An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver.
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.
Li
F
F
Division of Clinical Cell Biology, MRC Clinical Research Centre, Harrow, Middx., U.K.
Lim
C K
CK
Peters
T J
TJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010088:Oxidoreductases / Q000032:analysis*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains
D011524:Protoporphyrins; C011539:protoporphyrinogen; C028025:protoporphyrin IX; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase