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| Section: Biotechnology Methods » Enzymology |
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| Addition of Enzyme Inhibitors |
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Materials
- Enzyme extract
- 8 mM L-DOPA
- 8 mM benzoic acid
- 8 mM KCN
- 0.1 M citrate buffer, pH 6.6.
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Procedure
- Tyrosinase is inhibited by compounds that complex with copper, as well
as by benzoic acid and cyanide. To determine the inhibitory effects of
benzoic acid and cyanide, set up a series of tubes as indicated.
- Using one tube at a time, add 0.5 mL of the enzyme dilution previously
calculated to yield 10 micromoles of dopachrome in 2–3 minutes. For each
tube, measure the time required to convert 10 micromoles of DOPA to
dopachrome. Enter those times in the table below. Compute the reaction
velocity for each substrate concentration:
- Calculate the values for 1/s and 1/ν for each of the corresponding s and
&nu in the table. Plot 1/&nu versus 1/s for the presence of benzoic acid and a
second plot for the presence of KCN. Compute the values of Vmax and Km
for the presence of each inhibitor. Determine whether these inhibitors are
competitive, noncompetitive, or uncompetitive.
| |
KCN Inhibition |
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Tube number |
8 mM DOPA |
8 mM KCN |
Buffer |
12 |
1.8 |
0.5 |
0.2 |
13 |
1.6 |
0.5 |
0.4 |
14 |
1.4 |
0.5 |
0.6 |
15 |
1.2 |
0.5 |
0.8 |
16 |
1.0 |
0.5 |
1.0 |
17 |
0.8 |
0.5 |
1.2 |
18 |
0.6 |
0.5 |
1.4 |
19 |
0.4 |
0.5 |
1.6 |
20 |
0.2 |
0.5 |
1.8 |
21 |
0 |
0.5 |
2.0 |
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