Activity 1: Virtual Lab: Enzyme Activity Assay
- Go to the following link to complete the virtual lab: https://biomodel.uah.es/en/lab/abs/activ_enz.htm#
- In this virtual lab you will be manipulating pH and temperature to observe their effects on enzyme activity.
- The enzyme we will be using will be using is 6-O-α-L-ramnosyl-D-glucosidase. This enzyme converts our substrate, hesperidin, into the products of rutinose and hesperetin.
- The amount of rutinose can be quantified by using an assy. The assay we will be using is performed by adding the chemical DNS to our solution containing the rutinose and hesperetin. The DNS binds to the rutinose to form a red product. The absorbance of this product can be measured by using a spectrophotometer at a wavelength of 540 nm. The higher the absorbance reads indicates a high level of product and thus a high level of rutinose. More details about the reaction can be found by clicking the “Fundamentals of the assay” button near the bottom of the webpage.
- To perform the experiment, do the following:
- Adjust the pH/Temperature as needed by sliding the scale on the left-hand side of the page.
- Once you have your pH/Temperature adjusted, add the buffer and substrate to the cuvette by pressing the “add” button.
- Next, add the enzyme to the cuvette by pressing the “add” button that appears next to the word “sample”. A pop-up window will appear while the sample is being incubated. Once the incubation period is over a green checkmark will appear.
- Afterwards, add the DNS to the cuvette by pressing the “add” button that appears next to the word “DNS”. Once the incubation period is over a green checkmark will appear.
- Finally, click the “take measurement” button that is on top of the spectrophotometer and a reading will be displayed. Enter the absorbance values into the table below.
Table 1: Absorbance (540 nm) of products from the virtual enzyme activity assay.
|Absorbance (540 nm)||pH|
Questions (All questions are worth 1 point unless otherwise noted)
- Perform the experiment and fill in the missing data in Table #1. (Worth 2 points)
- Using the data from Table 1, create a graph using excel (or whatever other means you’d like). The X values will be the temperature and the Y values will be the absorbencies. Be sure to include all aspects of a properly constructed graph. You can insert the graph below this question or append it to the end of this document. (Worth 5 points)
- What was the optimal temperature?
- What was the optimal pH?
- What was the substrate?
- What was the enzyme?
- What was the purpose of letting the substrate and enzyme incubate for 1 hour?
- What purpose did the DNS serve?
Activity 2: Cellular Respiration
- In glycolysis, glucose is converted to
- CO2 and H2O
- Acetyl coA
- NAD+ and ADP
- The NET result of a single glycolysis run is the formation of
- 1 NADH and 1 ATP
- 2 NADH and 2 ATP
- 2 NADH and 4 ATP
- 4 NADH and 2 ATP
- 4 NADH and 4 ATP
- Under anaerobic conditions, the end-product of glycolysis is converted to
- CO2 and H2O
- Amino acids
- Lactic acid
- Hydrochloric acid
- Acetic acid
- During glycolysis, a 6-carbon sugar diphosphate molecule is split into two 3-carbon sugar phosphate molecules.
- Under aerobic conditions, the end-product of glycolysis is further reduced to yield more ATP.
- Before entering the Krebs cycle, pyruvate is converted to
- H2O and CO2
- Acetic acid
- Acetyl coA
- A single “turn” of the Krebs cycle will yield
- 1 ATP, 2 NADH and 1 FADH2
- 1 ATP, 2 NADH and 2 FADH2
- 1 ATP, 3 NADH and 1 FADH2
- 2 ATP, 2 NADH and 2 FADH2
- 2 ATP, 3 NADH and 2 FADH2
- The initial reaction of the Krebs cycle involves the addition of a
- 2-carbon molecule to a 4-carbon molecule
- 2-carbon molecule to a 5-carbon molecule
- 2-carbon molecule to a 6-carbon molecule
- 3-carbon molecule to a 4-carbon molecule
- 3-carbon molecule to a 5-carbon molecule
- The Krebs cycle occurs in the mitochondria.
- A single “turn” of the Krebs cycle involves four different decarboxylation reactions.
Electron Transport and ATP Formation
- The electron transport chain in bacteria is located
- In the cytoplasm
- In the mitochondria
- In the cell wall
- In the cell membrane
- In the plasmid
- During aerobic respiration, the last carrier protein transfers a pair of electrons to
- Coenzyme Q
- A proton (H+)
- The function of the enzyme ATP synthase is to
- accept a proton from inside the cell membrane as it accepts electrons.
- utilize the energy of the proton motive force to convert ADP to ATP.
- produce reduced coenzymes like NADH.
- transfer hydrogen to the electron transport chain.
- shuttle electrons from NADH to a terminal electron acceptor.
- Electrons enter the electron transport chain when NADH transfers them there along with protons in the form of hydrogen.
- The electron transport chain consists of a series of membrane-bound carriers that shuttle protons and electrons to NADH.
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