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Biophys J, October 2000, p. 2155-2161, Vol. 79, No. 4

Radiation Inactivation of Ribonucleotide Reductase, an Enzyme with a Stable Free Radical

Gordon Bolger,* Michel Liuzzi,* Richard Krogsrud,* Erika Scouten,* Robert McCollum,* Ewald Welchner,* and Ellis Kempnerdagger

 *Department of Biological Sciences, Boehringer Ingelheim (Canada) Limited, Bio-Méga Research Division, Laval, Québec H7S 2G5, Canada; and  dagger Laboratory of Physical Biology, National Institutes of Health, Bethesda, Maryland 20892 USA

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of ~210 kDa and the other of ~20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Biophys J, October 2000, p. 2155-2161, Vol. 79, No. 4
© 2000 by the Biophysical Society   0006-3495/00/10/2155/07  $2.00


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G. C. Ness, L. C. Pendleton, and M. J. McCreery
Target Size Analysis by Radiation Inactivation: The Use of Free Radical Scavengers
Experimental Biology and Medicine, July 1, 2005; 230(7): 455 - 463.
[Abstract] [Full Text] [PDF]


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