INVESTIGATIONS ON THE LIGHT-EMITTING SPECIES IN THE REACTION OF METMYOGLOBIN AND METHEMOGLOBIN WITH HYDROGEN-PEROXIDE

The formation of a compound I type ferryl complex in the reaction of m ethemoglobin (MetHb) and metmyoglobin (MetMyo) with hydrogen peroxide is accompanied by strong chemiluminescence. An approach to identify th e nature of the light-emitting species was made by the use of quencher s and sensitizers reacting with singlet oxygen and compounds interferi ng in the formation and reactivity of other reactive oxygen species. S inglet oxygen is not the source of light emission. This could be concl uded from the results obtained using the specific singlet oxygen trap 9,10-anthracenedipropionic acid (ADPA) in combination with high-perfor mance liquid chromatography (HPLC) analysis. The singlet oxygen adduct of ADPA was not formed in the incubation systems (MetHb or MetMyo/H2O 2). Instead, ADPA was oxidized by the ferryl ion to a different oxidat ion product, which was characterized by HPLC and IR spectroscopy. In t he case of MetHb-related chemiluminescence, light emission does not re sult from a single source. Both, SH-groups and O-2(radical-anion) radi cals are involved because blocking of thiol-groups with N-ethylmaleimi de (NEM) and scavenging of O-2(radical-anion) (by superoxide dismutase ) suppressed chemiluminescence by 50% and 30%, respectively. Developme nt of MetMyo-related chemiluminescence is not dependent on thiol group s (which are not present in the globin moiety) and also O-2(radical-an ion) is not involved. Although generation of chemiluminescence in MetH b and MetMyo seems to follow different mechanisms, both types of light -emitting species are sensitive to antioxidants, such as uric acid and ascorbate. The detection of the respective free radicals by means of ESR demonstrates that both MetHb- and MetMyo-mediated chemiluminescenc e is associated with a strong one-electron oxidizing species, which se ems to be identical with the light-emitting source itself. Also desfer al, which was originally used to exclude the involvement of a Fenton-t ype reaction, was readily oxidized to the nitroxide free radical assoc iated with a strong decrease of chemiluminescence. This quenching effe ct was not dependent on iron complexation because the addition of iron was ineffective. In summary, chemiluminescence is not restricted to a single chemical process but is related to different one-electron tran sfer reactions from globin residues to the oxo-heme center.