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Pre-Lab Questions

 

 

1. What is the name of the enzyme found in barley seeds that you’ll be studying during this lab investigation?
2. What is the function of this enzyme?
3. Iodine is an indicator of what substance?  What color results when iodine combines with the substance?

Part I: Does a Barley Seed Contain Amylase?

1. Why was the barley seed cut in half and printed on the starch indicator paper?

   

    

    

2. You needed exactly 20.0 mL of diluted iodine yet the stock solution of iodine was 10x.  How did you perform the dilution?                                                                                                                                                                                                        

3. Why did you need to allow your seed print to dry 5-10 minutes before submerging it in iodine?
4. Why did you submerge the starch paper with seed print in a solution of iodine?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
5. How do you account for the white spot on the stained starch paper?                                                                                                                            

Part IIA: Preparation of a Standard Curve

1. Why did you prepare two sets of test tubes for your dilution series?                                                                                                          
2. You needed to prepare exactly 27.0 mL of diluted buffer.  Since the stock solution was 10x, how did you make the diluted buffer?
3. You used a 1% starch solution.  How did Mrs. Annee prepare this solution? 
4. What is the role of starch in this experiment? 
5. Why are the test tubes (with amylase and starch) incubated at 37 degrees Celsius for five minutes?
6. Which of your six experimental test tubes (4 U/mL, 3 U/mL, 2 U/mL, 1 U/mL, 0.5 U/mL, or 0.25 U/mL) contained the darkest shade of purple?  Which one contained the lightest shade of purple?  Why?
7. What is the purpose of the control?
8. Observe the picture below.  What is the name of the machine?  What is its purpose in this lab?                                                                                                                 
9. What is the purpose of making a standard curve?

Part IIB: Determination of Specific Activity in Malted Barley

1. Observe the picture below.  What is the average mass of a malted barley seed?                                                                                                                
2. You needed exactly 15.0 mL of diluted buffer (the stock solution was 10x).  How did you prepare the diluted buffer?
3. Why was the seed crushed?
4. You transferred 1.8 mL of seed extract to a test tube and then added 0.1 mL of a 1% starch solution to the 1.8 mL of seed extract.  How did Mrs. Annee make the 1% starch solution?  What is the purpose of the starch solution?                                                                                         
5. According to the protocol, hydrochloric acid stops the reaction.  How does HCl accomplish this? 
6. Why did you add iodine to the seed extract and starch solution after the hydrochloric acid was added?
7. Consider #6 of the protocol (“Add 0.1 mL of 1% starch, mix, and incubate in a 37 degree Celsius waterbath for 5 minutes.”).  Imagine the bell rings…You are in a hurry to get to your next class so you place parafilm on the test tube and leave it at room temperature.  When you arrive to biotech class the next day, you add hydrochloric acid to the solution followed by iodine.  What color do you expect to see once the iodine is added?  Why?
8. Observe the picture below.  How did you quantify the amount of starch in the sample?

                                                             

Part IIC: Enzyme Activity Related to Time 

1. What is the purpose of the “blank”?
2. You stopped the reaction between amylase and starch at time zero, and after 1, 2, 3, 5, 7, and 10 minutes.  What was the purpose of performing this protocol?                              
3. Which test tube (0, 1, 2, 3, 5, 7, or 10) should contain the most starch?  The least starch?  Have the greatest absorbance value?  Have the least amount of amylase activity?

Part IID:  Enzyme Activity Related to Temperature

1. You tested amylase activity for five minutes at five separate temperatures: 4 degrees Celsius, 23 degrees Celsius, 37 degrees Celsius, 60 degrees Celsius, and 100 degrees Celsius.  At which temperature(s) did the greatest amount of amylase activity occur?  Did you expect this result?  Why or why not?