An HPLC assay for rat liver ferrochelatase activity

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Abstract
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 μM, for protoporphyrin IX 8.8 μM and for zinc 6.0 μM. 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.