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Characterization of globin domains: heme binding to the central exon product
Insights
The central exon of the human beta-globin gene encodes a functional domain that tightly binds heme. This domain, when isolated, demonstrates specific heme-binding properties crucial for hemoglobin structure and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The human beta-globin gene comprises three exons, each encoding specific regions of the globin protein.
- Understanding the functional contribution of individual exons is key to deciphering protein structure-function relationships.
Purpose of the Study:
- To isolate and characterize peptide fragments of human beta-globin corresponding to its three exons.
- To investigate the heme-binding properties of the fragment encoded by the central exon.
- To explore the role of exon-encoded fragments in the structural integrity of hemoglobin.
Main Methods:
- Enzymatic digestion of human beta-globin using the arginine-specific protease clostripain.
- Isolation and purification of peptide fragments.
- Spectroscopic analysis (Soret and visible absorption bands) to assess heme binding.
- Circular dichroism spectroscopy to evaluate secondary structure.
Main Results:
- The central exon fragment (beta(o) 31-104) was isolated as a mixture with a smaller internal fragment (beta(o) 41-104).
- This fragment mixture bound heme stoichiometrically and tightly, exhibiting characteristic Soret and visible absorption bands similar to intact hemoglobin.
- Preliminary studies with alpha-globin fragments also indicated specific heme binding.
- Circular dichroism data suggested that fragments from side exons contribute to native hemoglobin's three-dimensional structure.
Conclusions:
- The central exon product of the globin gene functions as a complete domain capable of tight and specific heme binding.
- Non-covalently associated fragments may refine the heme pocket's fit, enhancing binding precision.
- Exon products play a role in achieving the native three-dimensional structure of hemoglobin.
Abstract:
We have prepared and isolated the peptide fragments coded for by the three exons of the human beta-globin gene, 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 arginine at position 40. This site was less susceptible to cleavage than the two sites that correspond to the exon-intron boundaries, and the isolated central fragment was an approximately equimolar mixture of the entire central fragment, beta(o) (31-104), and the somewhat smaller fragment contained within it, beta(o) (41-104). This central fragment mixture bound heme stoichiometrically and tightly at micromolar concentrations, generating a strong Soret absorption band as well as a characteristic absorption band in the visible spectrum. The Soret band occurred at the same wavelength and had the same shape as in hemoglobin, 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 have also been undertaken with alpha globin. A clostripain digest containing alpha(o) (1-31) and alpha(o) (32-141) 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 three-dimensional structure of hemoglobin. These experiments confirm a prediction of W. Gilbert that the product of the central exon of the globin gene is a complete functional domain that binds heme tightly and specifically.