Investigations towards understanding the thermal denaturation of lactoperoxidase

Abstract

The heat induced structural changes of lactoperoxidase were evaluated using a combination of methods. The presence of two structurally distinct species was evidenced through fluorescence spectroscopy, with a midpoint transition at 65 °C. Further details on the mechanisms of thermal denaturation were obtained by molecular modelling studies and specific activity measurements. The kinetic studies showed that thermal inactivation of lactoperoxidase follows a biphasic kinetics model. Only 5.2% of the enzyme activity was preserved after 5 min of treatment at 70 °C. The highest conformational variability with temperature increase was obtained in the β-sheet motifs, mainly located on the protein surface. A revealing comparison between lactoperoxidase–substrate complexes at 25 °C and 70 °C was also provided after performing automated docking procedure; the decrease of enzyme affinity at high temperature was explained by narrowing the channel, which connects the protein surface to the binding pocket.