Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

• Autores: Rosa V. Flores, Hilda M. Solá, Juan C. Torres, Rafael E. Torres, Ernick E. Guzmán
• Localización: Journal of chemical education, ISSN 0021-9584, Vol. 90, Nº 2, 2013, págs. 248-251
• Idioma: inglés
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• Resumen

  • A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence, which led them to conclude that its fluorescence, denaturation, and renaturation was pH dependent. The importance of temperature control in fluorescence spectroscopy, and of control samples in experiments, was also stressed. This experiment is suitable for an upper-level biochemistry laboratory, as an extension of experiences involving bacterial transformation and hydrophobic interaction chromatography to purify the protein. It is also appropriate for an instrumental analysis laboratory using a provided protein sample.