Exercise 5.11 - Advanced Analysis of Enzyme Parameters


In the extraction procedure for tyrosinase, only one precipitation was employed utilizing ammonium sulfate. This procedure yields a rapid means for obtaining the enzyme, but results in increased contamination and lower yield. The following improved procedure uses a serial increase in ammonium sulfate.

After blending the potato, add 1/2 volume of ammonium sulfate to yield a 33% solution as opposed to 50%. Collect the precipitate and label it as P1. Add another 1/2 volume of ammonium sulfate to raise the concentration to 50% and collect and label the resulting precipitate as P2. Finally add a third 1/2 volume to raise the concentration to 67% and collect and label the precipitate as P3. Use each of these to measure the specific activity as well as the total activity in each fraction. The step-wise addition of ammonium sulfate will increase the specific activity. If pure enzyme is desired, the steps can be made even smaller, going from 30% to 70% in 10% increments.

If you wish further purification, then subject the resulting precipitated protein to electrophoretic separation and analyze the resulting bands for their tyrosinase activity. While these procedures can yield pure enzyme fractions, they are more pertinent to biochemical studies.

A more interesting analysis can be made by comparing tyrosinase extracted from differing biological materials (mushrooms, bananas, melanocytes) for the values of Km and for their optimum pH and temperature. Based on those differences, might it be possible to predict the darkening of fruit under varying conditions of pH and temperature?

Finally, the enzyme can be studied in a structure linked mode by combining aspects of cell organelle isolation (Chapter Three) to the activity of the enzyme. The presence (or absence) of tyrosinase can be monitored within the organelles of various cells. Melanocytes would be an excellent source of cellular tyrosinase. You could then correlate the activity of tyrosinase with the production of the pigment melanin.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu