Facile and Selective Determination of Dipeptides Using 3-Methylcatechol as a Novel Fluorogenic Reagent
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.
KeywordsPeptides 3-Methylcatechol Fluorescence Selectivity
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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Conflict of interest
The authors declare that they have no conflict of interest.
Research Involving Human and Animal Participants
This article does not contain any studies with human participants or animals performed by any of the authors.
- Ishida J, Kai M, Ohkura Y (1986) High-performance liquid chromatography of tyrosine containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene [4,5-dimethoxy-o-phenylenediamine]. J Chromatogr 356:171–177CrossRefPubMedGoogle Scholar
- Roberti ML, Ricottini L, Capponi A, Sclauzero E, Vicenti P et al (2014) Immunomodulating treatment with low dose interleukin-4, interleukin-10 and interleukin-11 in psoriasis vulgaris. J Bio Regul Homeost Agents 28(1):133–139Google Scholar
- Yang J, Saggiomo V, Velders AH, Cohen Stuart MA, Kamperman M (2016) Reaction pathways in catechol/primary amine mixtures: a window on crosslinking chemistry. PLoS ONE 11(12):1–17Google Scholar