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 ap ple cultivars (Malus domestica Borkh. cvs. Braeburn, Gala, Jonagold, McInto sh, Red Delicious, and Spartan). Activity of CAT was determined for fresh a nd frozen tissue of the same fruit. Freezing resulted in reductions of 50 t o 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 red uction of CAT activity, with slower freezing procedures leading to greater losses in activity. Six additives to the extraction buffer were tested to e valuate their potential to reduce the inactivation of CAT from frozen tissu e, but only EDTA and Tween 20 showed any benefit. However, EDTA and Tween 2 0 provided only partial recovery in CAT activity. In contrast, crude extrac ts prepared from fresh tissue showed no appreciable loss in CAT activity af ter frozen storage for two weeks at -80 degreesC. Gel electrophoresis and i mmunological analysis indicated that the loss in CAT activity from tissue f reezing could be attributed to loss of both the tetrameric CAT enzyme struc ture and total CAT protein. The implications of using freezing to preserve apple tissue samples prior to catalase activity analysis is discussed.