Investigations on Radiolabeling Substance P Derivatives and Reversed-Phase Liquid Chromatographic Separation of the Products

Abstract

In investigations of radiolabeling substance P_1–11 (SP) with 125I isotope, its [Tyr8]-derivative was subjected to the lactoperoxidase (LPO) technique, whereas the native substance P undecapeptide was coupled with 125I-tyrosin-N-hydroxy-succinimidyl ester to the corresponding 125I-Bolton-Hunter SP derivative. For separation of the desired components from their by-products, labeled [Tyr8]-SP was pre-purified by ion-exchange chromatography (IEC) on carboxymethyl cellulose and subsequently investigated by gradient high performance reversed-phase liquid chromatography (gRP-HPLC) with 0.1 M triethyl-ammonium phosphate in acetonitrile (pH 2.5) on an octadecylsilyl silica gel (RP-18) stationary phase. When the total amount of labeled peptide affording a specific activity of approx. 2 × 106 Ci/M was checked by gRP-HPLC, the LPO technique yields about an equimolar mixture of 125I-[Tyr8]-(Met11)-SP and 125I-[Tyr8]-(Met11O)-SP. However, in contrast, substantial enrichment of 125I-[Tyr8]-(Met11)-SP was observed when more retained IEC fractions were analyzed.

The 125I-Bolton-Hunter reagent was coupled in about 54 % yield to the SP undecapeptide at slightly alkaline conditions and separated from by-products only by gRP-HPLC yielding a specific activity of approx. 2 × 106 Ci/M. Both radiolabeled derivatives of SP are further characterized by their binding properties to a specific antibody raised in rabbits against synthetic SP_1–11. When individual fractions of either radioiodinated [Tyr8]-SP or the corresponding 125I-Bolton-Hunter derivative were investigated with respect to their binding properties to the antibody, only those fractions coinciding with the maximum amount of radioactivity showed a satisfactory ratio of specific vs. non-specific antibody-binding, especially in the case of the 125I-Bolton-Hunter derivative. Therefore, the observed results are in good accordance with the existence of the desired target components.