Ovine lutropin subunit isolation. Comparison of salt precipitation and countercurrent distribution procedures.

A series of comparative studies on the chemical and biological characterizations of the ovine lutropin subunits and their recombinants prepared by a countercurrent distribution (CCD) procedure and a salt precipitation method is presented. The salt precipitation Method B (0.1 M acetate and 3 M NaCl) gave a low yield of pure subunits and the subunits thus prepared recombined poorly to form a product with very low biological activity. The properties of subunits from the CCD procedure were about the same as those from salt precipitation Method A (0.01 nacetate and 3 M NaCl). In general, the CCD subunits recombined better than the salt precipitation Method A subunits, whereas the recombinant of the latter was more active in the in vitro bioassay than the former. However, all recombinants failed to recover 100% of the original lutropin activity. The circular dichroic spectra of the recombined lutropin preparations were different from those of intact lutropin. The recombinant from CCD subunits gave higher ellipticity at the 235 nm extremum, while that from the salt precipitation subunits gave poor pattern recovery in the region of 275 to 280 nm. Scatchard analyses of the 12”I-labeled intact and recombined lutropin indicated that all recombined lutropin preparations belong to a population that is different from the intact lutropin. The intact lutropin has a higher affinity for the receptors than the recombined lutropin preparations. This unique affinity was irreversibly disrupted by the separation and recombination of the subunits regardless of which method was used for subunit preparation. All the LY subunits thus prepared had extraneous lysine at their NH, termini (determined by the dansyl chloride method) when compared with the original lutropin. After reduction, LY subunit preparations dissociated into three bands in sodium dodecyl sulfate-gel electrophoresis. It is concluded that the a subunit might be damaged by the acid dissociation of the hormone used for subunit isolation. Based on the present studies, a method was devised for subunit isolation from ovine lutropin avoiding acidic condi-