Inquiry of trace amount of bioactive unknown peptides by specific chemiluminescent derivatization detection

特异性化学发光衍生化检测探究微量生物活性未知肽

• 批准号: 10672020
• 负责人: KAI Masaaki
• 金额: $ 2.11万
• 依托单位: NAGASAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672020/
• 关键词: Peptide; Selective detection; Chromatography; Chemiluminescence; Sensitive detection; Inquiry; Derivatization; Tryptophan; クロマドグラフィー; 化学発光法; 高感度; 誘動体化反応; 誘導体化反応

We found that an N-terminal Trp-containing peptide reacts with glyoxal under weakly acidic conditions (pH 3-4) with heating at 80℃ for 5 min, and then a fluorescent and chemiluminescent compounds are formed. The produced chemiluminophore in the reaction mixture emitted a light in the presence of HィイD22ィエD2OィイD22ィエD2 when pH of the mixture was changed to pH 8.5-9.5 by adding an alkaline borate solution. On the basis of the above reactions we established manual quantitative methods for N-terminal Trp-containing peptides and HィイD22ィエD2OィイD22ィエD2 showing determinable lower limits of ca. 0.5 nM and ca. 3 μM, respectively. The molecular weights of the luminescent substances in the reaction mixture were measured by liquid chromatographic mass spectrometry. Consequently, we estimated that the chemical structures of the luminescent substances have each new ring in which 2 molecules of glyoxal are self condensed and followed by binding with the amino and imino nitrogens of N-terminal Trp. Next, a sensitive chemiluminescent detection system with adaptation of the glyoxal reaction was constructed for high-performance liquid chromatography of peptides. The chemiluminescent detection method indicated 5-60 times more sensitive than conventional UV or fluorescent detection. Two unknown N-terminal Trp-containing peptides in a Japanese soy source were easily detected by the detection system. We will elucidate structures of the unknown peptides and bioactivity in tissues and cells.

研究发现，在弱酸性条件下（pH 3-4），80℃加热5 min，N端含色氨酸的多肽与乙二醛反应，生成荧光和双荧光化合物。当通过加入碱性硼酸盐溶液将混合物的pH改变至pH 8.5-9.5时，在反应混合物中产生的荧光团在H ↓ [2] D22 ↓ [2] D2 O ↓ [2] D22 ↓ [2] D2存在下发光。在上述反应的基础上，我们建立了N-末端含Trp肽和H_（22）Trp D_2O_（22）Trp D_2的手工定量方法，显示了ca. 0.5 nM和ca. 3 μM。通过液相色谱质谱法测量反应混合物中发光物质的分子量。因此，我们估计发光物质的化学结构具有每个新的环，其中2个乙二醛分子自缩合，然后与N-末端Trp的氨基和亚氨基氮结合。其次，构建了一个灵敏的荧光检测系统与乙二醛反应的适应肽的高效液相色谱。荧光检测法的灵敏度比传统的紫外或荧光检测法高5-60倍。两个未知的N-末端含色氨酸的肽在日本大豆源很容易检测系统。我们将阐明未知肽的结构和在组织和细胞中的生物活性。