Circular dichroic spectroscopy of Arg46-nicked ovine lutropin α and derived fragments
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Theα subunit of ovine lutropin can be nicked with the endoproteinase Arg-C to give a single cleavage of the Arg46-Ser47 peptide bond. Following reduction by sulfitolysis, the N-terminal (residues 1–46) and C-terminal (residues 47–96) fragments can be separated and then recombined and reoxidized to yield a reconstituted nickedα that binds to theβ subunit but exhibits only 2–3% of the receptor-binding potency of intact lutropin. We have investigated nickedα, the two separated fragments, and reconstituted nickedα by circular dichroic spectroscopy and compared the spectra with those of intactα and reduced, reoxidized intactα. Between 200 and 225 nm the spectra of the two intact preparations are similar, as are the spectra of the two nicked preparations. However, the extremum negative ellipticities of the nicked preparations are substantially less than those of the intact preparations between 210 and 220 nm, indicating a loss in secondary structure accompanying cleavage of the Arg46-Ser47 bond. The sum of the spectra of the two fragments is significantly different from that of reconstituted nickedα, showing that the secondary structures in the isolated fragments are quite different from that of the reconstituted nicked protein. Reduced receptor binding by lutropin preparations containing a nickedα subunit may be attributable in part to the loss of secondary structure, probably helicity.
Key wordsCircular dichroism lutropinα Arg-C cleavage
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