Determine the amount of PPO enzyme activity in your extract. Enzyme activity is
ID: 225632 • Letter: D
Question
Determine the amount of PPO enzyme activity in your extract. Enzyme activity is reported in units (), which is defined as the amount of enzyme capable of converting 1 micromole (mol) of substrate to product per minute at 25°C.
Phosphate Coomassie Standard Final Protein Blue Reagent /extract Conc. (ug/ml) A585 Tube Buffer 0.05 m (A) 1000 1.005 1 ml 1.5 m 2 1 ml 1.5 m 0.05 m (B) 750 0.736 3 1 ml 1.5 m 0.05 m (C) 500 0.05 m (D) 250 0.05 m (E) 125 0.420 0.267 1 ml 1.5 m 1.5 m 0.124 1 m 0.02 1.5 m (F) 25 1 m 0.05 m 1 ml 1.5 m 0.05 m (G) 0 (Blank) 0 0.015 m (extract) 1 ml 1.5 m 0.55 Vmax 0.1 mM minn-1 Enzyme assay 0.004 L Absorbance VS Protein concentration 1.2 concentration for extract- 0.8 0.6 absorbance 0.4 y 0.0009x 0.0274 near (absorbance) 0.2 200 400 600 800 1000 1200 Protein concentration (LLg/mExplanation / Answer
Answer:
From the calibration curve:
x = =(0.55-0.0274)/0.0009 = 581 ug/ml
Volume of extract used = 0.015ml and Volume of reaction = 4ml, therfore dilution factor = 4/0.015 = 266.7
Concentration of the enzyme in undiluted sample = 581ug/ml x 266.7 = 154933ug/ml = 154.9mg/ml
Enzyme activity = Vmax x volume = 0.1mM/min x 4 ml = 0.4mM/min = 400uM/min
Total amount of protein present in extract sample in assay = 581ug/ml x 4ml = 2324ug = 2.324mg
Specific activity of enzyme = Enzyme activity / mg protein = 400uM/min / 2.324mg = 172.11 uM/min/mg = 172units/mg
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