CIRCULAR DICHROIC SPECTROSCOPY OF ARG(46)-NICKED OVINE LUTROPIN-ALPHAAND DERIVED FRAGMENTS

The alpha subunit of ovine lutropin can be nicked with the endoprotein ase Arg-C to give a single cleavage of the Arg(46)-Ser(47) peptide bon d. Following reduction by sulfitolysis, the N-terminal (residues 1-46) and C-terminal (residues 47-96) fragments can be separated and then r ecombined and reoxidized to yield a reconstituted nicked alpha that bi nds to the beta subunit but exhibits only 2-3% of the receptor-binding potency of intact lutropin. We have investigated nicked alpha, the tw o separated fragments, and reconstituted nicked alpha by circular dich roic spectroscopy and compared the spectra with those of intact alpha and reduced, reoxidized intact alpha. Between 200 and 225 nm the spect ra of the two intact preparations are similar, as are the spectra of t he two nicked preparations. However, the extremum negative ellipticiti es of the nicked preparations are substantially less than those of the intact preparations between 210 and 220 nm, indicating a loss in seco ndary structure accompanying cleavage of the Arg(46)-Ser(47) bond. The sum of the spectra of the two fragments is significantly different fr om that of reconstituted nicked alpha, showing that the secondary stru ctures in the isolated fragments are quite different from that of the reconstituted nicked protein. Reduced receptor binding by lutropin pre parations containing a nicked alpha subunit may be attributable in par t to the loss of secondary structure, probably helicity.