Studies of the Specific Role of the Subunits of Choriogonadotropin for Biological, Immunological and Physical Properties of the Hormone

Digestion of the isolated α‐subunit of choriogonadotropin by carboxypeptidase A results in the release of the five C‐terminal amino acids, 88–92 (‐Tyr‐Tyr‐His‐Lys‐Ser‐OH) [des(88–92)‐α‐subunit] indicating their superficial arrangement. By reduction of incubation time, temperature and diminution of the enzyme to protein ratio, sequential release of C‐terminal amino acids can be observed. A modified α‐subunit was obtained at which serine‐92 was split off (1.0 mol) and lysine‐91 was partially removed (0.6 mol) (des‐Lys 91 , Ser 92 ‐α‐subunit), and another one at which 0.5 mol serine was liberated (des‐Ser 92 ‐α‐subunit). The C‐terminus of the isolated β‐subunit seems to be resistant to the action of carboxypeptidase A. Using conditions which permit complete removal of the residues 88–92 at the isolated α‐subunit, the hormone seems not to be digested by carboxypeptidase A. By prolongation of the incubation time, a slow liberation of amino acids only from the C‐terminal region of the α‐subunit can be achieved. The ability of the α‐subunit to recombine with the native β‐subunit remains unaffected by the modification. Digestion of the hormone with carboxypeptidase A did not cause its dissociation. The recombination product des‐Lys 91 ,Ser 92 ‐α‐subunit + native β‐subunit displays a biological activity of 30% in comparison to hormone recombined from native subunits. Removal of further amino acids [des(88–92)‐α‐subunit + native β‐subunit] results in a complete loss of biological activity in vivo . Biological activity in vivo (rat prostate assay) and in vitro binding to the receptor of rat Leydig cells (radioligand‐receptor assay) are diminished to the same extent. An immunological determinant of the hormone, which lacks the isolated subunits seems to be completely restored at the recombination product des(88–92)‐α‐subunit + native β‐subunit as judged by means of immunodiffusion methods. The ability of choriogonadotropin to enhance the 1‐anilino‐8‐naphthalenesulfonate (ansyl) fluorescence is reduced by removal of amino acids from the C‐terminus of the α‐subunit. The biologically inactive recombination product des(88–92)‐α‐subunit + native β‐subunit shows 26% ansyl fluorescence in comparison to choriogonadotropin recombined from the native subunits. Ansyl fluorescence, however, seems not to be decreased due to a loss of binding sites for ansyl, because ansyl‐induced aggregation of choriogonadotropin is unaffected, as could be shown by investigation of the recombination products of a modified α‐subunit and the native β‐subunit by means of gel permeation chromatography on Biogel P 200 thin‐layers in presence of 1 mmol ansyl/l. It is concluded that diminution of ansyl fluorescence very sensitively reports differences in the conformation between native and modified choriogonadotropin. As demonstrated by means of CD measurements, conformation is changed when amino acids are removed from C‐terminus of the isolated α‐subunit. Liberation of the residues 88–92 results in an increase of the CD at 224 nm, which likely corresponds to formation of α‐helix structure. The data presented in this paper indicate that the C‐terminal amino acids 88–92 on the whole are responsible for maintaining the native conformation of the α‐subunit. When this conformation is lost the modified α‐subunit is suggested to be unable to alter the conformation of β‐subunit effectively. Thus, the receptor binding site of the hormone is presumably not organized into its active state.