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

我们发现了一种含有N-终端Trp-肽反应，在弱酸性条件（pH 3-4）下加热80 ° C，5分钟，然后形成荧光和化学化合物。在反应混合物中产生的化学物质在H-D22-D2 O-D22-D2的存在中产生的化学物质磷，当混合物的pH值被改变为pH值8.5-9.5时，通过添加一种碱性硼酸溶液。On the basis of the above reactions we established manual quantitative methods for N-terminal Trp-containing peptides and H-D22-D2showing determinable lower limits of ca。0.5 NM和CA。3 μM， respectively。The Molecular Weights of the Luminescent substances in the reaction mixture were measured by liquid chromatographic mass spectrometry.从某种意义上说，我们估计，发光物质的化学结构在每一个新的环中都有两个分子的糖基是自我约束的，并由N-终端Trp绑定的氨基和氨基硝基化合物组成。下一个是一个敏感的化学胺检测系统，用于肽的高性能液体色谱法的适应。化学物质检测方法被确定为比常规紫外线或荧光检测更敏感的5-60次。两个未知的N-终端Trp--在日本人的来源中很容易被检测到。我们将在结核病和细胞中回收未知肽和生物活性的结构。

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.