The syntheses of four trifunctional reagents for alkylating sulfhydryl groups in proteins are described: (N-\gamma)-maleimidobutyrylbiocytinyl-tyramine (compound I) and its sulfone, (N) (\gamma)-maleimidobutyrylbiocytinyltyrosine (compound II), and (N^{\alpha}-4)-maleimidobutyrylbiocytinamido- (2^{\prime})-[p-(hydroxyphenyl)-propionamido] ethane (compound III). Each reagent contains a maleimide function capable of reacting with (\mathrm{SH}) groups, a (p)-hydroxyphenyl group that can be iodinated, and a "biotin handle" to facilitate purification of the clerivatized proteins or peptides derived from them by biotin-avidin affinity chromatography. Detailed conditions for obtaining the pure di-iododerivatives of the compounds have been developed. The biotin is attached to all the reagents via the (\varepsilon)-amino group of lysine (biocytin) to provide sufficient space for optimum binding to avidin. The half-times (\left(t_{1 / 2}\right)) for dissociation of compound I from succinoyl avidin ( 36.7 days), its monoiodo ( 26.1 days) and di-iodo derivatives ( 21.4 days), and compound I sulfone ( 29.8 days), demonstrate that iodination does not significantly interfere with binding of the biotin residue to succinoyl avidin and that these reagents can be used effectively as affinity ligands. Remarkably, all the reagents can be iodinated without loss of their sulfhydryl alkylating capacity. Alkylation of highly purifitd human placental insulin receptor with the di-iodo derivatives of the reagents resulted in sizgnificant incorporation of ({ }^{125} \mathrm{I}) into the (\beta)-subunit of the receptor and the alkylation was prevented by prior exposure of the receptor to NEM. The advantages of these reagents over those previously available are that the parent molecules (1) are inexpensive to prepare. (2) are solids that can be stored indefinitely without degradation, (3) and can be radiolabeled to specific activity levels over seventy times higher with ({ }^{13!} \mathrm{I}) than the specific activity available for ({ }^{3} \mathrm{H}) derivatives. 1995 Academic Press. Inc.