Limitations in the use of tritiated methyltrienolone for the photoaffinity labelling of androgen receptor proteins.

In the absence of photoexcitation and under conditions of low ionic strength, the native form of the androgen receptor in rat prostate sediments as a large, 9.2S complex with tritiated androgens, including [3H]methyltrienolone. On photoexcitation, the configuration of labelled receptor complexes changes to a form of lower sedimentation coefficient, 4.2S. Initial experiments indicated that photoaffinity labelling of the androgen receptor protein may be readily achieved and with extensive covalent attachment of [3H]methyltrienolone. However, from ultracentrifugation analyses conducted under denaturing conditions it was established that, at best, only 5-8% of available [3H]methyltrienolone is covalently attached to the receptor protein. The remaining [3H]methyltrienolone is presumably adsorbed or entrapped within the receptor protein and may resist extraction into organic solvents, but it is not authentically bound in an irreversible, covalent manner. Our findings raise doubts concerning the efficiency and usefulness of [3H]methyltrienolone as a photoaffinity reagent for androgen receptor proteins. An additional problem is that photoillumination of [3H]methyltrienolone leads to the attachment of radioactivity to non-receptor proteins present in human plasma.