Covalent attachment of a progestational steroid to chick oviduct progesterone receptor by photoaffinity labelling

Abstract

The chick oviduct progesterone receptor has been characterised and purified by us^1–3and shown to consist of a mixture of two hormone-binding proteins^4,5. These two proteins have equivalent ligand-binding sites, to which progesterone and other progestational steroids bind with high affinity (k_diss = 10⁻⁹ M)^6,7. Due to the noncovalent nature of this interaction, analysis of the proteins in denaturing or nonequilibrium conditions has always been hampered by ligand–protein dissociation during the experiments (t_1/2 = 12 h at 0 °C, 25 min at 37 °C)⁶. Efforts to use covalent attachment of steroids by alkylation or photoactivation have been largely unsuccessful, both for this protein and for other receptors⁸. However, covalent labelling of steroid isomerases⁹ and an androgen-binding protein¹⁰ has been obtained in reasonable yields using photoactivation of keto steroids containing multiple double bonds in conjugation with the ring-A ketone. We now report the first successful covalent attachment of a radiolabelled steroid to its receptor protein. The ligand chosen was a synthetic progestin, 17α,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020; Roussel-Uclaf)¹¹. R5020 is available as the tritiated 17α-methyl derivative (≥50 Ci mmol⁻¹) and has been used extensively for measuring mammalian progesterone receptors in normal^12,13 and neoplastic tissues^14,15. This steroid was chosen for the photoaffinity study because its absorption maximum is at 320 nm, thereby allowing its activation by UV light at wavelengths above 300 nm, where protein damage would be minimised. The method should be generally applicable to other progestin receptors.