1. Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami
M Patthy, D H Schlesinger, J Horvath, M Mason-Garcia, B Szoke, A V Schally Proc Natl Acad Sci U S A. 1986 May;83(9):2969-73. doi: 10.1073/pnas.83.9.2969.
Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.
2. Proton: a major factor for the racemization and the dehydration at the cyclization/cleavage stage in the Edman sequencing method
H Matsunaga, T Santa, T Iida, T Fukushima, H Homma, K Imai Anal Chem. 1996 Sep 1;68(17):2850-6. doi: 10.1021/ac951253r.
The racemization of the liberated 7-[(N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl)-thiazoli none (DBD-TZ) amino acid during the cyclization/cleavage reaction with trifluoroacetic acid (TFA) in the Edman sequencing procedure has been carefully investigated, and evidence is presented to show conclusively that the racemization is caused by the replacement of a hydrogen atom by TFA. The fluorescent reagent 7-[N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) was used for amino acid sequencing, and DBD-TZ amino acid was used for sequence and configuration determination. DBD-thiocarbamoylated peptides were cyclized and cleaved with deuterated TFA, and the protonated pseudomolecular ions (M-d1 + H)+ of DBD-TZ amino acids were detected by LC/MS. Furthermore, in the reaction kinetics study, we confirmed that the replacement reaction by TFA correlated sufficiently with the racemization of DBD-TZ amino acids. For the purpose of retaining D/L-amino acid configuration in sequencing, we used an aprotic acid, i.e., the Lewis acid boron trifluoride (BF3), for the cyclization/cleavage reaction. When we used BF3, the derivatized DBD-TZ amino acid was scarcely racemized under cyclization/cleavage conditions. Using this method, amino acid sequencing of D-Phe-Met-Arg-Phe-amide could be performed, retaining the D/L-configuration of the amino acid residues.
3. Separation of 17 DL-amino acids and chiral sequential analysis of peptides by reversed-phase liquid chromatography after labeling with R(-)-4- (3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole
T Toyo'oka, N Tomoi, T Oe, T Miyahara Anal Biochem. 1999 Dec 1;276(1):48-58. doi: 10.1006/abio.1999.4307.
Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid.
