Biosynthetic preparation of isotopically labeled heme.
An efficient method for the preparation of isotopically enriched heme has been developed. This method utilizes a commercially available bacterial host and plasmid, into which a synthetic gene encoding for rat liver outer mitochondrial membrane cytochrome b5, a heme-binding protein, has been inserted. The method described in this report utilizes the efficient synthesis of the cytochrome b5 polypeptide together with the enhanced biosynthesis of heme brought about by addition of the first committed precursor in heme biosynthesis, delta-aminolevulinic acid. Apocytochrome b5 sequesters heme as the macrocycle is being synthesized in order to form holocytochrome b5, thus avoiding toxic concentrations of free macrocycle in the cell. Relatively high concentrations of free heme in the cell have been shown to stimulate excretion of heme precursors such as coproporphyrinogen and uroporphyrinogen (W. F. Harris III, R. S. Burkhalter, W. Lin and R. Timkovich, (1993) Bioorg. Chem. 21, 209-220), therefore causing isotopic dilution of the labeled material. The heme obtained using this methodology was determined to be > 85% enriched. Because the heme in cytochrome b5 is not covalently attached to the polypeptide, it can be extracted and used in other applications. Use of glutamate, a precursor of delta-amino-levulinate biosynthesis in Escherichia coli, did not result in high levels of isotopic incorporation into heme, thus pointing out to the importance of using a labeled precursor that is committed to heme biosynthesis in order to obtain high levels of isotopic labeling.
Rivera
M
M
Department of Chemistry, Oklahoma State University, Stillwater 74074, USA.
Walker
F A
FA
D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes; D015786:Cytochromes b5 / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D006418:Heme / Q000096:biosynthesis*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D051381:Rats
D002247:Carbon Isotopes; D018698:Glutamic Acid; D006418:Heme; D000622:Aminolevulinic Acid; D015786:Cytochromes b5