Physicochemical characterization of native and asialo human chorionic gonadotropin.

Human chorionic gonadotropin (HCG) was desialized by treatment with neuraminidase to yield asialo-HCG, and comparative physicochemical studies were carried out on HCG and asialo-HCG. The E:trn value at 276 nm was 5.47 for HCG and 5.72 for asialo-HCG. Intrinsic viscosities of HCG and asialo-HCG at near isoionic point were estimated to be 3.4 and 3.2 ml per g, respectively. HCG had an isoionic point of 5.02 as compared with that of 6.13 for asialoHCG. Acid-base titration curves of the two proteins were different, the difference corresponding to a loss of strongly acidic groups, the number of which equals approximately that of neuraminic acid residues removed from HCG. The electrostatic interaction factor obtained for side chain carboxyl groups of asialo-HCG showed good agreement with that calculated for the same group of a rigid sphere model of the same molecular weight. Only 4 of the 7 tyrosyl residues of HCG were reversibly titratable tinder normal conditions with an apparent pK of 10.3, and complete ionization of the tyrosyl groups was possible only in the presence of 5.4 M guanidine hydrochloride or after exposure of the protein to pH 12.8 at 5” for 6 weeks. In asialo-HCG the ionization was time-dependent and irreversible under normal conditions and all the seven tyrosyl groups were titratable when the sample was allowed to stand for 1 week at 5” and pH 12.8. The intrinsic pK value and the interaction factor estimated for the four tyrosyl groups of HCG suggested that these groups are normally reactive. The circular dichroism spectra of HCG and asialo-HCG in the ultraviolet region were essentially the same at near-neutral or high pH and did not show the presence of any secondary structure in the molecule. A comparison of the near-ultraviolet spectra in neutral and alkaline media indicated that microconformational changes took place at high pH. These results indicate that both HCG and asialo-HCG are compact, nearly spherical molecules and have a similar gross conformation. However, tyrosyl residues of asialoHCG are more easily accessible to solvent molecules than those of HCG, suggesting that desialization of HCG results in minor conformational changes to reduce the stability of the molecule at alkaline pH. It is established that AcNeul residues of the HCG molecule are essential for the biological but not for the immunological activity of the hormone (l-6). The loss of biological activity of HCG after removal of the AcNeu residues has recently been attributed to rapid removal of the desialized product (asialo-HCG) from the circulation by the liver (7). Also it has been shown that with in vitro systems asialo-HCG elicits biological activity to nearly the same extent as intact HCG (8, 9). However, little information is available as to the effect of desialization of HCG on its conformation. This report describes the results of a series of experiments in which AcNeu was selectively removed from HCG by treatment with neuraminidase and comparative physicochemical studies were conducted on HCG and desialized HCG with such parameters as viscosity, acid-base titration, spectrophotometric titration, and CD.