Preparation of apo-cytochrome b5 utilizing heme transfer to apo-myoglobin

OBJECTIVES: Cytochrome b 5 (cyt b 5 ), 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 b 5 , but also that of apo-cyt b 5 . To prepare apo-cyt b 5 , heme transfer from native cyt b 5 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 b 5 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 b 5 at pH 4.2 that is the optimal pH for heme transfer from cyt b 5 into apo-myoglobin. The apo-cyt b 5 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 b 5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b 5 . Oxidized and reduced forms of the apo-b 5 reconstituted with heme exhibit the same absorbance spectra as native cyt b 5 . The prepared apo-cyt b 5 reconstituted with heme can receive electrons from NADPH:CYP reductase. CONCLUSION: A biologically active apo-cyt b 5 was prepared using transfer of heme from cyt b 5 to horse heart apo-myoglobin by the procedure described here.