An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.

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