1. Studies on peptides. CXXVIII. Application of new heterobifunctional crosslinking reagents for the preparation of neurokinin (A and B)-BSA (bovine serum albumin) conjugates
N Fujii, Y Hayashi, S Katakura, K Akaji, H Yajima, A Inouye, T Segawa Int J Pept Protein Res. 1985 Aug;26(2):121-9.
A decapeptide corresponding to the entire amino acid sequence of neurokinin A, a porcine spinal cord peptide, was synthesized in a conventional manner using protecting groups removable by 1 M TFMSA-thioanisole in TFA. The HS-CH2CH2CO group was introduced onto the synthetic neurokinin A by reaction of 3-(S-acetyl-thiopropionyl)-thiazolidine-2-thione, followed by deacetylation with hydroxylamine. 2,4-Dinitrophenyl-p-(beta-nitrovinyl)-benzoate trapped the above HS-CH2CH2CO-neurokinin A derivative in acidic media, then BSA in basic media in nearly quantitative yield. A similar decapeptide, neurokinin B, was also synthesized and conjugated onto BSA using an alternative SH-introducing reagent, 3-(S-p-methoxybenzyl-thiopropionyl)-thiazolidine-2-thione, and the above heterobifunctional conjugating reagent.
2. Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure
N S Cheung, S Basile, B G Livett Neuropeptides. 1993 Feb;24(2):91-7. doi: 10.1016/0143-4179(93)90026-7.
Comparison of data based on the reverse-phase HPLC with two ion-pairing reagents, trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA), together with the use of two antibodies, has allowed us to identify the various tachykinins in the bovine adrenal medulla. The results show that substance P-like, neurokinin B-like, and neurokinin A-like (including its extended forms, neuropeptide K and neuropeptide gamma) immunoreactivity are present in the bovine adrenal medulla. The concentration of SP-like immunoreactivity in the adrenal medulla was found to be substantially higher than that of NKA-like and NKB-like immunoreactivity. The strategy described here, using radioimmunoassay combined with HPLC employing TFA and HFBA as the ion-pairing reagents, should be useful for the identification of tachykinins and other peptides in the central and peripheral nervous system.
3. Synthesis and immunological evaluation of N-terminal, noncrossreactive tachykinin antigens
W Neugebauer, P Elliott, A C Cuello, E Escher J Med Chem. 1988 Oct;31(10):1907-10. doi: 10.1021/jm00118a007.
The N-terminal hexa- or pentapeptide sequences of the three mammalian tachykinins substance P, neurokinin A, and neurokinin B have been synthesized by the conventional solid-phase procedure with 6-aminocaproyl-S-(acetamidomethyl)cysteine as a C-terminal spacer and attachment function. A fourth sequence, with an additional N-terminal 6-aminocaproyl residue on the substance P-hapten sequence, was cyclized N- to C-terminally. For this purpose, a four-level protection scheme has been applied: BOC-TFA for N-terminal protection and cleavage; TFA-stable but HF-labile anchoring function and side-chain protection; S-acetamidomethyl for semipermanent thiol protection. The side chain amino function of Lys was protected with NO2Z, stable against HF but readily cleaved with hydrogenation. The hapten sequences were coupled to maleimidated BSA, after the Acm group was removed by mercury/hydrogen sulfide treatment. Mice immunized with the three linear hapten sequences produced sera that were specific in enzyme-linked immunosorbant assay for the presented hapten and the respective tachykinin but displayed no crossreactivity at all toward the other haptens nor to one of the other tachykinins. It is concluded that this approach produced antisera, specific and selective for its respective mammalian tachykinins.
