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

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.