Comparison of various methods to determine kinetic constants for β‐galactosidase in soluble and immobilised states

Abstract

AbstractThe graphical methods of Lineweaver—Burk, Eadie, Hanes and the ‘direct linear plot’ have been used to determine the kinetic constants of β‐galactosidase in soluble and immobilised states. Because some of these methods require a reciprocal concentration scale, the activity values obtained from dilute substrate solutions are weighted more than when using more concentrated substrates. However, in the case of immobilised enzymes the values from dilute substrate are more affected by diffusion from the bulk solution into the pores where the enzyme is fixed, and vice versa. Therefore it, seems preferable to use methods with direct substrate and velocity scales. The direct linear plot method is recommended because it does not require any transformation of the measurements, the area of intersection points indicates the accuracy of the measurement and runaway points are easily detected.