Purification of cytochrome P-450 from n-hexadecane-grown Acinetobacter calcoaceticus.
A convenient procedure for the purification of cytochrome P-450 from an n-alkane-assimilating strain of the bacterial species Acinetobacter calcoaceticus has been elaborated. The cytochrome P-450 of n-hexadecane-grown cells was found to be distributed in cell-free extracts among particulate and soluble subcellular fractions. For isolation, cytochrome P-450 was extracted from particulate fractions by addition of Triton X-100 to the buffer. Subsequently, purification to an apparently homogeneous state was achieved by chromatography on DEAE-cellulose, Sepharose CL-6B and hydroxylapatite cellulose. A Mr of 52,000, an isoelectric point of 4.7 and the amino acid composition were determined. The Soret bands of absolute absorption spectra showed maxima at 417 nm for the oxidized form, at 408 nm for the reduced form and at 448 nm for the carbon monoxide compound of the reduced cytochrome. Difference spectra with octylamine showed maxima at 432 nm and minima at 410 nm indicating the predominance of the low-spin state. Conversion to the high-spin state could not be obtained. The isolated cytochrome P-450 was stable in the presence of Triton X-100 under neutral pH conditions. Removal of the detergent or change of the pH to values higher than 8.0 or lower than 6.0 resulted in the destruction of the cytochrome P-450.
Müller
R
R
Department of Biochemistry, Karl Marx University Leipzig, GDR.
Asperger
O
O
Kleber
H P
HP
D000150:Acinetobacter / Q000254:growth & development / Q000378:metabolism*; D000473:Alkanes / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002474:Cell-Free System; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D013347:Subcellular Fractions / Q000378:metabolism
D000473:Alkanes; D000596:Amino Acids; D006882:Hydroxyapatites; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; C007932:n-hexadecane