Characterization of globin domains: Heme binding to the central exon product

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Abstract
The peptide fragments coded for by the 3 exons of the human .beta.-globin gene were prepared and isolated using the arginine-specific protease clostripain (EC 3.4.22.8). The region encoded by the central exon (amino acid residues 31-104) contains an Arg at position 40. This site was less susceptible to cleavage than the 2 sites that correspond to the exon-intron boundaries, and the isolated central fragment was an approximately equimolar mixture of the entire central fragment, .beta.o31-104, and the somewhat smaller fragment contained within it, .beta.o41-104. This central fragment mixture bound heme stoichiometrically and tightly as micromolar concentrations, generating a strong Soret absorption band and a characteristic absorption band in the visible spectrum. The Sorbet band occurred at the same wavelength and had the same shape as in Hb, exhibiting an intensity greater than 2/3 that achieved when native intact .beta.-globin is reconstituted with heme. Nearly the full intensity was regained when an equivalent of heme was added to the unfractionated digest, suggesting that the noncovalently associated side fragments add precision to the fit of the heme pocket. Three controls were used in establishing the specificity of heme binding to the central fragment mixture. Similar, but preliminary, experiments were also undertaken with .alpha.-globin. A clostripain digest containing .alpha.o1-31 and .alpha.o32-141 bound heme, yielding a Soret band identical to that observed in .alpha. subunits reconstituted from the native globin chains and heme. Measurements of circular dichroism spectra as indices of secondary structure suggested a role for the side exon products in the acquisition of the native 3-dimensional structure of Hb. These experiments confirm a prediction of Gilbert that the product of the central exon of the globin gene is a complete functional domain that binds heme tightly and specifically.