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enzymekineticslab - Chem 365 Biochemistry Team Project#2...

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Chem 365 Biochemistry – Team Project #2 Enzyme Characterization: Analysis Of Red Potato Extract For Monophenol Monooxygenase Activity References: Boyer, R.F. J. Chem. Educ. , 1977 , 54(9) , pp 585-586. Friedman, M.E.; Daron, H.H. J. Chem. Educ. , 1977 , 54(4) , pp 256-257. This project involves analyzing a simple extract from red potatoes for activity of the enzyme tyrosinase (monophenol monooxygenase EC Tyrosinases present in both animal and plant cells catalyze the hydroxylation of various monophenols, and the aerobic oxidation of diphenols leading to the production of melanin pigments. The specific activity of the extract and determination of kinetic parameters based on the simple Michaelis-Menten enzyme model will be determined. The reaction, which will be studied spectrophotometrically, is the conversion of the substrate 3,4-dihydroxyphenylalanine (DOPA) to the product dopachrome which is an orange colored o-quinone. Enzyme units are obtained by recording the increase in absorbance of a buffered solution of DOPA and potato extract at 475 nm for three minutes. The initial rate of reaction in m moles dopachrome formed per minute is equivalent to the slope of the linear portion of the absorbance vs. time plot early in the reaction (times less than 3 minutes). Multiplying this slope by the total solution volume (0.00300 L), and divided by the molar absorptivity of dopachrome in a 1 cm cuvet at 475 nm, 3.60 X 10 -3 m M -1 , gives enzyme units in m moles dopachrome formed per minute. Solutions: 1) Prepare 250 mL of a 0.1 M sodium phosphate buffer, pH 6.8. (pKa = 6.82) Use handbook instructions given on last page. 2) Prepare 15.0 mL of a 20.0 mM DOPA(197.19 g/mol) solution in buffer 1. 3) Bradford reagent. Laboratory: A) Preparation of Potato Extract 1) Peel and chop a potato and put about 50 g of potato and 50 mL of buffer 1 in a blender cup. Blend for about 1 minute.
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