Use of Cross-linking to Assess Subunit Interaction of Recombinant Human Coproporphyrinogen Oxidase

Jason R Stephenson,Marjorie A Jones,Jon A Friesen,Nancy E Thomas

doi:10.3844/ajbbsp.2005.103.106

Jason R Stephenson, Marjorie A Jones + Show 2 more

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https://doi.org/10.3844/ajbbsp.2005.103.106

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Abstract

To provide further evidence for a dimeric form of coproporphyrinogen oxidase reported using the conventional hydrodynamic methods, bifunctional cross-linkers were incubated with purified, recombinant human coproporphyrinogen oxidase to determine subunit interaction in solution. The use of cross-linkers provides an effective way to demonstrate subunit association and allows for assessment of activity upon covalent cross-linking. Following incubation with selected cross-linkers, enzyme apparent molecular weight was evaluated using SDS-PAGE and enzymatic activity was monitored by spectroscopy following HPLC. The predominate multimeric form of coproporphyrinogen oxidase observed had a mass that corresponded to a dimer, indicating that coproporphyrinogen oxidase most likely functions as a homodimer in solution.

Highlights

Coproporphyrinogen oxidase is the sixth enzyme in the heme biosynthetic pathway[1]
Yoshinaga and Sano[6] purified copro’gen oxidase from bovine liver and determined the molecular structure to be a 71.6 kDa monomer, whereas Kohno et al.[7] reported the protein from bovine liver to be a homodimer, with an apparent molecular weight of 74 kDa
Due to the selectivity of the cross-linkers, we can probe the abundance of lysine or cysteine residues at the subunit interface. We report both the apparent molecular weight and the enzymatic activity of copro’gen oxidase with and without incubation with selected cross-linkers

Summary

Introduction

Coproporphyrinogen oxidase (copro’gen oxidase [E.C. 1.3.3.3]) is the sixth enzyme in the heme biosynthetic pathway[1]. Batlle et al.[2] purified copro’gen oxidase from rat liver and determined it to be an 80 kDa monomer. Copro’gen oxidase purified from S. cerevisiae was shown to be a homodimer by Camadro et al.[3], whereas Poulson and Polglase[4] determined the protein from the same species to be a monomer.

Results

Conclusion