Lab Enzymes

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Full Name:

 

UMUC Biology 102/103

Lab 4: Enzymes

INSTRUCTIONS:

 

        On your own and without assistance, complete this Lab 4Answer Sheet electronically and submit it via the Assignments Folder by the date listed intheCourse Schedule (underSyllabus).

        To conduct your laboratory exercises, use the Laboratory Manual located under Course Content. Read the introduction and the directions for each exercise/experiment carefully before completing the exercises/experiments and answering the questions.

        Save your Lab 4Answer Sheet in the following format:  LastName_Lab4 (e.g., Smith_Lab4).

        You should submit your document as a Word (.doc or .docx) or Rich Text Format (.rtf) file for best compatibility.

 

Pre-Lab Questions

 

1.      How could you test to see if an enzyme was completely saturated during an experiment?

 

2.      List three conditions that would alter the activity of an enzyme. Be specific with your explanation.

 

3.      Take a look around your house and identify household products that work by means of an enzyme. Name the products, and indicate how you know they work with an enzyme.

 

 

Experiment 1: Enzymes in Food

This experiment tests for the presence of amylase in food by using Iodine-Potassium Iodide, IKI. IKI is a color indicator used to detect starch. This indicator turns dark purple or black in color when in the presence of starch. Therefore, if the IKI solution turns to a dark purple or black color during the experiment, one can determine that amylase is not present (because presence of amylase would break down the starch molecules, and the IKI would not change color).

 

Materials

(1) 2 oz. Bottle (Empty)

(1) 100 mL Graduated Cylinder

30 mL Iodine-Potassium Iodide, IKI

Permanent Marker

Ruler

2 Spray Lids

30 mL Starch (liquid)

*Cutting Board

  

*2 Food Products (e.g., ginger root, apple, potato, etc.)

*Kitchen Knife

*Paper Towel

*Saliva Sample

*Tap Water

*You Must Provide

 

Procedure:

1.      Remove the cap from the starch solution. Attach the spray lid to the starch solution.

2.      Rinse out the empty two ounce bottle with tap water. Use the 100 mL graduated cylinder to measure and pour 30 mL of IKI into the empty two ounce bottle. Attach the remaining spray lid to the bottle.

3.      Set up a positive control for this experiment by spraying a paper towel with the starch solution. Allow the starch to dry for approximately one hour (this time interval may vary by location).

4.      In the mean time, set up a negative control for this experiment. Use your knowledge of the scientific method and experimental controls to establish this component (hint: what should happen when IKI solution contacts something that does not contain starch?) Identify your negative control in Table 1.

Note: Be sure to space the positive and negative controls apart from each other to prevent cross-contamination.

5.      When the starch solution has dried, test your positive and negative controls. This step establishes a baseline color scale for you to evaluate the starch concentration of the food products you will test in Steps 7 – 11. Record your results in Table 1.

6.      Select two food items from your kitchen cabinet or refrigerator.

7.      Obtain a kitchen knife and a cutting board. Carefully cut your selected food items to create a fresh surface.

Figure 3: Sample set-up.

8.      Gently rub the fresh/exposed area of the food items on the dry, starch-sprayed paper towel back and forth 10 – 15 times. Label where each specimen was rubbed on the paper towel with a permanent marker (Figure 3).

9.      Wash your hands with soap and water.

10.  Take your finger and place it on your tongue to transfer some saliva to your finger. Then, rub your moistened finger saliva into the paper towel. Repeat this step until you are able to adequately moisten the paper towel.

Note: You should always wash your hands before touching your tongue! Alternatively, if you do not wish to put your hands in your mouth, you may also provide a saliva sample by spitting in a separate bowl and rubbing the paper towel in the saliva. Be sure not to spit on the paper towel directly as you may unintentionally cross-contaminate your samples.

11.  Wait five minutes.

12.  Hold the IKI spray bottle 25 – 30 cm away from the paper towel, and mist with the IKI solution.

13.  The reaction will be complete after approximately 60 seconds. Observe where color develops, and consider what these results indicate. Record your results in Table 1.

Table 1: Substance vs. Starch Presence
Substance Resulting Color Presence of Starch?
Positive Control: Starch    
Negative Control: Student Must Select    
Food Product:    
Food Product:    
Saliva:    

 

Post-Lab Questions

1.      What were your controls for this experiment? What did they demonstrate? Why was saliva included in this experiment?

 

2.      What is the function of amylase? What does amylase do to starch?

 

3.      Which of the foods that you tested contained amylase? Which did not? What experimental evidence supports your claim?

 

4.      Saliva does not contain amylase until babies are two months old. How could this affect an infant’s digestive requirements?

 

5.     There is another digestive enzyme (other than salivary amylase) that is secreted by the salivary glands. Research to determine what this enzyme is called. What substrate does it act on? Where in the body does it become activated, and why?

 

6.       Digestive enzymes in the gut include proteases, which digest proteins. Why don’t these enzymes digest the stomach and small intestine, which are partially composed of protein?

 

 

Experiment 2: Effect of Temperature on Enzyme Activity

Yeast cells contain catalase, an enzyme which helps convert hydrogen peroxide to water

Figure 4: Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.

and oxygen. This enzyme is very significant as hydrogen peroxide can be toxic to cells if allowed to accumulate. The effect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2 H2O2 → 2 H2O + O2).

In this lab you will examine the effects of temperature on enzyme (catalase) activity based on the amount of oxygen produced. Note, be sure to remain observant for effervescence when analyzing your results.

 

Materials

(2) 250 mL Beakers

3 Balloons

30 mL 3% Hydrogen Peroxide, H2O2

Measuring Spoon

Permanent Marker

Ruler

20 cm String

  

3 Test Tubes (Glass)

Test Tube Rack

Thermometer

Yeast Packet

*Hot Water Bath

*Stopwatch

*You Must Provide

 

Procedure

1.      Use a permanent marker to label test tubes 1, 2, and 3. Place them in the test tube rack.

2.      Fill each tube with 10 mL hydrogen peroxide. Then, keep one of the test tubes in the test tube rack, but transfer the two additional test tubes to two separate 250 mL beakers.

3.      Find one of the balloons, and the piece of string. Wrap the string around the uninflated balloon and measure the length of the string with the ruler. Record the measurement in Table 2.

4.      Create a hot water bath by performing the following steps:

1.      Determine if you will use a stovetop or microwave to heat the water. Use the 100 mL graduated cylinder to measure and pour approximately 200 mL of water into a small pot or microwave-safe bowl (you will have to measure this volume in two separate allocations).

2.      If using a stovetop, obtain a small pot and proceed to Step 4c. If using a microwave, obtain a microwave-safe bowl and proceed to Step 4e.

3.      If using a stove, place a small pot on the stove and turn the stove on to a medium heat setting.

4.      Carefully monitor the water in the pot until it comes to a soft boil (approximately 100 °C). Use the thermometer provided in your lab kit to verify the water temperature. Turn the stove off when the water begins to boil. Immediately proceed to Step 5.

CAUTION: Be sure to turn the stove off after creating the hot water bath. Monitor the heating water at all times, and never handle a hot pan without appropriate pot holders.

5.      If using a microwave, place the microwave-safe bowl in the microwave and heat the water in 30 second increments until the temperature of the water is approximately 100 °C. Use the thermometer provided in your lab kit to verify the water temperature. Wait approximately one minute before proceeding to Step 5.

5.      Place Tube 1 in the refrigerator. Leave Tube 2 at room temperature, and place Tube 3 in the hot water bath.

Important Note: The water should be at approximately 85 °C when you place Tube 3 in it. Verify the temperature with the thermometer to ensure the water is not too hot! Temperatures which exceed approximately 85  °C may denature the hydrogen peroxide.

6.      Record the temperatures of each condition in Table 2. Be sure to provide the thermometer with sufficient time in between each environment to avoid obscuring the temperature readings.

7.      Let the tubes sit for 15 minutes.

8.      During the 15 minutes prepare the balloons with yeast by adding ¼ tsp. of yeast each balloon. Make sure all the yeast gets settled to the bulb of the balloon and not caught in the neck. Be sure not spill yeast while handling the balloons.

9.      Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube.

10.  Attach the neck of a balloon you prepared in step 8 to the top of Tube 2 (the room temperature test tube) making sure to not let the yeast spill into the test tube yet. Once the balloon is securely attached to the test tube lift the balloon and allow the yeast to enter the test tube. Tap the bulb of the balloon to ensure all the yeast falls into the tube.

11.  As quickly and carefully as possible remove the Tube 1 (cold) from the refrigerator and repeat steps 9 – 10 with Tube 1 using a balloon you prepared in step 8.

12.  As quickly and carefully as possible remove Tube 3 (hot) from the hot water bath and repeat steps 9 – 10 with Tube 3 using a balloon you prepared in step 8.

13.  Swirl each tube to mix, and wait 30 seconds.

14.  Wrap the string around the center of each balloon to measure the circumference. Measure the length of string with a ruler. Record your measurements in Table 2.

Table 2: Balloon Circumference vs. Temperature
Tube Temperature (°C) Balloon Circumference (Uninflated; cm) Balloon Circumference (Final; cm)
1 – (Cold)      
2 – (RT)    
3 – (Hot)    

 

 

Post-Lab Questions

1.      What reaction is being catalyzed in this experiment?

2.      What is the enzyme in this experiment? What is the substrate?

3.      What is the independent variable in this experiment? What is the dependent variable?

4.      How does the temperature affect enzyme function? Use evidence from your data to support your answer.

 

5.      Draw a graph of balloon diameter vs. temperature. What is the correlation?

 

6.      Is there a negative control in this experiment? If yes, identify the control. If no, suggest how you could revise the experiment to include a negative control.

 

7.      In general, how would an increase in substrate alter enzyme activity? Draw a graph to illustrate this relationship.

 

8.      Design an experiment to determine the optimal temperature for enzyme function, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use?

 
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Micro Bio Case Study

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Texas Southern University*** Clinical Laboratory Science *** CLSC 369 – Case Studies I

Name: _______________________________________________ Score: ________/50

Case 1:

This organism was isolated from blood-tinged, nasal discharge of a diabetic, 65-year-old male.

Growth was detected after 2 days of incubation at 25ºC on Sabouraud dextrose agar (SDA), with no growth on Mycosel agar.

image1.jpg

1. Identify this fungus.

2. This fungus belong to the class _____________________

3. This class of fungus has __________ hyphae and an asexual reproductive structure called a ___________________.

4. Macroscopically, this fungus’ colony is described as:

5. This class of fungi is the causative agent of ________________which is associated with infection of the __________________________.

Case 2:

This fungus was isolated from the lung of a 35-year-old female lymphoma patient. Growth was detected after 3 days of incubation at 25ºC on SDA, with no growth on Inhibitory mold agar.

image2.jpg

1. Identify this fungus.

2. What test(s) would you performed to identify your named fungus?

3. Microscopic characteristics of this fungus include: _______________________________________________________________________

4. Some species of this genus are the etiologic agents of __________________________

Case 3:

This fungus was isolated from the sputum of a 35-year-old male. A brown colony was detected at 72 hours of incubation at 25ºC on SDA, with no growth on Mycosel agar.

image3.jpg

1. Identify this fungus.

2. Microscopic characteristics of this fungus include:

3. Clinically, this fungus is considered to be a/an:

Case 4:

This organism was isolated from an inflamed cornea of a 14-year-old male contact lens wearer.

Growth was detected on SDA on day 3 of incubation at 25ºC with no growth on Mycosel agar.

image4.jpg

1. Identify this fungus.

2. Clinically, this fungus is considered to be a/an:

3. A medical student submitted to the laboratory a Mycosel slant inoculated at bedside with corneal scrapings he had collected from a 20-year-old female inpatient. He indicated that the physician was trying to rule out mycotic keratitis. What is the next best step?

Case 5:

Shown is a Periodic-Acid-Schiff (PAS) stain of a lung biopsy from a 58-year-old male who complained of a chronic, productive cough. He smoked two to three packs of cigarettes a day for the last 35-40 years. Upon x-ray, a homogeneous mass was noted in his left lung. The wet preparations are of the organism growth after 12 days of 35ºC incubation.

 

1. Identify this fungus.

2. The microscopic morphology of this organism’s growth at 25° C characteristically exhibits __________________________________________________________

Case 6:

A 24-year old male forestry student from Washington State developed an infection on

his left third finger, which drained serous fluid. Shortly thereafter he developed painful

hyperpigmented nodules up the dorsal left arm. On examination he had 2 nonfluctant nodules that were palpable along this line. Two pustules were present over the left biceps muscle and no axillary lymphadenopathy was present. A diagnosis of staphylococcal cellulitis was made and the patient was treated without resolution of the lesions. The patient returned home to Rochester, Minnesota and went directly to St. Mary’s Hospital Emergency Department. A history of handling sphagnum moss was given by the patient. A nodule was aspirated and the exudate sent for culture. The patient was placed on itraconazole therapy and sent home.

Slide-1 Slide-2 Slide-3
image7.jpg image8.jpg image9.jpg
Slide-4 Slide-5 Slide-6
image10.jpg image11.jpg image12.jpg

image13.jpg

1. Identify this fungus.

2. Describe the characteristics, macroscopic and microscopic morphology of the fungus growth at 25° C.

Page 3 of 3

 
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Biology 121 Lab

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1. A.  Briefly, outline the main steps of the photosynthetic pathway.  Be sure to indicate how the light-dependent and light-independent reactions are coupled.

B. Why is Chlorophyll-a central to the light-dependent reactions?  AND – What important role(s) do accessory pigments play in this process?

2. Consider last week’s laboratory exercise concerning carbon-fixation in an aquatic plant.  Recall, we used BTB to monitor pH of the surrounding medium as a proxy for CO2 concentration.    Why might we expect to see a DECREASE in pH in the plant/dark tube?  (i.e., What metabolic process might contribute to this result?)
3.  If a farm pond, stocked with fish and plants, were measured for pH at sunrise and sunset what would be the results?  Why?
4. Describe the redox reaction that was the subject of the “Hill reaction”.  What is normally the final electron acceptor and what did we use as a substitute? Why did we use what we used?

CELLULAR RESPIRATION

 

 

What is CELLULAR respiration?

chemical E (glucose) + O2 → “biochemical currency” (ATP)

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + E

  Oxygen (O2) is ESSENTIAL for AEROBIC respiration…

  4 main steps…1 is common to both aerobic AND anaerobic respiratory pathways…

 

 

Aerobic vs. Anaerobic Respiration

  Aerobic   requires O2   4 main steps   yields up to 38 ATP glucose-1   obligate aerobes, facultative anaerobes

  Anaerobic   NO O2 required   1 main step   yields 2 ATP glucose-1   obligate anaerobes

 

 

The Mitochondrion

•  Glucose is broken down in the cytoplasm •  Kreb’s Cycle occurs in the matrix •  Electron transport occurs in/on the cristae

(envelope)

 

 

Aerobic Respiration

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + E

Step 1: Glycolysis (“glyco” “lysis”; cytoplasm)*

glucose → 2 pyruvate + 2 ATP + 2 NADH2 (6C) (3C)

cytoplasm

glycolysis

 

 

Step 2: Pyruvate Oxidation (mito matrix)

2 pyruvate → 2 Acetyl CoA + 2 CO2 + 2 NADH2 (3C) (2C)

pyruvate oxidation

matrix

 

 

Step 3: Kreb’s Cycle (aka TCA Cycle or Citric Acid Cycle; mito matrix)

2 Acetyl CoA + 2 Oxaloacetic Acid (2C) (4C)

2 Citric Acid + 4 CO2 + 2 ATP + 6 NADH2 + 2 FADH2 (6C)

Kreb’s Cycle

matrix

 

 

Step 4: Oxidative Phosphorylation (aka e- transport; mito cristae)

  NADH2 + FADH2 are involved with e – transport

  donate e- to carriers in the transport chain   pumping of H+ ions → [ ] gradient   generation of ATP

  O2 is the final e- acceptor oxidative phosphorylation

cristae

 

 

out

in

mito matrix

intermembrane space

NADH2 & FADH2

ATP synthase

 

 

Summary   3 ATP per NADH2 (x 10) (= 30; steps 1-3)   2 ATP per FADH2 (x 2) (= 4; step 3)   4 “substrate-level” ATP (= 4; steps 1 & 3)

38 TOTAL glucose-1

  Theoretical maximum = 38 ATP…no system is perfect!

∴ this number is rarely [if ever] achieved…

 

 

Anaerobic Respiration

glucose → 2 pyruvate + 2 ATP + 2 NADH2 (6C) (3C)

2 lactate 2 ethanol + 2 CO2 (= fermentation) (3C) (2C)

– animals – plants – microbes – microbes

demand > O2 release of metabolic poison

 

 

5.1 Respiration in Peas Protocol 5.1: Germinating vs. Non-germinating

⇒  Atmospheric/background CO2 level = 350-400 ppm

1.  Obtain 25 germinating peas & blot dry 2.  Place the peas in the respiration chamber 3.  Place the CO2 sensor in the chamber 4.  Wait 1 minute → begin collecting data for 5 minutes 5.  Measure & record the weight (g) of the peas 6.  Place the germinating peas in a beaker and place on ice for 5 minutes 7.  Follow the instructions on pg. 4

  determine the rate of respiration (slope, m = rate; ppm CO2 min-1)   store the data for comparison with other measurements

15.  Rinse and dry chamber 16.  Place the CO2 sensor in the chamber with non-germinating peas 17.  Wait 1 minute → begin collecting data for 5 minutes 18.  Follow the instructions on pg. 4

  Use a notebook to “fan” (i.e., clear) the sensor for 1 minute, returning the CO2 level to 300-400 ppm between EACH measurement!

 

 

5.2 Respiration in Peas Protocol 5.2: Room vs. Cold Temperature

1.  Empty the chamber by PUTTING THE NON-

GERMINATING PEAS BACK ON THE SIDE BENCH, and “clear” it…

2. Rinse and dry chamber

3. Repeat steps 1-7 (Protocol 5.1) using COLD germinating peas

 

 

5.3 Respiration in Crickets Protocol 5.3: Room vs. Cold Temperature

1.  Obtain 5-8 crickets & place in the respiration chamber 2.  Place the CO2 sensor in the chamber 3.  Wait 1 minute → begin collecting data for 5 minutes 4.  Measure & record the weight (g) of the crickets 5.  Place the crickets in the chamber on ice for 5 minutes (or until static) 6.  Follow the instructions on pg. 4

  determine the rate of respiration (slope, m = rate; ppm CO2 min-1)   store the data for comparison with other measurements

7.  Repeat steps 1-6 using COLD crickets 8.  Rinse & dry the respiration chamber when finished

  Use a notebook to “fan” (i.e., clear) the sensor for 1 minute, returning the CO2 level to 300-400 ppm between EACH measurement!

 

 

Do the results support your predictions? Peas   Germinating vs. Non-Germinating

  germinating > non-germinating   WHY?

  Room vs. Cold Temperature   room > cold   WHY?

Germinating

Germinating/COLD

Non-germinating

 

 

Do the results support your predictions?

Crickets   Room vs. Cold Temperature

  room > cold   WHY?

  What about PEAS vs. CRICKETS??   Why is it important to “normalize” by some

biological parameter (= fresh weight) for comparison?

 
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Biology 301 Pamphlet Assignment

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Biology 301 Pamphlet Assignment

Pamphlet addressing non-scientific community and presentation

 

Addresses Learning Outcome:

Recognize risk factors leading to disease and identify preventive measures and treatments.

This assignment is designed for you to demonstrate your ability to communicate your knowledge of a disease to the lay (non-scientific and non-medical) public.

Botulism

The disease or disorder should not be a common (uncommon) chronic disease or a unique preventable disease that has already addressed in our course.  Common diseases include coronary artery disease, Alzheimer disease, arthritis, diabetes, AIDS, hypo- and hyper-thyroidism, hypertension, psoriasis, sleep apnea, Lyme’s Disease, sinusitis, allergic rhinitis, mononucleosis, asthma, urinary tract infections, many STDs (check with your instructor), irritable bowel disease, strep throat, MRSA, polio, tuberculosis, Lockjaw, anorexia nervosa, autism, Down syndrome, and many cancers (check with your instructor).  For any additions to this list, check with your instructor.

 

Process

Create an informative pamphlet about the chosen chronic or preventable disease for a non-scientific community. Ideally, the pamphlet should be in the form of a six-page pamphlet.

 

Content

The pamphlet should contain the following:

Content and language that targets a lay audience.  All terms should be explained and diagrams and graphics should be provided to clarify concepts and ideas.

A brief description of the disease, including its symptoms and signs

Explanation of the effects of the disease on healthy body function including cellular, tissue, organ and organ system levels

Risk factors contributing to the onset of the disease

Description of preventive steps to avoid the disease (if avoidable)

Available diagnostic and therapeutic tools

Outcomes of the disease (such as prognosis or recovery potential)

Information about how a person suffering from the disease can maintain a desirable quality of life, minimize or slow the progress of the disease

 

Body of the pamphlet

At a minimum, your pamphlet should contain the following required elements:

Introduction

Addresses signs, symptoms

Explains effects of the disease on healthy body functions, and relates this to signs and symptoms

Analyzes risk factors and preventive steps

Describes maintenance of quality of life

Discusses diagnostic and therapeutic tools

Explains expected outcomes and prognosis

Describes current areas of research into prevention, treatment or cure

Describes possible future areas of research into prevention, treatment or cure

Conclusion – summary of your findings

Final list of references included in pamphlet

Clarity, proper grammar, punctuation, spelling

 

List all references at the end of your pamphlet. You must use at least five references from credible, scientifically rigorous sources and cite them using American Psychological Association (APA) style. Two or more references should be from the UMUC Library. The references cited should be no older than seven years (published 2004 – 2011).  References should be listed on a separate page that does not count toward the required length of the paper.

 

Format of the pamphlet

Single-spaced

Two tri-fold pages (if printed back to back this tri-fold would be one page printed on both sides)

All content written in your own words

Reference citations included in APA format (not part of the tri-fold; should be provided on a separate page)

 

 

Pamphlet and PPT presentation (10%): Explain a disease of the reproductive system that has a genetic basis — risk factors, prevention, maintenance of health, treatment, outcomes; chronic disease—maintenance of state (prevention/slow progression), quality of life. If none is to your liking, OK to choose another disease that you have not chosen before. Also explain the disease’s effects on the body.  Post the Pamphlet in your Assignments Folder but the PPT in the Discussion of week # 8.

 
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