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Isolation and characterization of the unique prosthetic group of a green hemoprotein from human erythrocytes.

1976; Elsevier BV; Volume: 251; Issue: 13 Linguagem: Inglês

10.1016/s0021-9258(17)33337-9

1083-351X

Donald E. Hultquist, Roger T. Dean, Dan Reed,

RNA modifications and cancer

Abstract A simplified and streamlined purification scheme has been developed for the large scale isolation of a green hemoprotein from human erythrocytes. The isolation procedure involves hypotonic lysis, freezing of the hemolysate at -60 degrees, centrifugation, direct chromatography of the supernatant on DEAE-cellulose, and subsequent cation exchange, anion exchange, and gel filtration chromatography. Approximately 2 mumol of this anionic hemoprotein were isolated per liter of packed erythrocytes. The previous inability to separate the prosthetic group from the protein by conventional solvent extraction procedures was shown to be a consequence of the highly polar character of the heme and not due to covalent linkage between heme and protein. The polar nature and marked lability of the heme necessitated development of techniques for the extraction, purification, and derivatization of the prosthetic group. The heme was separated from the protein by membrane filtration in the presence of pyridine and alkali or by disc gel electrophoresis in the presence of cyanide. The heme was methylated with trimethyloxonium tetrafluoroborate and further derivatized. The heme derivatives were purified on columns of Sephadex LH-20 or alumina. Chromatography of the heme, heme methyl ester, acetylated heme ester, and the corresponding porphyrin derivatives suggests that the heme contains three carboxyl groups and one or more polar, acetylatable functional groups, probably hydroxyl groups. Spectral characterization of these compounds, as well as the derivatives resulting from reaction of the heme with NH2OH, NaHSO3, and Na2S2O4, show that the prosthetic group is a previously undescribed, formyl-containing heme that can be clearly distinguished from heme a, Spirographis heme, and all other naturally occurring prosthetic groups.