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Purification and characterization of murine protoporphyrinogen oxidase
Abstract:
The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.
Insights
Protoporphyrinogen oxidase, crucial for heme synthesis, was purified from mouse liver mitochondria. This research details its biochemical properties and catalytic activity.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial Biology
Background:
- The heme biosynthetic pathway is essential for cellular function.
- Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in this pathway.
- Mitochondrial localization of this enzyme is critical for its function.
Purpose of the Study:
- To purify protoporphyrinogen oxidase from mouse liver mitochondria.
- To characterize the biochemical and kinetic properties of the purified enzyme.
- To understand the enzyme's role in heme biosynthesis.
Main Methods:
- Enzyme purification using sodium cholate solubilization, ammonium sulfate fractionation, and multiple chromatography techniques (Sepharose CL-6B, phenyl-Sepharose, DEAE-5PW).
- Molecular weight determination via gel filtration and SDS-PAGE.
- Enzyme kinetics measured using protoporphyrinogen IX as substrate.
- pH optimum and cofactor analysis.
Main Results:
- Purified protoporphyrinogen oxidase exhibits a molecular weight of approximately 65,000 Da.
- The enzyme shows high affinity (Km 5.6 µM) and activity (Vmax 2300 nmol mg-1 h-1) for protoporphyrinogen IX.
- A broad pH optimum around 7.1 was observed, with no requirement for divalent cations or sensitivity to sulfhydryl reagents.
- No chromophoric cofactor was detected in the purified enzyme.
Conclusions:
- Mouse liver protoporphyrinogen oxidase has been successfully purified and characterized.
- The enzyme's kinetic and biochemical properties provide insights into heme biosynthesis regulation.
- This purified enzyme serves as a valuable tool for further research into heme metabolism disorders.