PURIFICATION AND RECEPTOR BINDING PROPERTIES OF COMPLEXES BETWEEN LUTROPIN AND MONOVALENT ANTIBODIES AGAINST ITS α SUBUNIT

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Abstract
A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its .alpha. subunit, which is common to the glycoprotein hormones, was purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex was heterogeneous with respect to molecular size; 70-80% of the hormone was complexed with either 2 or 3 Fab fragments. The LH-Fab .alpha. complexes retained only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permitted direct measurement of competition between receptor and the bovine complex because the .alpha. portion of the human hormone did not cross react significantly with antibodies directed against bovine .alpha. subunits.) Complex formation did not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-.beta. immunoassay which distinguishes free .beta. subunit from intact LH. Complexing of LH with Fab-.alpha. fragments also caused little or no change in the affinity of the hormone''s .beta. subunit for anti-LH-.beta. antibodies indicating that significant changes in .beta. subunit conformation did not occur. At least 2 well-separated antigenic regions on the .alpha. subunit are exposed to the surface in the intact hormone. The loss of binding activity to receptor is evidently due to steric effects rather than to changes in conformation or dissociation, and there may be sites on the .alpha. subunit which interact directly with the receptor.

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