Doubly Radioiodinated Luteinizing Hormone: Preparation and Characterization*

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Abstract
Bovine [131I]iodo-.alpha.LH[luteinizing hormone]-[125I]iodo-.beta.LH (**LH) was prepared and shown to be physically and biologically equivalent to unmodified hormone. The .beta.-subunit was modified with 125I, purified by adsorption to concanavalin A-Sepharose and elution with methylmannoside, added to .alpha.-subunit, and allowed to reassociate to intact hormone. Iodination with 131I was then carried out in the reassociation mixture and **LH was isolated by gel filtration. Both gel electrophoresis and rechromatography on Sephadex G-100 showed that both radiolabels comigrated with unmodified hormone. Sodium dodecyl sulfate gel electrophoresis showed that 131I was in the .alpha.-subunit and 125I in the .beta.-subunit; this result is in agreement with studies by others which show that the tyrosines of the .beta.-subunit are nonreactive in intact hormone. In receptor-binding assays, both radiolabels were specifically displaced in a similar fashion by LH. Scatchard analysis showed high affinity binding (Ka .simeq. 1.5 .times. 1010 M-1) for both labels. Comparison of receptor-binding activity with steroidogenic activity showed that iodinated hormone molecules not only bound to receptor but also stimulated testosterone production. The demonstration that full biological activity is retained with iodination in both subunits shows that such doubly labeled LH can be used to monitor the disposition of both subunits simultaneously during interaction of the hormone with target cells.