Lab Report Lab Safety and Enzymes

After you complete this lab, you will be able to:

• Describe the function of the spectrophotometer
  • Explain the significance of the Lambert Beer Law
  • Describe what is being measured by the spectrophotometer
    • What does the diffraction grating do?
    • What is measured in nanometers?
    • What is absorbance?  How is it measured?  What is measured by the detector?
  • Define the cuvette
  • Describe the way in which the spectrophotometer measures absorbance
• Write a hypothesis based on textbook readings and lecture material
• Explore the relationship between the structure and function of proteins
  • Describe how the molecular structure of proteins is related to the function of enzymes
  • Define pH and use the definition to describe acid, base, and neutral
  • Using data, describe how temperature and pH changes affect enzyme activity
• Become familiar reaction set up for assessing enzyme activity
  • Define master mix
  • Identify the enzyme being measured, as well as the substrate
  • Determine the effect of increasing substrate concentration on the reaction rate of turnip peroxidase at a given enzyme concentration.
  • Explore what happens when you alter (do not worry about calculations—save that for chemistryJ )
    • The enzyme concentration
    • The substrate concentration
    • The pH and the temperature
      
      
• Analyze data

Introduction:

You will explore the laboratory equipment as you set up an experiment to explore the function of enzymes.  The concept of amino acids forming the primary structure of proteins, which leads to the complex three-dimensional enzyme molecule, is addressed in the lecture portion of the class.  In this experiment, you will investigate the enzyme, alcohol dehydrogenase.  We will observe the activity of the enzyme, as it catalyzes the conversion of the substrate (ethanol) to product (acetaldehyde) as NAD+ is reduced to NADH, using the spectrophotometer.You will explore the enzyme activity as you alter the substrate and enzyme.  You will continue collecting data as we investigate the impact of various temperature and pH levels on the activity of the enzymes.

The concept of enzyme activity as it relates to metabolism is foundational in the study of microbiology.  Enzymes are proteins that act as biological catalysts, allowing for the chemical reactions to maintain homeostasis. In this exercise we will measure the activity of the enzyme alcohol dehydrogenase using a spectrophotometer. This piece of equipment measures electromagnetic radiation (light).Compounds will emit or absorb or transmit light of different wavelengths. There is a relationship between the wavelength of light (emitted/absorbed) and frequency. If a particular compound is known to absorb or emit light at a particular wavelength, then measuring the amount of absorbance (transmittance) allow us to determine the concentration ofthe compound using Lambert-Beer Law.Beer’s Law states that the absorbance is directly proportional to the concentration.

The alcohol dehydrogenase added to ethanol and NAD+ causes the following reaction:

Ethanol + NAD+ → Acetaldehyde + NADH

In this exercise you will collect data under the following conditions:

• Altering the enzyme concentration
  • Altering the substrate concentration
•  room
• Adjusted temperature (ice bath, 50^o, or 80^o).
• Adjusted pH (pH 2, 5, 9 or 11)

In this exercise, you should consider the textbook selections and lecture material addressing topics of various pH levels on enzyme activity. pH determined by taking the opposite of the base 10 log of the molar concentration of hydrogen ions (pH = – log [H⁺]).   The pH scale (from approximately 0-14) is a measure of the amount of free hydrogen found in the solution.  Acids dissociate in water, releasing a hydrogen cation and an anion.  As a result of this dissociation, the hydrogen is ‘free’.   The higher the amount of free hydrogen the stronger the acid, the closer to zero on the pH scale.  Bases reduce the amount of free hydrogen available either directly (by binding the hydrogen ions, e.g. NH₃ + H⁺à NH₄) or indirectly (by neutralizing e.g. when dissociating in solution to release the hydroxide anion, OH ^–, which combines with H⁺ to form water).

Procedures: 

1. The easiest way to complete this lab report is to use the Labster simulations. If you are unable to use the simulations you will find all the information you need in learning sources 2-9 (at the top of this document). If you are able to use the labster simulations follow the steps below. 
2. Open the Labster Simulation titled Enzyme Kinetics
   1. Complete the simulation only through the pH and temperature optimization missions.
   1. The system is designed to gather data using the 340 nm wavelength on the Spectrophotometer.  Continue with the set-up of the test solutions. 
3. The experimental design was used to test the effects altering the substrate and enzyme concentrations, and altering physical conditions of temperature and pH on the enzyme. 
   1. Compare the graph of enzyme activity at pH 3, 5, 9, and 11 (all at 25^o C)
   1. Compare the graph of activity at the following temperatures: 0^o  ,25^o ,50^o ,70^o  (all at pH 8.5)
   1. Lab data will be provided on Canvas for your use in analyzing changes in the pH and temperature on for this enzyme activity.  Use this data for the lab report.  Note, the temperatures and pH levels are different than the simulations.
4. Open the Labster Simulation titled Protein Denaturation

Explore what happens to the proteins in solutions of hydrochloric acid, sodium hydroxide, ethanol, copper sulfate, and high and low salt solution

Fill out lab report questions and submit lab report before deadline.