Oxygen and radical scavengers in radiation-inactivation of trypsin in vitro

It has been known for about 30 years that the inactivation of biologically active macromolecules irradiated in vitro with X or gamma rays exhibit the dose-response phenomenon illustrated in a figure. As shown in the figure, in pure buffer (control) the inactivation proceeds more or less at the same rate in the absence or in the presence of oxygen, as is very frequently observed with biological macromolecule such as DNA and enzymes. When a thiol is added to the solution, a marked oxygen effect becomes apparent. The common interpretation of this finding is still that proposed in 1960 by Howard-Flanders in terms of the so-called radical repair model. According to this model the enhanced inactivation produced by oxygen in the presence of a thiol compound is due to the inhibition, by oxygen, of the repair of the transient radiation damage on the target molecule, by hydrogen donation from the thiol. In other words, the difference between the upper two curves in Fig. 1 would be the result of a competition between damage fixation by oxygen and damage repair by H donation, the former reaction being much faster than the latter. This interpretation, however, completely disregards the fact that thiyl radicals themselves react rapidly with oxygen to give, most likely, the corresponding thiol peroxyl radicals. The authors have reported evidence that this is actually the case and postulated that peroxyl radicals of glutathione, cysteine and penicillamine can be revealed in pulse radiolysis by their absorption spectra. They have also considered the possibility that the oxygen effect as shown in Fig. 1 might be due to the inactivation produced by GSOOsm-bullet. The present paper gives a preliminary account of studies carried out in order to resolve the ambiguity outlined in the previous paragraphs. In these studies gamma-ray inactivation of trypsin (Try) in dilute aqueous solutions containing reduced glutathione (GSH) or ethanol (EtOH) was investigated. 7 refs., 3 figs