Basic Biochemistry Laboratory (University of South Florida)
lOMoAR cPSD| lOMoAR cPSD|
Basic Biochemistry Laboratory (University of
South Florida)
Part 1: Determining the Concentration of Tyrosinase
Introduction and Problem
This part of the experiment was performed to determine the concentrat...
Basic Biochemistry Laboratory (University of
South Florida)
Part 1: Determining the Concentration of Tyrosinase
Introduction and Problem
This part of the experiment was performed to determine the concentration of Tyrosinase
in the experiment. Tyrosinase was the enzyme used throughout all five parts of the kinetics
experiment. The problem in part 1 was that the concentration of Tyrosinase was unknown. This
was a problem because the concentration must be known in order to determine the specific
enzyme activity.
Hypothesis
The hypothesis of this part of the experiment was that by using spectrophotometry with a sample
of tyrosinase, the absorbance reading could be used to calculate the E1% and further calculate
the concentration of tyrosinase.
Solution
To solve the problem, an A280 assay will be performed followed by using the E1% method to
determine the concentration of tyrosinase.
Variables
The independent variable in this part of the experiment was the concentration of Tyrosinase.
Although this was unknown, it was not changed during part 1. The dependent variable was the
, lOMoAR cPSD| 36278399
absorbance reading of the sample as this depended on the concentration. The control variable
was the blank potassium phosphate buffer used in the spectrophotometer.
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Materials
• 50Mm potassium phosphate buffer (pH 7)
• Tyrosinase sample
• 100-1000 uL pipettes + tips
• UV-vis spectrophotometer
• Wave scan computer software
• Cuvettes
Procedure
1. Set the spectrophotometer to 280nm
2. Blank the spec with a cuvette of 50mM potassium phosphate buffer
3. Prepare 1mL of tyrosinase in a cuvette and run on the spec to obtain the absorbance
4. Use the E1% method to solve for the concentration of tyrosinase
Observations
There were no qualitative observations in this part of the experiment as it was only an A280
assay being performed with tyrosinase. The quantitative observations are shown below in the
calculations. The concentration of tyrosinase was determined to be 4.39x10-7 M.
Calculations
A280 = .14
E1% = 24.9
ε = = = 318,720 M-1cm-1
A = εcl
.14 = 318,720 (c) (1)
C = 4.39x10-7 M
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