CHE 341 - Characterization of Tyrosinase Isozymes in the White Button Mushroom

Purpose

Over the next five weeks, you will undertake an open-ended investigation of the localization of tyrosinase isozymes in the common white button mushroom, Agaricus bisporus. Your job will be to apply common biochemical methods to learn as much as you can about these enzymes and communicate what you find in the style of a professional journal article. You will dissect mushrooms into anatomical sections, extract the soluble protein, measure the protein concentration, test latent and induced enzyme activity, qualitatively separate tyrosinase isozymes using native gel electrophoresis, measure the Michaelis-Menten kinetics parameters (Km, Vmax and kcat), and evaluate the mechanisms of tyrosinase inhibitors. This work will result in two lab reports.

This week, you will dissect mushrooms into separate tissues. After this, you will work in a team that will be assigned a tissue. Your team will create a buffered, crude extract of the proteins located within that tissue. Finally, you will measure the rate of tyrosinase activity within your extracted tissue. By measuring the rate of product formation via enzymatic assay, you will be able to calculate the amount of enzymatic units per gram of your tissue. You will then measure the activity of your tissue once the tyrosinase has been activated with 0.1% SDS (induced activity).

 

Objectives

  • Analyze the qualities of mushroom tissues that will be studied.
  • Dissect whole mushrooms in order to distribute the isolated portions according to each group's tissue assignment.
  • Extract protein from your tissue sample through homogenization and subsequent extraction. You will then turn in aliquots of your crude extract to be frozen so that they may be used during the following weeks.
  • Apply a written procedure to measure relative tyrosinase activity in sample through latent and activated saturating (Vmax) kinetics carried out spectrophotometrically using an L-Dopa (substrate) solution.
  • Construct meaning from data collected through calculations and interpretation.

 

Pre-lab Preparation

* Read the assigned reading (including this lesson) completely

* Complete the prelab assessment at least 24 hours before lab begins

* Outline the purpose, objectives, and procedures sections in your notebook.

Lecture Video

Lecture Slides