Protein Concentration/Enzyme Activity

Materials

• Commercially pure tyrosinase
• UV spectrophotometer or materials for Lowry or Bradford protein determination
• L-DOPA
• 0.1 M citrate buffer, pH 6.6

Procedure

1. Prepare a solution of 0.7 micrograms of commercially pure tyrosinase diluted to 4 mL with 0.1 M citrate buffer, pH 6.6.
2. 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.
3. Place both enzyme samples in a water bath at 30°C for 5 minutes to temperature equilibrate.
4. Turn on the spectrophotometer, set the wavelength to 475 nm, and blank the instrument using citrate buffer as the blank.
5. 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 475 nm.
6. Replace the tube in the water bath and wait exactly 5 minutes. Read the OD₄₇₅ immediately.
7. 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.
8. 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.

Notes
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.