Biosynthetic preparation of isotopically labeled heme

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An efficient method for the preparation of isotopically enriched home 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 heine-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 home brought about by addition of the first committed precursor in home biosynthesis, δ-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, 209220), therefore causing isotopic dilution of the labeled material. The heine obtained using this methodology was determined to be >85% enriched. Because the heine 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 δ-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. © 1995 Academic Press, Inc.

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Analytical Biochemistry

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