Enzyme Lab

Enzyme Student Training

By Quanina Quan UCSD ScienceBridge

Objective

To extract the enzyme Tyrosinase from a Portobello mushroom in order to test the reaction rate of the enzyme in normal conditions and compare it to conditions where the enzyme is exposed to inhibitors, increased temperature, or changes in pH.

The Enzyme Tyrosinase

Tyrosinase is an enzyme that causes pigmentation in apples, skin, hair, etc.

The Enzyme Tyrosinase

The Enzyme Tyrosinase

Albinism

The Reaction

General Enzyme-Substrate Reaction

Enzyme + Substrate = Product + Enzyme

General Tyrosinase ReactionTyrosinase+ L-DOPA = Dopachrome + Tyrosinase

+ =

The Lab

3 Main Parts of the Tyrosinase Enzyme Lab

1. Enzyme Extraction2. Control Reaction3. Experimental Reaction

The Lab – Enzyme Extraction

1a. Break open the cell to free Tyrosinase

The Lab – Enzyme Extraction

1b. Filter out the solid chunks of mushroom

The Lab – Control Reaction4 roles:- Mixer - Reader- Timer - Recorder

Tyrosinase+ L-DOPA = Dopachrome + Tyrosinase

+=

0.1 mlENZ CTL CTL

The Lab – Experimental Trials

3 Different Experimental Trials

- pH- Inhibitor- Temperature

The Lab – Experimental TrialpH

Tyrosinase+ L-DOPA = Dopachrome + Tyrosinase

+=

0.1 ml

- Test the pH of L-DOPA- Test the pH of L-DOPA after you add HCl- Start the reaction

HCl

?ENZ EXP EXP

The Lab – Experimental Trial

Inhibitor- Add Inhibitor- Start the reaction

Tyrosinase+ L-DOPA = Dopachrome + Tyrosinase

+=

0.1 ml

Inhibitor

?EXP EXPENZ

The Lab – Experimental TrialTemperature

-Heat 0.1 ml of L-DOPA in EXP tube for 1 min. in water

- Add L-DOPA and start the experiment

0.1 ml Tyrosinase

L-DOPA

=

?

EXP EXP

EXP

Graphing!

X = time in secondsY = mM of Dopachrome

10 30 50 80 120

160

240

360

0

0.5

1

1.5

2

2.5

Sample Control Enzyme Reaction

Control Enzyme Reaction

Why Study Enzyme Reaction Rates?

Myasthenia Gravis Dog Video Cone sea snails Video

(toxin inhibiting receptors)