- Commercially pure tyrosinase
- UV spectrophotometer or materials for Lowry or Bradford protein determination
- 0.1 M citrate buffer, pH 6.6
- Prepare a solution of 0.7 micrograms of commercially pure tyrosinase
diluted to 4 mL with 0.1 M citrate buffer, pH 6.6.
- Measure the OD280 of your sample and prepare a dilution of the enzyme
extract to a final concentration of 0.7 micrograms in 4 mL of citrate buffer.
- Place both enzyme samples in a water bath at 30°C for 5 minutes to
- Turn on the spectrophotometer, set the wavelength to 475 nm, and blank
the instrument using citrate buffer as the blank.
- Select the commercial preparation and add exactly 1.0 mL of L-DOPA
(4 mg/mL in citrate buffer) and immediately read the absorbance at
- Replace the tube in the water bath and wait exactly 5 minutes. Read the
- The molar absorbance coefficient for dopachrome is 3.7 ¥ 104. Use this
value to compute the specific activity of the commercial enzyme preparation.
Check this activity against that listed with the enzyme preparation.
- Repeat steps 5 and 6 with your extracted enzyme preparation. Compute
the specific activity (enzyme units of activity/mg protein) of your enzyme
preparation. Enzyme unit: The absorbance reading under the conditions specified
in this exercise is proportional to the enzyme concentration, where 1 unit of
enzyme activity yield a 0.81 OD change in readings.
The protein content can be measured by the Lowry or Biuret procedures or more
simply, by a single spectrophotometric measure of the absorbance of the sample
at 280 nm. Without going into mathematical detail, a 1% pure solution of
tyrosinase has an OD280 equal to 15.6/cm. The Beer-Lambert law can thus be
used to determine protein content in a nondestructive manner.