To determine the types of brain cells responsible for the production of amyloid beta peptides (A beta), as well as their carboxyl-terminal properties, we studied the secretion of A beta in rat neuronal, astrocytic, microglial and meningeal primary cell cultures. All four types of cells produced A beta, among which neurons secreted approximately 4 times more A beta than other cell types. The percentage of A beta42 ending at position 42 as a fraction of total A beta was similar between different cell types, ranging from 10 to 15%. These results suggest that neurons might be the most potent source for A beta production in the brain, although other non-neuronal type cells could also contribute to this process.

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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.

In this paper, a new method is described for N-terminal amino acid sequencing of peptides using the fluorescent reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS). Sequence determination is carried out by identifying thiazolinone (TZ) amino acids, which are generally unstable and difficult to detect. The employed system can easily and quickly derive TZ amino acids using the Edman reaction with DBD-NCS; these amino acids are also stable enough to be efficiently detected by high-performance liquid chromatography. Resultant detection limits for DBD-TZ amino acids range from 50 fmol to a sub-picomole level (S/N = 3). This system successfully analyzed sequences of Leu5-enkephalin (25 pmol) and angiotensin I (100 pmol) using fluorometric detection at 524 nm with excitation at 387 nm.

It is shown that an electron-withdrawing or -donating group at the para-position of aromatic isothiocyanate significantly affects the racemization of 2,1,3-benzoxadiazolylthiazolinone (TZ) derivatives of amino acids, derivatized with newly synthesized benzoxadiazolyl isothiocyanates in Edman sequence analysis. A linear relationship between the logarithms of the TZ-amino acid enantiomer ratio and the para-substituent constants (s p ) for the isothiocyanate moiety was obtained, and the D/L configuration of the amino acid residue was retained with an isothiocyanate containing an electron-donating group at the para-position. The para-substitution effect on the racemization of phenylthiohydantoin (PTH) amino acids was also confirmed by several para-substituted phenylisothiocyanate (PITC) reagents, including nitro-PITC, chloro-PITC, PITC, methyl-PITC and methoxy-PITC. The relationship between the fluorescence intensity of the 2,1,3-benzoxadiazolyl TZ amino acid and s p was also demonstrated. When the isothiocyanate containing an electron-donating group was used, the fluorescence intensity of the TZ-amino acid decreased while retaining the D/L configuration of the amino acid residues.

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The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/β-cyclodextrins, Ph/CD) were newly prepared by modification of β-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation (α ≥ 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a d-amino acid ([d-Thr(2)]leucine enkephalin-Thr).