Takafumi Ohta

Photochemical nitrosation of dimethylamine in aqueous solution containing nitrite

Dimethylamine was found to be nitrosated photochemically in aqueous solution containing nitrite both by the irradiation with a high pressure mercury lamp and by the exposure to sunlight to give nitrosodimethylamine, a well-known carcinogen. The nitrosation occurred more readily at alkaline pH than at acidic and neutral pH. These findings suggest that nitrosodimethylamine is produced photochemically under natural environmental conditions.

Formation of mutagens from tryptophan by the reaction with nitrite

Summary Tryptophan treated with nitrite under acidic conditions was found to be mutagenic to Salmonella typhimurium tester strains TA 100 and TA 98 both in the presence and in the absence of S-9 mix. Tryptamine, glycyltryptophan and indole were also mutagenic when treated with nitrite, suggesting that the appearance of mutagenic activity from tryptophan was attributable to the reaction of nitrite with the indole ring. Nitrite-treated arginine and proline were not mutagenic in the presence of S-9 mix.

Formation of a diazoquinone-type mutagen from acetaminophen treated with nitrite under acidic conditions

Acetaminophen (paracetamol) showed mutagenicity to Salmonella typhimurium TA100 and TA98 either with or without S9 mix when treated with excess nitrite in acidic solution. The mutagen was isolated and identified as 4-acetylamino-6-diazo-2,4-cyclohexadienone. Mutation tests and product analysis by high-performance liquid chromatography, however, suggested that this type of mutagen was hardly formed in the digestive tract of normal subjects.

Selective extraction of β-blockers from biological fluids by column-switching high-performance liquid chromatography using an internal-surface phenylboronic acid precolumn

A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of beta-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 microliters) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed briefly, the selectively retained beta-blockers were eluted with methanol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non-treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol.

Application of an anhydrotrypsin-immobilized precolumn for selectvie separation of peptides having arginine or lysine at theinr C-termini by column-switching high-performance liquid

A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol—silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loadd with water as a carrier solvent and the precolum was washed with 10–30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having d-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC separation of peptides.

Preparation of anhydrotrypsin-immobilized diol silica as a selective adsorbent for high-performance affinity chromatography of peptides containing arginine or lysine at their C-termini

SummaryAnhydrotrypsin (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with trifluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high-performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed was accomplished.

Application of a β-cyclodextrin sulfate-immobilized precolumn to selective on-line enrichment and separation of heparin-binding proteins by column-switching high-performance liquid chromatography

Abstract A column-switching high-performance liquid chromatography (HPLC) system which consisted of a β-cyclodextrin (β-CD) sulfate-immobilized hydrophilic vinyl–polymer gel precolumn and a reversed-phase analytical column was developed for the selective on-line enrichment and separation of heparin-binding proteins. Of 15 proteins investigated, 10 proteins having heparin-binding activity were retained on the β-CD sulfate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for basic fibroblast growth factor constructed at various sample volumes were nearly identical, indicating that the protein could be enriched by this system. The system was successfully used for the selective separation of lysozyme in egg white. The β-CD sulfate-immobilized precolumn showed no loss of analytical performance over 2 years during which about 400 samples were analysed.

Preparation of anhydrochymotrypsin diol silica as selective adsorbent for affinity chromatography of peptides with aromatic amino acids at C-termini

SummaryAnhydrochymotrypsin (AHC), a catalytically inert derivative of chymotrypsin in which the serine-residue active site has been converted chemically to a dehydroalanine residue, was immobilized on diol silica by activation with trifluoroethanesulfonyl chloride. A AHC-diol-silica column was used for high-performance affinity chromatographic separation of peptides with aromatic amino acids at their C-termini from other peptides. Faster separations were achieved.

The reaction of tryptophan with cystine during acid hydrolysis of proteins: Formation of tryptathionine as a transient intermediate in a model system

Under the conditions commonly used to hydrolyze proteins with 6 M HCl, tryptophan reacted with cystine to give a transient intermediate, which was isolated and identified as 2-(2-amino-2-carboxyethylthio)tryptophan (tryptathionine) by NMR studies, etc. Studies on the formation and degradation of the above compound showed that beta-3-oxoindolylalanine and cysteine, which were previously reported to be the main degradation products of tryptophan and cystine, respectively, are formed by the hydrolysis of this intermediate during the course of the reaction.

Spectrophotometric determination of N-nitroso compounds by flow injection analysis

A spectrophotometric method for the determination of N-nitroso compounds by flow injection analysis, based on a denitrosation reaction with hydrogen bromide-acetic acid followed by reaction of the liberated nitrite with the Griess reagent, is described. Under the optimum reaction conditions, the detection limit for N-nitrosopyrrolidine (NPYR) was 5 × 10–8M for a sample volume of 200 µl and the relative standard deviations (n= 9) for 1 × 10–7 and 5.4 × 10–6M NPYR were 3.8 and 0.5%, respectively. Various other N-nitroso compounds gave a similar level of response. Phenol, sorbic acid and methanol did not interfere with the determinations, whereas piperazine interfered seriously with the determination of nitrosoproline. The sampling rate was 25 samples h–1. The method was successfully applied to the analysis of two anti-cancer drug formulations, viz., nimustine hydrochloride and 1-(2-chloroethyl)-3-(methyl-α-D-glucopyranos-6-yl)-1-nitrosourea injections.