title: Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata.
abstract: The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: R S
authors.initials: RS
authors.lastname: Zafar
authors.forename: R E
authors.initials: RE
authors.lastname: Weber
authors.forename: P K
authors.initials: PK
authors.lastname: Sharma
authors.forename: S N
authors.initials: SN
authors.lastname: Vinogradov
authors.forename: D A
authors.initials: DA
authors.lastname: Walz
mesh_terms: D000818:Animals; D004912:Erythrocytes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011077:Polychaeta / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D010100:Oxygen