RADIATION INACTIVATION OF PROTEINS - TEMPERATURE-DEPENDENT INTER-PROTOMERIC ENERGY-TRANSFER IN OX LIVER CATALASE

The radiation-inactivation method is widely used to determine the olig omeric structure of enzymes without need for solubilization or purific ation. We have used purified ox liver catalase, a tetrametric enzyme i n solution, to study energy transfer between associated protomers resp onsible for oligomer inactivation. However, after freeze-drying the te tramer dissociates into an asymmetric dimer. In the present paper we c ompare both the radiationin-activation 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 protomer was fragmented after being hit by a gamma-ray and, as expected, this proto mer was also inactivated. This result was obtained when either catalas e was in tetrameric or in dimeric forms. However, at 38-degrees-C, eve n though a single monomer was fragmented as at -78-degrees-C, the whol e dimer was inactivated. This result suggests that, at the higher temp erature, there is a transfer of energy from the fragmented protomer to the other associated protomer, causing inactivation of the whole dime r. The inactivation of oligomeric enzymes is a two-step mechanism invo lving: (1) fragmentation of the hit monomer, followed by (2) temperatu re-dependent energy transfer from the fragmented towards the associate d protomer. Thus we conclude that the radiation-inactivation size refl ects the transfer of absorbed energy inside the oligomer which causes inactivation of one or several monomers.