High-performance liquid chromatographic assays for protoporphyrinogen oxidase and ferrochelatase in human leucocytes.
Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 +/- 0.96 microM (mean +/- S.D.). The mean (+/- S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 +/- 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn2+ as substrates, the Michaelis constants were 1.49 and 8.33 microM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn-protoporphyrin or 2.05 nM Zn-mesoporphyrin formed per h per mg protein.
Guo
R
R
Division of Clinical Cell Biology, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Lim
C K
CK
Peters
T J
TJ
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004795:Enzyme Stability; D005227:Fatty Acids; D005294:Ferrochelatase / Q000097:blood*; D005420:Flavoproteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008875:Middle Aged; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000097:blood*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011524:Protoporphyrins / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents; D015032:Zinc / Q000737:chemistry
D003902:Detergents; D005227:Fatty Acids; D005420:Flavoproteins; D024101:Mitochondrial Proteins; D011524:Protoporphyrins; D013439:Sulfhydryl Reagents; C011539:protoporphyrinogen; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase; D005294:Ferrochelatase; D015032:Zinc