The radiation-inactivation method is widely used to determine the oligomeric structure of enzymes without need for solubilization or purification. We have used purified ox liver catalase, a tetrameric enzyme in solution, to study energy transfer between associated promoters responsible for oligomer inactivation. However, after freeze-drying the tetramer dissociates into an asymmetric dimer. In the present paper we compare both the radiation-inactivation size (obtained by following the activity decay) and the target size (obtained by measuring the amount of remaining protein by SDS/PAGE) of catalase under various states of aggregation and temperature. At −78 degrees C, only one promoter was fragmented after being hit by a gamma-ray and, as expected, this protomer was also inactivated. This result was obtained when either catalase was in tetrameric or in dimeric forms. However, at 38 degrees C, even though a single monomer was fragmented as at −78 degrees C, the whole dimer was inactivated. This result suggests that, at the higher temperature, there is a transfer of energy from the fragmented protomer to the other associated protomer, causing inactivation of the whole dimer. The inactivation of oligomeric enzymes is a two-step mechanism involving: (1) fragmentation of the hit monomer, followed by (2) temperature-dependent energy transfer from the fragmented towards the associated protomer. Thus we conclude that the radiation-inactivation size reflects the transfer of absorbed energy inside the oligomer which causes inactivation of one or several monomers.
- © 1994 The Biochemical Society, London