Studies on ferrochelatase. The effects of thiols and other factors on the determination of activity.

1. Haems are unstable under aerobic conditions in the presence of thiols, which are used to activate the ferrochelatase enzyme; catalase inhibits this degradation of haem. In addition, thiols interfere with the determination of protohaem as its pyridine haemochromogen derivative. 2. Three ferrochelatase assays are described that minimize interference by these two reactions. Two of these assays involve measurement of porphyrin utilization, one spectrophotometrically and the second spectrofluorimetrically. The third assay measures haem formation by a pyridine haemochromogen technique. Results obtained with these three methods were in close agreement at a GSH concentration of 4mm. 3. The stimulatory effect of GSH on ferrochelatase has been confirmed. The spectrum of the haem formed is dependent on GSH concentration; at high GSH concentrations (20mm) the haem is in the reduced state, but at low concentration (4mm) the spectrum of the product resembles that of an oxidized haemoprotein such as ferrihaemoglobin. 4. The inhibitory effect of oxygen on ferrochelatase activity has been confirmed by spectrophotometric assay of porphyrin disappearance.