Abstract
A novel fluorometric method was developed for the sensitive and selective detection of Pro-Gly (PG) and Pro-Gly-Pro (PGP) using 4-chlorobenzene-1,2-diol (4-CBD) as a fluorogenic reagent. The reaction was performed at 37°C for 30 min in the presence of a borate buffer (pH 7.0) and sodium periodate. The resulting fluorescence intensity was measured using a spectrofluorometer with excitation and emission wavelengths of 450 nm and 535 nm. To obtain a stable fluorescent signal and maximise its intensity, different reaction conditions such as the concentrations of the reagents, the reaction time, and the pH were optimised. Under the optimised conditions, a linear relationship was obtained between fluorescence intensity and peptide concentrations from 1.0–40.0 µmol L−1 with a limit of detection of 1.0 µmol L−1 (S/N = 3). Both PG and PGP generated a strong signal out of all the peptides tested and no other biogenic substances such as amino acids or proteins produced any fluorescence. The reaction thus developed is simple, rapid, selective, and sensitive. It can be applied to the determination of peptides as biomarkers or substrates.
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Baker, A. M., Batchelor, D. C., Thomas, G. B., Wen, J. Y., Rafiee, M., Lin, H., & Guan, J. (2005). Central penetration and stability of N-terminal tripeptide of insulin-like growth factor-I, glycine-proline-glutamate in adult rat. Neuropeptides, 39, 81–87. DOI: 10.1016/j.npep.2004.11.001.
Gaggar, A., Jackson, P. L., Noerager, B. N., O’Reilly, P. J., McQuaid, D. B., Rowe, S. M., Clancy, J. P., & Blalock, J. E. (2008). A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. The Journal of Immunology, 180, 5662–5669. DOI: 10.4049/jimmunol.180.8.5662.
Gentilucci, L. (2004). New trends in the development of opioid peptide analogues as advanced remedies for pain relief. Current Topics in Medicinal Chemistry, 4, 19–38. DOI: 10.2174/1568026043451663.
Ioudina, M., & Uemura, E. (2003). A three amino acid peptide, Gly-Pro-Arg, protects and rescues cell death induced by amyloid β-peptide. Experimental Neurology, 184, 923–929. DOI: 10.1016/s0014-4886(03)00314-5.
Kabashima, T., Yu, Z. Q., Tang, C. H., Nakagawa, Y., Okumura, K., Shibata, T., Lu, J. Z., & Kai, M. (2008). A selective fluorescence reaction for peptides and chromatographic analysis. Peptides, 29, 356–363. DOI: 10.1016/j.peptides.2007.11.014.
Lyapina, L. A., Pastorova, V. E., Samonina, G. E., & Ashmarin, I. P. (2000). The effect of prolil-glycil-proline (PGP) peptide and PGP-rich substances on haemostatic parameters of rat blood. Blood Coagulation & Fibrinolysis, 11, 409–414. DOI: 10.1097/00001721-200007000-00002.
Pan, W., & Kastin, A. J. (2007). From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides. Peptides, 28, 2411–2434. DOI: 10.1016/j.peptides.2007.10.006.
Pickart, L., Freedman, J. H., Loker, W. J., Peisach, J., Perkins, C. M., Stenkamp, R. E., & Weinstein, B. (1980). Growth-modulating plasma tripeptide may function by facilitating copper uptake into cells. Nature, 288, 715–717. DOI: 10.1038/288715a0.
Ramesh, C. V., Jayakumar, R., & Puvanakrishnan, R. (1995). In vitro studies on a novel micelle-forming peptide with anticoagulant activity. International Journal of Peptide and Protein Research, 45, 386–390. DOI: 10.1111/j.1399-3011.1995.tb01053.x.
Sara, V. R., Carlsson-Skwirut, C., Bergman, T., Jörnvall, H., Roberts, P. J., Crawford. M., Håkansson, L. N., Civalero, I., & Nordberg, A. (1989). Identification of Gly-Pro-Glu (GPE), the aminoterminal tripeptide of insulin-like growth factor 1 which is truncated in brain, as a novel neuroactive peptide. Biochemical and Biophysical Research Communications, 165, 766–771. DOI: 10.1016/s0006-291x(89)80032-4.
Sizonenko, S. V., Sirimanne, E. S., Williams, C. E., & Gluckman, P. D. (2001). Neuroprotective effects of the N-terminal tripeptide of IGF-1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury. Brain Research, 922, 42–50. DOI: 10.1016/s0006-8993(01)03148-1.
Toyo’oka, T., Ishibashi, M., & Terao, T. (1994). Sensitive determination of N-terminal prolyl peptides by high-performance liquid chromatography with laser-induced fluorescence detection. Journal of Chromatography A, 661, 105–112. DOI: 10.1016/0021-9673(94)85182-4.
Yasmin, H., Shibata, T., Rahman, M. S., Kabashima, T., & Kai, M. (2012). Selective and sensitive determination of peptides using 3,4-dihydroxyphenylacetic acid as a fluorogenic reagent. Analytica Chimica Acta, 721, 162–166. DOI: 10.1016/j.aca.2012.01.035.
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Yasmin, H., Rahman, M.S., Shibata, T. et al. Sensitive and selective determination of peptides, PG and PGP, using a novel fluorogenic reagent 4-chlorobenzene-1,2-diol. Chem. Pap. 69, 504–509 (2015). https://doi.org/10.1515/chempap-2015-0057
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DOI: https://doi.org/10.1515/chempap-2015-0057