Our laboratory is interested in factors affecting the activity of phenylalanine ammonia lyase an enzyme that catalyzes the conversion of phenylalanine to trans cinnamic acid (TCA). We therefore needed an assay to quantify TCA.

We developed an HPLC method with a mobile phase of 25 mM Phosphate buffer (pH 6.5): Acetonitrile in a 92:8 ratio. The mobile phase was pumped at a flow rate of 1.5 mL/min and the eluent was separated on a Zorbax Eclipse Plus C18 4.6 x 150 mm, 3.5 m column. TCA was detected using UV absorbance at a wavelength of 270 nm. The retention time of TCA in this system was 7.4 mins.

Listed above are absorbance (expressed as peak area) versus concentration data for a set of TCA standards using our system.

1. Plot the data on a linear-linear scale with peak area (absorbance) on the y-axis and concentration on the x-axis. Using linear regression, calculate the extinction coefficient of trans cinnamic acid. Please include its units.

2. In the same analytical system, the peak area (absorbance) of TCA in a sample produced from the reaction of 4 mM phenyalanine with phenylalanine ammonia lyase is 409045 units. Using the data in the table above and your linear regression results, calculate the concentration of TCA in the sample.

3. If the concentration of TCA in question 2 was produced over 7 mins by 10 microgram of phenylalanine ammonia lyase in the reaction mixture, calculate the velocity of the enzyme.

Note: Velocity of an enzyme is normally expressed as product formed per unit time. The amount of enzyme used in the reaction mixture also needs to be accounted for in the expression of enzyme velocity.