Notes on Enzymes and Experimental Lab Procedures

Background on Enzymes

• Chemical Reaction Requirements:
  • Molecules must collide with the right orientation to break/break bonds.
  • Low probability of collisions in a cell can lead to extremely slow reaction rates.
  • Some reactions may take thousands to millions of years without intervention.
• Role of Enzymes:
  • Enzymes are proteins that act as catalysts, increasing reaction rates without being consumed.
  • Lower activation energy by binding substrates in the correct orientation.
  • Active Site:
  • Specific region on an enzyme that binds substrates; often specific to a few chemical species.
• Michaelis-Menten Kinetics:
  • Enzyme velocity vs. substrate concentration plotted in a graph.
  • Y-axis: enzyme velocity (moles of product/time), X-axis: substrate concentration in Molarity.
  • Key Concepts:
  • Vmax: maximum enzyme velocity.
  • Km: substrate concentration at half Vmax.
• Factors Affecting Enzymes:
  • Reaction velocity influenced by temperature, pH, and inhibitors.
• Experiment Focus:
  • Using Catechol Oxidase to convert Catechol to Benzoquinone affecting the browning of fruits/vegetables.

Materials Required

• Buffers:
  • pH 4 Phosphate buffer
  • pH 6.8 Phosphate buffer
  • pH 9.0 Phosphate buffer
• Other Chemicals:
  • 1.50mM Phenylthiourea (PTU, inhibitor)
  • 0.30M Catechol
• Equipment:
  • Blender, Cheese Cloth, Beakers, Brown Glass Vials/Jars, Graduated cylinders, Logger Pro software, Spectrophotometer, Micropipettors, Water Baths, Ice.

Experimental Procedures

A. Catechol Oxidase Extract Preparation

1. Ingredients: Ice-cold pH 6.8 phosphate buffer, peeled potato.
2. Blend potato in buffer for 1 minute.
3. Filter through cheesecloth until light brown in color.
4. Wait 10 minutes to allow debris to settle, then aliquot to brown glass vials on ice.

B. Logger Pro Setup

1. Prepare Logger Pro with appropriate settings for Absorbance @ 400nm vs. Time.
2. Calibrate with RO/DI water in the cuvette.

C. Control Reactions

• Conduct the following three reaction controls:

1. Normal Reaction: Mix complete solution as fast as possible; record absorbance at specified intervals.
2. Absence of Substrate: Use only buffer and RO/DI to assess effects without substrate.
3. Absence of Enzyme: Use buffer and substrate to verify no reaction due to lack of enzyme.

D. Reaction with Inhibitor

1. Prepare and conduct reactions with PTU to observe inhibition impact.
2. Increase substrate concentration in subsequent reactions to analyze effects on reaction rate.

E. Temperature Variation Reactions

1. Prepare a master mix.
2. Conduct reactions at different temperatures (4C, 37C, 70C, 90C) to assess temperature's effect on enzyme activity.

F. pH Variation Reactions

1. Conduct reactions using phosphate buffers at pH 4.0 and 9.0 to observe pH impact on enzyme function.

Data Analysis

• Create scatter plots for absorbance @ 400nm vs. time for all reactions analyzed in Parts C-F.
• Include all relevant time points in the graphs and compare with the normal reaction data.

Discussion Points

• Analyze how each variable affected the normal reaction rate.
• Discuss the characteristics of PTU as an inhibitor.
• Identify any potential sources of error that could have influenced the data.