STUDY OF THE OLIGOMERIC STATUS OF COPROPORPHYRINOGEN OXIDASE (CPO) USING ANALYTICAL ULTRACENTRIFUGATION (AU)

Ivan Mikula1, Borries Demeler2 and Pavel Martásek1

1Dept. of Pediatrics, 1st School of Medicine, Charles Univ., Prague, Czech Republic, 2 Dept. of Biochemistry, Univ. of Texas, San Antonio, Texas, USA

CPO catalyzes the sixth enzymatic step of the biosynthesis of the critical cofactor heme. The CPO deficiency in human causes a severe metabolic disorder, hereditary coproporhyria. CPO is active as a homodimer.

We purified human CPO using GST Fusion System (Pharmacia) to electrophoretic homogeneity (>95%). The data from the sedimentation equilibrium experiments were analyzed with the new version of UltraScan software. Monte-Carlo analysis was used to determine 95% confidence intervals of all parameter estimates. All scans were fitted to a global model describing either a single ideal species, or a reversibly self-associated monomer-dimer or monomer-dimer-tetramer system.

All three models strongly suggest that CPO is almost exclusively present in the dimeric form. We found the monomer-dimer-tetramer model as the most accurate, with the monomer MW = 37.6 kDa. Using this model, the KD for monomer-dimer was calculated to be 4.31e-7 M and for monomer-tetramer 1.39e-17 M, respectively.

AU proved to be powerful tool to address CPO oligomerization behaviour. Our results suggest monomer only being present in low nanomolar range, while minuscule amount of tetramer being present only in the highest concentrations examined. Disease causing CPO mutants will be tested now using AU to address their effect on self-association of the enzyme.

Supported by the grant GAUK 25/04 (IM & PM) and by the NSF grant DBI-9974819 (BD).