Loss of Bioreactivity and Preservation of Immunoreactivity of Iodothyrotropin-Releasing Hormone*

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Abstract
Radioiodinated TRH [thyrotropin releasing hormone] binds to antibodies raised against noniodinated TRH and it has been suggested but not proven that iodo-TRH may possess biological activity. To test the properties of iodo-TRH, the hormone was iodinated with variable amounts of both stable (127I) and radioactive (125I or 131I) iodide using chloramine-T. Depending upon the molar ratio of TRH to I, various proportions of diiodinated TRH (peak I) and monoiodinated TRH (peak II) could be synthesized. Separation of iodo-TRH (peak II) from TRH could be achieved by elution of multiple small fractions from Sephadex G-10 column. The position of TRH was identified by Pauly''s reagent. The mean partition coefficients (Kav) .+-. SD were: TRH = 0.31 .+-. 0.05; iodo-TRH (peak II) = 0.75 .+-. 0.09; and iodide = 2.02 .+-. 0.03. After repurification, iodo-TRH devoid of noniodinated TRH could be prepared. [125I]- and [131I]TRH, bound to TRH antibodies, and 95-105% of the iodinated TRH still reacted with anti-TRH serum after incubation for 3 h at 37.degree. C with fresh normal human or rat serum. TRH displaced [125I]TRH from the antibodies before but not after incubation with serum (95% inactivation after incubation for 3 h at 37.degree. C). In contrast, [127I]TRH displaced [125I]TRH from the antibodies equally well before and after incubation with serum. Addition of iodide to TRH produced an extinction of the reactivity with Pauly''s reagent and loss of the bioreactivity, as measured in vivo by the stimulation of TSH and PRL [prolactin] release or in vitro by displacement of [3H]TRH from rat pituitary cells (GH3) grown in tissue culture. Immunologically, iodo-TRH is indistinguishable from TRH and is resistant to inactivation by fresh serum. Iodo-TRH is devoid of bioreactivity and does not react with Pauly''s reagent. The presence of an unencumbered histidyl residue on TRH is important for the expression of its bioreactivity, enzymatic degradation, and reactivity with Pauly''s reagent. In contrast, iodide addition to the histidyl residue of TRH does not alter its immunoreactivity. Results obtained from experiments using radioiodinated TRH in vivo or in the presence of serum in vitro should be interpreted with great caution.

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