Ligand-induced self-association of human chorionic gonadotropin. Positive cooperativity in the binding of 8-anilino-1-naphthalenesulfonate

Abstract

Human chorionic gonadotropin (hCG) self-associates to form higher molecular weight species in the presence of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium and fluorescence titration data have been analyzed in terms of a monomer-dimer-tetramer model in which the various oligomers have different affinities and/or capacities for the ligand. The results indicate that the ligand affinities are in the order tetramer greater than dimer greater than monomer whereas the numbers of ligand binding sites per mole of hCH are in the reverse order. Consequently, addition of ANS first shifts the equilibrium from monomer to tetramer and gives rise to positive cooperativity in the titration curves. At sufficiently high ANS concentration (approximately 0.5 mM), the equilibrium shifts back to the dimer because of its greater binding capacity. This is manifested by a second phase in the titration curve and a decrease in the polarization of ANS fluorescence. The results are discussed in terms of the general problem of ligand controlled protein association and are contrasted to results reported to the previous paper for the homolgous protein, human luteinizing hormone.

Kinetics of recombination of the subunits of human chorionic gonadotropin. Effect of subunit concentration

“…The concentration of ANS required for half-maximal saturation was ~20 µ . However, subsequent studies of this interaction revealed that hCG self-associates to form dimers ( 2ß ) and tetramers (onfif) in the presence of ANS and that enhancement of ANS fluorescence was due to binding by these oligomers (Ingham et al, 1975). This finding compelled us to confirm that ANS fluorescence is enhanced only by the native intact form of the hormone and that the presence of ANS in the recombination mixture does not influence the rate of that reaction.…”

“…This conclusion is further supported by the agreement between the rates of recombination as measured by the two assays (Figure 2). The fact that hCG self-associates to form dimers (o^/Sa) and tetramers (a^f) in the presence of ANS (Ingham et al, 1975) was utilized in the present report as an aid in the purification of commercial hCG by gel chromatography (see Methods). A similar approach might be useful for relieving subunit preparations of contamination by intact hormone.…”

“…A second source of hCG was a commercial preparation (Sigma) which was purified to a potency comparable to the CR117 preparation by gel chromatography (see below). Concentrations of hCG were based on the absorbance at 276 nm using e 1.2 X 104 l./mol/cm (Ingham et al, 1975).…”

“…After adding 30 µ of a solution of ANS in acetone (10 mg/ml), the sample was reapplied to the same column which was equilibrated and eluted with 100 µ ANS (0.01 M phosphate, pH 7.3, no KC1). Under these conditions, hCG self-associates to form higher oligomers (Ingham et al, 1975) and the partition coefficient, Kav, was reduced from 0.31 to 0.11. This allows hCG to be separated from other proteins of similar molecular weight which do not self-associate in the presence of ANS.…”

Studies of the Specific Role of the Subunits of Choriogonadotropin for Biological, Immunological and Physical Properties of the Hormone. Digestion of Choriogonadotropin and Its Isolated Subunits with Serine Carboxypeptidase

“…Some diminution is observed in the recombination product α-native-+ des-( 143-145 )-|3-subunit. Ans induces aggregation of choriogonadotropint 25 '. This was used to investigate whether the reduced Ans-choriogonadotropin fluorescence of recombination products containing a modified a-and a native (3-subunit might be caused by a loss of binding ability for Ans by a portion of the recombined hormone.…”