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 bact erial host and plasmid, into which a synthetic gene encoding for rat l iver outer mitochondrial membrane cytochrome b(5) a heme-binding prote in, has been inserted. The method described in this report utilizes th e efficient synthesis of the cytochrome b(5) polypeptide together with the enhanced biosynthesis of heme brought about by addition of the fi rst committed precursor in heme biosynthesis, delta-aminolevulinic aci d. Apocytochrome b(5) sequesters heme as the macrocycle is being synth esized in order to form holocytochrome b(5), thus avoiding toxic conce ntrations of free macrocycle in the cell. Relatively high concentratio ns 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 labele d material. The heme obtained using this methodology was determined to be >85% enriched. Because the heme in cytochrome b(5) is not covalent ly attached to the polypeptide, it can be extracted and used in other applications. Use of glutamate, a precursor of delta-aminolevulinate b iosynthesis in Escherichia coli, did not result in high levels of isot opic incorporation into heme, thus pointing out to the importance of u sing a labeled precursor that is committed to heme biosynthesis in ord er to obtain high levels of isotopic labeling. (C) 1995 Academic Press , Inc.