Protoporphyrlnogen Oxidase Guo et al 1991

Protoporphyrinogen oxidase catalyzes the six-electron oxidation of protopor-phyrinogen IX to protoporphyrin IX. Application of the assay to leukocytes provides a more convenient enzyme source than liver tissues in the diagnosis of porphyrias.

The product, protoporphyrin IX, is separated from mesoporphyrin, the internal standard, on an ODS-Hypersil column (5 mm X 250 mm). The mobile phase was 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Fluorescence detection was used with excitation at 400 nm and emission at 618 nm.

Protoporphyrinogen IX was prepared fresh by reduction of protoporphyrin IX with sodium and amalgam. The incubation mixture contained 100 fiL of leukocyte suspension (about 0.5 mg of protein) and 50 fiL of 0.25 M Tris-HC1 buffer (pH 8.6) containing 5 mM EDTA, 5 mM glutathione, and 1% Tween-20 (w/v). After preincubation at 37°C in the dark for 5 minutes, the reaction was started by adding 100 fiL of approximately 35 ¡xM protoporphyrinogen IX substrate. After 10 minutes, the reaction was stopped by adding 1 mL of ice-cold methanol-DMSO (8:2, v/v) containing 42 nM mesoporphyrin as internal standard. The mixture was centrifuged, and the resulting supernate was flushed with nitrogen before HPLC analysis. A boiled leukocyte suspension was used in a parallel assay to correct for formation of protoporphyrin IX by autoxidation. Protoporphyrin IX formation was linear up to 10 minutes and was proportional with leukocyte protein up to 1 mg.

Enzyme activity was measured in leukocytes isolated from heparinized blood.

Figure 9.61 shows chromatogram for this assay.

Figure 9.61 HPLC for the determination of protoporphyrinogen oxidase activity in human leukocytes, (a) Enzyme incubation mixture, (b) Blank incubation with boiled enzyme. Column ODS-Hypersil (250 mm X 5.0 mm i.d.); eluent, 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Flow rate, 1.5 mL/min; fluorescence detection, excitation at 400 nm and emission at 618 nm. Peaks: 1, mesoporphyrin (internal standard); 2, protoporphyrin IX. (From Guo et al., 1991.)

Figure 9.61 HPLC for the determination of protoporphyrinogen oxidase activity in human leukocytes, (a) Enzyme incubation mixture, (b) Blank incubation with boiled enzyme. Column ODS-Hypersil (250 mm X 5.0 mm i.d.); eluent, 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Flow rate, 1.5 mL/min; fluorescence detection, excitation at 400 nm and emission at 618 nm. Peaks: 1, mesoporphyrin (internal standard); 2, protoporphyrin IX. (From Guo et al., 1991.)

time (mini

time (mini

Was this article helpful?

0 0

Post a comment