title: Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates.
abstract: Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen.
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan.
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Kawano
authors.forename: Kouji
authors.initials: K
authors.lastname: Uda
authors.forename: Ryuji
authors.initials: R
authors.lastname: Otsuki
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D017931:DNA Primers; D009211:Myoglobin