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2001
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In the Laboratory
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The Effect of Organic Solvents and Other Parameters on Trypsin-Catalyzed Hydrolysis of Na-Benzoyl-arginine-p-nitroanilide. A Project-Oriented Biochemical Experiment
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L. C. Correia, A. C. Bocewicz, S. A. Esteves, M. G. Pontes, L. M. Versieux, S. M. R. Teixeira, M. M. Santoro, and M. P. Bemquerer
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Pampulha-Belo Horizonte-M.G. 31270-901, Brazil
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November 2001
Vol. 78 No. 11
p. 1535
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| Abstract |
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The study of enzymatic catalysis is a classical biochemistry experiment for undergraduate classes. We propose the utilization of the serine protease trypsin to discuss several parameters affecting enzyme catalysis. Hydrolysis of the chromogenic substrate Na -benzoyl-arginine-p-nitroanilide (BApNA) was followed by spectrophotometric monitoring. The optimal pH and temperature values were found to be 8.0 and 40 °C, respectively. Km and Vmax values were obtained by adjustment to Michaelis-Menten, Lineweaver-Burke, and Hanes equations. We then investigated the effect of organic solvents (a series of alcohols) on the hydrolysis of the chromogenic substrate. The reaction rate was reduced in the presence of methanol and further reduced by ethanol, 1-propanol, and 2-propanol, when compared to the data obtained with buffer. Finally the students were asked to measure the molar absorptivity of p-nitrophenol in the presence of the alcohols employed for the kinetic experiments. Thus they could learn that the value of this parameter varies with the solvent. These experiments were designed as a project-oriented approach to teach biochemistry methodologies and theoretical aspects of enzyme kinetics. They took about four months with four to six hours per week spent in the laboratory.
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| Supplement |
Some further experimental observations and data are available.
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 Contents |
JCE2001p1535W.doc (Microsoft Word)
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| More Information |
Citation |
Correia, L. C.; Bocewicz, A. C.; Esteves, S. A.; Pontes, M. G.; Versieux, L. M.; Teixeira, S. M. R.; Santoro, M. M.; Bemquerer, M. P. J. Chem. Educ. 2001 78 1535.
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Keywords |
Biochemistry; Enzymes; Laboratory Instruction; Solutions / Solvents; UV-Vis Spectroscopy
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History |
Created:
Last Updated: |
October 8, 2001
August 31, 2005
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| Home > JCE Print > Journal of Chemical Education > Issues >
2001
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November
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1535
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