title: Immobilization and kinetics of lactate dehydrogenase at a rotating nylon disk.
abstract: 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 von Karman-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(-1/2), where f is the speed of rotation of the disk. Extrapolation of f(-1/2) 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 degrees C, in phosphate buffer (pH 7.5) using the extrapolation method was 84 muM. This value was in good agreement with the previously published value of 87 muM, obtained with LDH attached to the inner surface of a nylon tubing. However, when compared to the K(m) for a free enzyme system, the 84 muM was about nine times larger, indicating an inherent reduction in the activity of the bound LDH. Since, at extrapolated infinite rotation speeds, diffusion effects were assumed eliminated, the drop in the activity was thought to be due to sterric hinderances imposed on the substrate NADH as a result of having LDH bound to another polymer.
authors.affiliation: Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Quebec, Canada H3G 1Y6.
authors.forename: J N
authors.initials: JN
authors.lastname: Daka
authors.forename: K J
authors.initials: KJ
authors.lastname: Laidler
authors.forename: R
authors.initials: R
authors.lastname: Sipehia
authors.forename: T M
authors.initials: TM
authors.lastname: Chang