2000 年 128 巻 3 号 p. 455-461
A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarhii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys (Acm)7, 24. 27 Lys (Boc)19]-Prc-MIH (1-39)-SCH2CH2CO-Nle-NH2 and H-[Cys (Acm)40. 44, 53 Lys (Boc)42, 51, 67]-Prc-MIH (40-75)-NH2, were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH (1-75)-NH2. This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys 7 and Cys 44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys 24 and Cys 40 and between Cys 27 and Cys 53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.