An HPLC assay for rat liver ferrochelatase activity.
A rapid, reliable, sensitive and reproducible HPLC method was developed for the assay of ferrochelatase activity in rat liver. The assay was carried out aerobically with Zn2+ and mesoporphyrin or protoporphyrin IX as substrates. Zn-porphyrins formed were extracted with dimethyl sulphoxide/methanol (30:70, v/v) containing Zn-deuteroporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for mesoporphyrin was 5.9 microM, for protoporphyrin IX 8.8 microM and for zinc 6.0 microM. The specific activities were 33.1 +/- 5.0 nmol Zn-mesoporphyrin or 13.4 +/- 2.0 nmol Zn-protoporphyrin formed per hour per mg of protein for mitochondria and 12.3 +/- 2.2 nmol Zn-mesoporphyrin or 4.6 +/- 0.9 nmol Zn-protoporphyrin per hour per mg of protein for liver homogenate.
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D005294:Ferrochelatase / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000032:analysis*; D008190:Lyases / Q000032:analysis*; D011166:Porphyrins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D011166:Porphyrins; D008190:Lyases; D005294:Ferrochelatase