusion-free K//m for NADH obtained at 25 degree C, in phosphate buffer (pH 7. 5) using the extrapolation method was 84 mu M.