Permissible discontinuity region of the alpha-chain of hemoglobin: noncovalent interaction of heme and the complementary fragments alpha 1-30 and alpha 31-141.
Generation of a fragment-complementing system of the alpha-chain on limited proteolysis with Staphylococcus aureus V8 protease has been investigated. Digestion of the alpha-chain (0.4 mM) of hemoglobin with V8 protease in phosphate buffer at pH 6.0 and 37 degrees C is limited to the peptide bonds of Glu-23, Glu-27, Glu-30, and Asp-47. Gel filtration of a V8 protease digest of the alpha-chain on a Sephadex G-50 column did not release any heme to the low molecular weight region, though some peptides were released from the protein. The filtration studies revealed the presence of two heme-containing components in the digest, the major one eluting at the alpha-chain position and the minor one eluting slightly ahead of the alpha-chain position. Reversed-phase high-performance liquid chromatography and amino-terminal sequence analysis demonstrated that the component eluting at the alpha-chain position contains species generated by the noncovalent interactions of heme and the complementary fragments alpha 1-30 and alpha 31-141. In dilute solutions (0.04 mM) the V8 protease digestion occurred exclusively on the carboxyl side of Glu-30(alpha). This high selectivity was also observed at pH 4.0 and pH 7.8. The visible spectra and the ultraviolet circular dichroic spectra of the digest reflect the native-like structure of the noncovalent fragment system. The dissociation constant of alpha 1-30 appears to be in the range of 10(-8) M. In tetrameric hemoglobin A the peptide bond of Glu-30-Arg-31 of the alpha-chain is not accessible to V8 protease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D010450:Endopeptidases; D006418:Heme / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism*; D006801:Humans; D006902:Hydroxymercuribenzoates; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D012697:Serine Endopeptidases
D000596:Amino Acids; D006902:Hydroxymercuribenzoates; D046911:Macromolecular Substances; D010446:Peptide Fragments; C030601:4-hydroxymercuribenzoate; D006418:Heme; D006441:Hemoglobin A; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase