Permissible discontinuity region of the α-chain of hemoglobin: Noncovalent interaction of heme and the complementary fragments α1-30 and α31-141

Ramnath Seetharam, Andy Dean, K. Subramonia Iyer, A. Seetharama Acharya

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Abstract

Generation of a fragment-complementing system of the α-chain on limited proteolysis with Staphylococcus aureus V8 protease has been investigated. Digestion of the α-chain (0.4 mM) of hemoglobin with V8 protease in phosphate buffer at pH 6.0 and 37°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 α-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 α-chain position and the minor one eluting slightly ahead of the α-chain position. Reversed-phase high-performance liquid chromatography and amino-terminal sequence analysis demonstrated that the component eluting at the α-chain position contains species generated by the noncovalent interactions of heme and the complementary fragments α1-30 and α31-141. In dilute solutions (0.04 mM) the V8 protease digestion occurred exclusively on the carboxyl side of Glu-30(α). 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 nativelike structure of the noncovalent fragment system. The dissociation constant of α1-30 appears to be in the range of 10-8 M. In tetrameric hemoglobin A the peptide bond of Glu-30-Arg-31 of the α-chain is not accessible to V8 protease digestion. The quaternary interactions of the α-chain and β-chain lead to the stabilization of this region, which represents a part of the α1β1 contact region of the tetramer.

Original languageEnglish (US)
Pages (from-to)5949-5955
Number of pages7
JournalBiochemistry
Volume25
Issue number20
Publication statusPublished - 1986
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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