LIFE 212 Lecture Notes - Tyrosinase, Reaction Rate Constant, Turnover Number
30 Mar 2014
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Reaction rates vary with changes to [substrate] and [enzyme] Generate standard curve (y = mx + b) Optimum [enzyme] req. to convert 2 moles of dopa to dopachrome in 3 to 5 minutes (= 2 mm dopa) Determined ph effects on velocity (at 1. 5 min) For serial dilution series: 5 different [substrate: plot product (y axis) vs minutes (x axis) Calc. slope m = velocity (x1, y1) (x2, y2) m = y2 y1 x2 x1: determine v for each dopa concentration. Plot velocity (y axis) vs [dopa] (x axis) Use lineweaver-burk plot to estimate the km, vmax and. Vi = and when km = [s], vi = Prepare 2 fold dilution series of [dopa] in citrate buffer ph 4. 8. Once the enzyme extract has been added, each dilution will be used to collect absorbance (a475) readings every 30 seconds for 5 minutes. Blank: 0. 8 ml of citrate buffer + 0. 2 ml enzyme extract.
