Isolation and partial purification of cytochrome oxidases from Bacillus thuringiensis subsp. israelensis HD-567

Analyses of the cell membrane fractions by spectral absorbance revealed the presence of cytochrome c and three cytochrome oxidases in Bacillus thuringiensis subsp. israelensis HD-567-cytochromes a+a3, d, and o. A modified procedure was used to purify the cytochrome c:o complex from this organism. The oxidase complex was first solubilized from a sonic-disrupted cell membrane fraction (R3 fraction) using deoxycholate and KCl. The resulting soluble fraction was further purified by Sephadex G-50 gel filtration and DEAE ionexchange chromatography. TMPD oxidase specific activity and cytochrome concentration were assayed to monitor the purification procedures. The F7 fraction (obtained after G-50 chromatography) contained cytochrome c (0.44 nmole/mg protein), cytochrome a+a3 (0.26 nmole/mg protein), and cytochrome o (0.38 nmole/mg protein), which had 6-fold, 3.4-fold and 18.9-fold increase, respectively, by comparison with the original R3 fraction. The TMPD oxidase specific activity of the F7 fraction also increased 4.8 fold. The F8 fraction (obtained after the final DEAE chromatography) contained a cytochrome c:o complex only (cytochrome c, 0.46 nmole/mg protein; cytochrome o, 0.16 nmole/mg protein), but no cytochrome a+a3 was found. Both the TMPD oxidase specific activity and the cytochrome o were attenuated greatly in comparison with the F7 fraction. SDS-PAGE analysis revealed that the F7 fraction contained numerous protein components, while the F8 fraction contained only a prominent major protein of 113.5 kD thought to be the cytochrome c:o complex, and a minor polypeptide (MW = 65.8 kD). Although the final DEAE procedure removed many undesired polypeptides, TMPD oxidase activity and cytochrome o component were also lost greatly. Kinetic studies of this cytochrome c:o complex is in progress in our laboratory.