Suppose you were interested in experimentally determining the turnover number (k

Question

Suppose you were interested in experimentally determining the turnover number (kcat) of tyrosinase. kcat is commonly defined as Vmax / [E]T, where [E]T refers to the total concentration of enzyme.

Write a general protocol for this experiment. Include the following:

o Equipment list
o Reagent lists (including approximate volumes and concentrations)
o General procedure (note: you do not need to give a step by step direction for using computer programs)

o Data collection and calculations required

o Citations for any references used

Explanation / Answer

Equipment list - Calorimeter to determine the velocity

Reagent lists - Tyrosinase enzyme 500 - 1000 U/ml, 1mM Tyrosine substrate, 50 mM potassium phosphate Buffer

Procedure - To add enzyme to substrate solution and determine the velocity of the reaction using absorbance at 280 nm.

Data collection and calculation required - r A280nm/min Test - r A280nm/min Blank) (df) Units/ml enzyme = (0.001) (0.1)

Determine Vmax by using double reciprocal plot. and divide it by enzyme Conc., to determine Kcat.

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