Tyrosinase Characterization

Materials
-Tyrosinase Solution (1 mg/ml in Phospate Buffer with pH 7.4 (0.05 M); stored FROZEN)
-Substrate solution (Catechol solution)
-Cell Lysate
-Citrate-Phosphate Buffer
-Water Bath

Methods

1. Lysate-Tyrosinase Ratio

Prepare a fixed amount of tyrosinase stock solution (depending on assay to be used) and add lysate in the following lysate/tyrosinase ratios:

-0.5
-1
-1.5
-2.0
-2.5
-3.0
-3.5
-4.0

Once a coarse plotline is established, refine as needed.

2. Tyrosinase pH Optimization

Profile tyrosinase activity according to pH, pH range: 4.0 to 7.0 in increments of 0.4 pH, using citrate-phosphate buffer.

Once the optimized value is discovered, follow experiments with the addition of lysate, using the optimized ratio value found in Method 1 against the pH range defined above.  Adjust pH range as needed once a coarse plotline is established.

3. Tyrosinase Temperature Optimization

Profile tyrosinase activity according to temperature, temperature range: 20 deg C to 40 deg C in increments of 2 deg C.

Once the optimized value is discovered, follow experiments with the addition of lysate, using the optimized ratio value found in Method 1 and the pH value found in Method 2 against the temperature range defined above.  Adjust temperature range as needed once a coarse plotline is established.

Procedure

Use the spectrophotometric methods established in Haghbeen and Tan's "Direct spectrophotometric assay of..." Analytical Biochemistry 312 (2003) 23-32.