Selective determination of peptides in mixtures or biological samples requires specific techniques for analysis. Herein, we report 3-methylcatechol (3-MC) as a novel fluorogenic reagent for the selective determination of dipeptides by a simple fluorescence (FL) derivatization reaction. After extensive screening of 31 different catechol analogues, 3-MC was found to generate FL with peptides. The reaction was performed at 100 °C for 10 min in the presence of borate buffer (pH 7) and sodium periodate. The resulting FL intensities were measured by spectrofluorometer at excitation wavelengths of 380 nm and emission wavelengths of 500 nm. Different reaction conditions such as concentration of sodium periodate, reaction time and pH of the borate buffer were studied to determine the optimum reaction conditions. Linearity was obtained between FL intensity and peptide concentrations from 10 to 160 µM with a lower detection limit of 10 µM (S/N = 3). Dipeptides containing Ala, Phe, Leu and Val at the N-termini generated significant FL in comparison to the reagent blank (**p < 0.005, ***p < 0.0005). The reaction is simple, rapid, selective and sensitive which can be applied for the determination of the dipeptides as biomarkers or to determine enzyme activity.
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This work was partially supported by Grants-in-aid for Scientific Research form the Ministry of Education, Culture, Sports and Technology of Japan.
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The authors declare that they have no conflict of interest.
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Supplementary Figure 1 Concentration range of AG used to determine the limit of detection (LOD) and the limit of linearity (LOL). Error bars represents standard uncertainty, n = 3 (PPTX 36 KB)
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Yasmin, H., Rahman, M.S. & Shibata, T. Facile and Selective Determination of Dipeptides Using 3-Methylcatechol as a Novel Fluorogenic Reagent. Int J Pept Res Ther 25, 583–589 (2019). https://doi.org/10.1007/s10989-018-9703-z