Preparation of apo-cytochrome b5 utilizing heme transfer to apo-myoglobin.
OBJECTIVE
Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized.


METHODS
Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose.


RESULTS
The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase.


CONCLUSIONS
A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here.
Mrazova
Barbora
B
Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Martinek
Vaclav
V
Martinkova
Marketa
M
Sulc
Miroslav
M
Frei
Eva
E
Stiborova
Marie
M
D000042:Absorption; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002074:Butanones; D002845:Chromatography; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D010084:Oxidation-Reduction; D011817:Rabbits; D013057:Spectrum Analysis
D001059:Apoproteins; D002074:Butanones; D009211:Myoglobin; C010857:apomyoglobin; D006418:Heme; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase