Preparation of apo-cytochrome b5 utilizing heme transfer to apo-myoglobin.
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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.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.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.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.Keywords:
Cytochrome b5
Hemeprotein
Sepharose
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Cytochrome b(5) (cyt b(5)), 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 b(5) on this enzymatic system has not been investigated in details, because preparation of cyt b(5) as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b(5) 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 b(5) as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b(5) and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3-6 (10 mM KH(2)PO(4), 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 b(5) into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b(5) was successfully prepared using procedure described here.
Hemeprotein
Cytochrome b5
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Cytochrome b5
Aptamer
Hemeprotein
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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.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.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.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.
Cytochrome b5
Hemeprotein
Sepharose
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Proteins encapsulated within sol-gel matrices (SG) have the potential to fill many scientific and technological roles, but these applications are hindered by the limited means of probing possible structural consequences of encapsulation. We here present the first demonstration that it is possible to obtain high-resolution, solution NMR measurements of proteins encapsulated within a SG matrix. With the aim of determining the breadth of this approach, we have encapsulated three paramagnetic proteins with different overall charges: the highly acidic human Fe3+ cytochrome b5 (cyt b5); the highly basic horse heart cytochrome c (cyt c); and the nearly neutral, sperm whale cyanomet-myoglobin. The encapsulated anionic and neutral proteins (cyt b5; myoglobin) undergo essentially free rotation, but show minor conformational perturbations as revealed by shifts of contact-shifted peaks associated with the heme and nearby amino acids.
Hemeprotein
Cytochrome b5
Sperm whale
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Cytochrome b5
Hemeprotein
Protein Engineering
Hydrophobic effect
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Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The iron atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.
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Cytochrome b5
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The transient complex of bovine myoglobin and cytochrome b5 has been investigated using a combination of NMR chemical shift mapping, 15N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b5 amide resonances upon complex formation with either metmyoglobin (FeIII) or carbon monoxide-bound myoglobin (FeII) are more than 10-fold smaller than in other transient redox protein complexes. From 15N relaxation experiments, an increase in the overall correlation time of cytochrome b5 in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b5 titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a Ka in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b5, with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.
Metmyoglobin
Hemeprotein
Docking (animal)
Chemical shift
Proton NMR
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Hemeprotein
Resonance Raman spectroscopy
Cytochrome b5
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