Formation of zinc protoporphyrin IX from myoglobin in porcine heart extract

The formation of zinc protoporphyrin IX (ZPP) in porcine heart extract was investigated by visible absorption and fluorescent spectral analysis. Characteristic absorption peaks of ZPP were observed at 417, 546, and 584 nm after anaerobic incubation of the extract with metmyoglobin and ZnCl2 in the dark at 30 degrees C for 72 h. In the fluorescent spectra (excitation: 410 nm), a strong peak due to ZPP was obtained at 589 nm. The time-course study showed that ZPP formation greatly increased after a lag period, during which metmyoglobin was reduced. ZPP formation was significantly facilitated by the use of oxymyoglobin. Zinc chelatase activity in the extract was estimated as 138 mU/mL-extract and its specific activity was 2.95 mU/mg-protein. The difference in activities with aerobic and anaerobic incubation was not significant. Measurements at pH 5.5-7.0 showed that activities were higher at lower pH. The activity of zinc chelatase was stimulated by the presence of ATP, and the highest activity was obtained at an ATP concentration of 2.5 mg/mL.