Regeneration and the kinetics of peroxidase inactivation

The objective of this work was to investigate why non-linear first-order kinetics of inactivation are observed when high-purity horseradish peroxidase (HRP) is heated under mildly acidic conditions. On heating HRP at 70 degrees C in acetate, pH 5.6, strong activity regeneration was found after short heating times whilst weak regeneration was observed after more extensive heating. This variation in regeneration is proposed as the major cause of the non-linear inactivation behaviour. Support for this proposal was the finding that simple first-order kinetics are followed when no regeneration occurred after heating the enzyme under neutral conditions. Using capillary electrophoresis, the major isoenzyme was shown to reform during activity regeneration. Under neutral conditions, new protein species were formed as a result of heating but these did not revert to the original isoenzyme on cooling. (C) 1997 Elsevier Science Ltd.