Optimalization of preparation of apocytochrome b5 utilizing apo-myoglobin
Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3-6 (10 mM KH2PO4, pH 3-6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here.
Altuve A, Lijun Wang L, Benson DR and Rivera M. (2004) Mammalian mitochondrial and microsomal cytochromes b5 exhibit divergent structural and biophysical characteristics. Biochem Biophys Res Comm314: 602-609.
Hargrove MS, Barrick D and Olson JS (1996) The Association Rate Constant for Heme Binding to Globin Is Independent of Protein Structure. Biochemistry35: 11293-11299.
Hargrove MS, Singleton EW, Quillin ML, Ortiz LA, Phillips GN, Jr., Olson JS and Mathews AJ (1994) His64- (E7)-Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. J Bio Chem269: 4207-4214.
Miksanova M, Igarashi J, Minami M, Sagami I, Yamauchi S, Kurokawa H and Shimizu T (2006) Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition. Biochemistry45: 9894-905.
Rossi-Fanelli A, Antonini E, Caputo A. (1959) Studies on the structure of hemoglobin. II. Properties of reconstituted protohemoglobin and protoporphyringlobin. Biochim Biophys Acta35: 93-101.
Shenkman JB and Jansson I. (2003) The many roles of cytochrome b5. Pharm Therap97: 139-152.
Stiborová M, Martínek V, Schmeiser HH and Frei E. (2006) Modulation of CYP1A1-mediated oxidation of carcinogenic azo dye Sudan I and its binding to DNA by cytochrome b5. Neuro Endocrinol Let27 (Suppl. 2): 35-39.
Wiechelman KJ, Braun RD, Fitzpatrick JD. (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem175: 231-237.
Yamazaki H, Shimada T, Martin MV and Guengerich FP. (2001) Stimulation of cytochrome P450 reactions by apo-cytochrome b5: evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase. J Biol Chem276: 30885-30891.