INVESTIGATIONS ON RADIOLABELING SUBSTANCE-P DERIVATIVES AND REVERSED-PHASE LIQUID-CHROMATOGRAPHIC SEPARATION OF THE PRODUCTS

In investigations of radiolabeling substance P1-11 (SP) with I-125 iso tope, its [Tyr(8)]-derivative was subjected to the lactoperoxidase (LP O) technique, whereas the native substance P undecapeptide was coupled with I-125-tyrosin-N-hydroxy-succinimidyl ester to the corresponding I-125-Bolton-Hunter SP derivative. For separation of the desired compo nents from their by-products, labeled [Tyr(8)]-SP was pre-purified by ion-exchange chromatography (IEC) on carboxymethyl cellulose and subse quently investigated by gradient high performance reversed-phase liqui d chromatography (gRP-HPLC) with 0.1 M triethyl-ammonium phosphate in acetonitrile (pH 2.5) on an octadecylsilyl silica gel (RP-18) stationa ry phase. When the total amount of labeled peptide affording a specifi c activity of approx. 2 x 10(6) Ci/M was checked by gRP-HPLC, the LPO technique yields about an equimolar mixture of I-125-[Tyr(8)]-(Met(11) )-SP and I-125-[Tyr(8)]-(Met(11)-->O)-SP. However, in contrast, substa ntial enrichment of I-125-[Tyr(8)]-(Met(11))-SP was observed when more retained IEC fractions were analyzed. The I-125-Bolton-Hunter reagent was coupled in about 54 % yield to the SP undecapeptide at slightly a lkaline conditions and separated from by-products only by gRP-HPLC yie lding a specific activity of approx. 2 x 10(6) Ci/M. Both radiolabeled derivatives of SP are further characterized by their binding properti es to a specific antibody raised in rabbits against synthetic SP1-11 W hen individual fractions of either radioiodinated [Tyr8]-SP or the cor responding I-125-Bolton-Hunter derivative were investigated with respe ct to their binding properties to the antibody, only those fractions c oinciding with the maximum amount of radioactivity showed a satisfacto ry ratio of specific vs. non-specific antibody-binding, especially in the case of the I-125-Bolton-Hunter derivative. Therefore, the observe d results are in good accordance with the existence of the desired tar get components.