Enzyme Lab

CHEM 4422: Enzymes Dr. Stone Spring 2016 (Protein Purification + Kinetics = 100 points)
I. Isolation of phenol oxidase from Potatoes, extraction, protein quantitation,
II. Enzyme kinetics, determine Km and Vmax for several substrates
III. Purification of phenol oxidase (increase specific activity)
IV. Determine purity using Gel Electrophoresis

Part I: Isolation of phenol oxidase from Potatoes, extraction, protein quantitation

Use the following literature paper: A Spectrophotometric Assay of Polyphenoloxidase, to extract polyphenol oxidase from potatoes. Before you test it for enzyme activity, measure the amount of protein in your extract using the Bradford assay.

Bradford Assay:

(1) Add 10-20 μl of the protein extract to 1 ml of the diluted Bradford (prepared for you) reagent and mix. Measure the blue color formed at the wavelength 595nm. If using cuvettes, use the disposable plastic cuvettes; the set of microcell cuvettes can read as little 100μl.

(2) Prepare a standard curve using a serial dilution series (0.1-1.0 mg/ml) of a known protein sample concentration; e.g., BSA dissolved in whatever buffer your protein is in.

Your final report will include your standard curve (with R2 and the equation of the line). Report the amount of protein in your extract. You will use this number to determine the specific activity of your enzyme solution. (activity/mg protein)

Part II: Enzyme Kinetics

Next, use the procedure in the Phenol oxidase paper to determine the Km and Vmax for the substrate, dopamine.

Part III: Purification to Increase specific activity

Skip this step and prepare your crude extract for electrophoresis

Part IV: Electrophoresis

Electrophoresis of Potato Phenol Oxidase

To prepare your sample for electrophoresis, put these four solutions, in this order in an Eppendorf tube:

Some volume of protein = 60 ug of protein,
Some volme of water (protein vol + water vol = 65uL)
10 uL of 10% 2-sulfanyl ethanol, 50 mM sodium phosphate, pH 7.2
5uL of 2% SDS (final concentration 0.1%, 0.1ug/100uL)

Put the above solutions an Eppendorf tube. Heat to 100°C. Cool. Add 20 uL 0.25% bromphenol blue with 80% glycerol and spin in a micro centrifuge.

Total volume is 100 uL

50uL will be loaded on the gel

We will be using a 12.5% polyacrylamide gel with SDS. The running buffer has been prepared for you.

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