Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide.

Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(II) catalyze hydroxyl free radical formation from H2O2. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of OH produced from H2O2 as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from H2O2 over the low level obtained in the buffer system only. The cations, Mg2+ and Ca2+, even at much higher than physiological levels and much higher than the level of added Fe(II), did not cause a substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H2O2 to yield OH. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated OH formation from H2O2 demonstrated that Fe(II) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(II) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing OH formation from H2O2; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H2O2 catalysis to yield OH. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when H2O2 is present with ferrous-nucleotide complexes.