Kazuya Nakagomi

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma.

We previously described a novel angiotensin‐I‐converting enzyme (ACE) inhibitory peptide, designated Acein‐1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein‐2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed‐phase gradient and isocratic high performance liquid chromatography. Acein‐2 was found to be a tripeptide, Leu‐Ile‐Tyr, which is thought to correspond to f(518–520) of human α2‐macroglobulin. The synthetic tripeptide showed a potent dose‐dependent inhibition of ACE, with an IC50 value of 0.82 μmol/l. Lineweaver–Burk plots suggested that Acein‐2 as well as the previously described Acein‐1 are non‐competitive inhibitors.

High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization.

An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.

Acein-1, a novel angiotensin-I-converting enzyme inhibitory peptide isolated from tryptic hydrolysate of human plasma

A novel angiotensin‐I‐converting enzyme (ACE) inhibitory peptide, designated acein‐1, was isolated from the tryptic hydrolysate of human plasma. Gel filtration and cation exchange chromatography were performed to purify this peptide, followed by reversed‐phase gradient and isocratic high‐performance liquid chromatography. Acein‐1 was found to be a heptapeptide, Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala‐Arg, corresponding to f(138–144) of human serum albumin. The synthetic heptapeptide, hexapeptide (Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala, des‐7R acein‐1) and octapeptide (Tyr‐Leu‐Tyr‐Glu‐Ile‐Ala‐Arg‐Arg, acein‐1R) showed dose‐dependent inhibitions of ACE, and their IC50 values were 16 μmol/l, 500 μmol/l and 86 μmol/l, respectively. Acein‐1 might be a non‐competitive inhibitor, while acein‐1R may be an uncompetitive inhibitor, as shown by Lineweaver‐Burk plots.

Inhibitory effects of pectic substances on activated hyaluronidase and histamine release from mast cells

In this paper, we report the effect of pectic substances and D-galacturonic acid, the main constituent of pectic substances, on activated hyaluronidase and histamine release from mast cells. Although D-galacturonic acid itself showed no inhibition, IC50 values of hyaluronidase inhibition were correlated with the D-galacturonic-acid content of pectic substances. It was thought that the polymerization of D-galacturonic acid was necessary for inhibition of activated hyaluronidase. This type of inhibition was suggested to be non-competitive by the Lineweaver-Burk plot. Furthermore, pectic substances, including those purified from Gymnema sylvestre, inhibited histamine release from isolated rat peritoneal mast cells, which had been induced by the antigen. These results suggest that pectic substances may have anti-allergic activities.

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography-electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 micromol/L (UMP and AMP) to 1.3 micromol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.

Enantioseparation of nicotine alkaloids in cigarettes by CE using sulfated β-CD as a chiral selector and a capillary coated with amino groups

Nicotine (NC) and its related compounds (cotinine (CN), nornicotine (NN), anatabine (AT) and anabasine (AB)) were simultaneously enantioseparated by CE using a capillary with amino groups and sulfated β‐CD as a chiral selector. The optimum running conditions were found to be 30 mM acetate buffer (pH 5.0) containing 8% sulfated β‐CD with an applied voltage of +15 kV at 30°C using direct detection at 260 nm. Using a capillary coated with amino groups, the EOF migrates toward the positive pole. However, when sulfated β‐CD was added to the BGE, it was found that the EOF migrated toward the negative pole due to ionic adsorption of sulfated β‐CD to amino groups on the capillary inner wall. All the cationic analytes migrated as anions, suggesting that they formed stable anionic complexes with sulfated β‐CD. With this system and a simple pretreatment with mini‐cartridges, NC alkaloids in five cigarette samples were enantioseparated. As a result, each of the compounds except for CN was detected. In the case of NC, only (S)‐NC was detected (more than 99.9%), but in the case of NN, AT and AB, the ratios of (S)‐isomer to total isomers were in the ranges 58–70, 81–85 and 59–65%, respectively. On the other hand, only NC was detected in cigarette smoke and the ratio of (S)‐ and (R)‐NCs was 96:4. The amounts of NC alkaloids in cigarettes suggest that the production of (R)‐NC resulted from racemization due to the high temperature/burning of the cigarette.

Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLC

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Protein domain of chicken α1-acid glycoprotein is responsible for chiral recognition

Abstract Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31–32% identities to rabbit and human α1-acid glycoproteins (α1-AGPs). Thus, OGCHI should be the chicken α1-AGP. Second, the recombinant chicken α1-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken α1-AGP is responsible for the chiral recognition.

Analysis of Purine in Purine-Rich Cauliflower

Purine is a general term for purine nucleotides, nucleosides, bases, and nucleic acid. The amount of purine nucleotides, nucleosides, and bases in purine-rich cauliflower was determined with the use of LC-MS and HPLC, and the ratio of these molecules were compared with in raw and in heated condition. Total purine content of raw and heated cauliflower was 42.6 and 43.2 mg/100 g, respectively. Nucleotide content was increased from 0.02 to 50.8 μmol/100 g, and nucleoside content was decreased from 12.4 to 7.7 μmol/100 g, by heating.

Purine contents of soybean-derived foods and selected Japanese vegetables and mushrooms.

Purine contents of soybean-derived food and various other Japanese foods were quantitatively determined by high-performance liquid chromatography (HPLC). Purine contents were as follows: soybean-derived foods, 21.9–172.5 mg/100 g or 100 mL; Japanese vegetables, 2.3–171.8 mg/100 g; Japanese mushrooms, 9.5–142.3 mg/100 g. Since purine levels in these foods did not exceed 200 mg/100 g, we recommend that eating of them should be adopted and good dietary habits followed.