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

We found that an N-terminal Trp-containing peptide reacts with glyoxal under weakly acidic conditions(pH3-4)with heating at 80℃for 5min，and then a fluorescent and chemiluminescent compounds are formed.The produced chemiluminophore in the reaction mixture emitted a light in the presence of H I D22 ii D 2 when pH of the mixture was changed to pH8.5-9.5 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 I D22文件D2O I D22文件D2 showing determinable lower limits of ca.0.5 nM and ca.3µM，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 which2molecules 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 5-60times 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。

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.