Effect of freezing on the activity of catalase in apple flesh tissue

Catalase (CAT, EC 1.11.1.6) activity was measured in flesh tissue of six apple cultivars (Malus domestica Borkh. cvs. Braeburn, Gala, Jonagold, McIntosh, Red Delicious, and Spartan). Activity of CAT was determined for fresh and frozen tissue of the same fruit. Freezing resulted in reductions of 50 to 90% in CAT activity compared with the activity measured in crude extracts from fresh tissues. The rate of freezing had an impact on the level of reduction of CAT activity, with slower freezing procedures leading to greater losses in activity. Six additives to the extraction buffer were tested to evaluate their potential to reduce the inactivation of CAT from frozen tissue, but only EDTA and Tween 20 showed any benefit. However, EDTA and Tween 20 provided only partial recovery in CAT activity. In contrast, crude extracts prepared from fresh tissue showed no appreciable loss in CAT activity after frozen storage for two weeks at -80 degrees C. Gel electrophoresis and immunological analysis indicated that the loss in CAT activity from tissue freezing could be attributed to loss of both the tetrameric CAT enzyme structure and total CAT protein. The implications of using freezing to preserve apple tissue samples prior to catalase activity analysis is discussed.