Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod ( G. morhua) using a 96-well microplate technique

Abstract

Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0–40 °C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 °C with high stability and accuracy in any data acquisition mode. At 4 °C the accuracy of the temperature control was ±0.1 °C and temperature homogeneity across the microplate was ±0.3 °C. As examples, analyses of the temperature dependence of Michaelis–Menten ( K ′PYR m) and substrate inhibition ( K si PYR) constants for pyruvate, of the maximal rate of reaction ( V ′ max), of the apparent Arrhenius activation energy ( E A), and of the Gibbs free-energy change ( ΔG ‡ ) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 °C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.