REDUCTION AND REOXIDATION OF EQUINE GONADOTROPIN ALPHA-SUBUNITS

Ovine (o) and equine (e) LH alpha-subunits were reduced and reoxidized using conditions known to be effective for bovine and human alpha-subunits. The major product of oLHalpha refolding was alpha-subunit monomer. In contrast, eLHalpha formed a 121,000 mol wt aggregate. Monomeric eLHalpha was recovered, but in greatly reduced yield. To test the effects of carbohydrate variation on the aggregation of equine alpha-subunits, all of the equine gonadotropin alpha-subunits (eFSHalpha, eCGalpha, eLHalpha, and free alpha-subunit) were reduced and reoxidized. In each case, the major product was the 121,000 mol wt aggregate accompanied by monomeric equine alpha. Removal of carbohydrate by trifluoromethane sulfonic acid hydrolysis accentuated the tendency to aggregation during reoxidation. Most reduced-reoxidized deglycosylated eLHalpha did not enter a 12% sodium dodecyl sulfate-polyacrylamide gel. The highest LH receptor-binding activities were found in the alpha-subunit preparations, eLHalpha itself and pituitary free alpha-subunit. Operationally, the latter was separated from eLH in the last step of the eLH purification procedure; thus, LH contamination in this preparation is likely. Reduction and reoxidation reduced the LH receptor-binding activity of these two preparations to the level of LH activity observed in the eFSHalpha and eCGalpha preparations. We concluded that the majority of the LH receptor-binding activity observed in equine alpha-subunit preparations was due to contamination with eLH. We also obtained preliminary evidence that the amino-terminal and carboxy-terminal fragments of proteolytically "nicked" equine alpha-subunits refolded properly to form alpha monomer.