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