Purification and characterization of cytochrome b5 from the housefly (Musca domestica)

Isopropanol treatment of Musca domestica (house fly) microsomes extracted cytochrome b5 and resulted in a minimum 5.2-fold purification. 2. Cytochrome b5 was highly purified from the isopropanol extract using ion exchange and reverse phase high performance liquid chromatogrphy. 3. A polyclonal antiserum was produced against the purified cytochrome b5. 4. Purified house fly cytochrome b5 had a molecular mass of 19.7 kDa, slightly higher than those reported for rabbit and rat microsomal cytochrome b5 (16.6–16.7 kDa), but lower than cytochrome b5 from Mediterranean fruit fly (21 kDa). 5. Reduced house fly cytochrome b5 absorbed maximally at 424 nm, similar to other cytochromes b5. 6. Amino acid analysis of house fly cytochrome b5 produces at higher percentage of hydrophobic amino acid residues and less acidic residues than other cytochromes b5. 7. In immuno-blot analysis, house fly cytochrome b5 shared antigenic epitope(s) with the dipteran species Musca autumnalis (face fly), Stomoxys calcitrans (stable fly) and to a lesser extent, Drosophila melanogaster (fruit fly). 8. Anti-house fly cytochrome b5 did not recognize cytochrome b5 from Apis mellifera (honey bee), Tricoplusia ni (cabbage looper), Tetranychus urticae (two-spotted spider mite) or Rattus rattus (rat).