- 8 mM DOPA, pH 6.6
- Enzyme extract, diluted to yield 10 micromoles of dopachrome in 3–5
- Spectrophotometer and cuvettes
- Prepare a reaction blank in a clean cuvette containing 2.5 mL of citrate
buffer and 0.5 mL of enzyme extract. Use this blank to adjust your
spectrophotometer for 100% transmittance. Remove the blank and save it.
- Add 2.5 mL of 8 mM DOPA, pH 6.6, to a clean cuvette.
- Add 0.5 mL of appropriately diluted enzyme extract. Shake well and immediately
insert the tube into the spectrophotometer. Record the absorbance
or transmittance as quickly as possible. Designate this reading as time 0.
- At 30-second intervals, read and record the transmittance until a transmittance
value of 10% (absorbance = 1.0) is reached. Complete the following
- Plot time in minutes (x-axis) versus the amount of dopachrome formed