Lab Two
Perform experiments 9-19:
After you are done with experiment #8, your TA will assign the remaining experiments (#9-19). Your TA will give you instructions for the experiments. Record your results on the board/overhead in front of class. After completion of the experiments, record the results from both your experiments as well as the other people in class, and then fill out the worksheet.
Notes:
P20 set to 100 = 10 µl (the last digit is a decimal and will be in red)
P200 set to 025 = 25 µl
P1000 set to 1000 = 1000 µl (or 1ml)
Also, note that some of the experiments use buffer alone, or ADP solution, and not the ATP solution.
Chart of Experiments Quick Hints:
Use 1 ml of the ATP, ADP, or buffer and if required add this to cuvette first
Use 10 µl of the appropriate enzyme(s) and/or substrate(s)
Mix reagents and incubate 5 minutes BEFORE adding 25 µl of the firefly extract
Treatment/Experimental Condition Result (Light/No Light) Duration of Light
1. ATP alone
2. Hexokinase/ATP
3. D-Glucose/ATP
4. Hexokinase/D-Glucose/ATP
5. Buffer Alone (no ATP)
6. ADP alone (no ATP)
7. Phos.Creatine+CreatPhoskinase + ADP (no ATP)
8. Hex/D-Glu + PhCreat+CrPhosKin +ATP
Treatment/Experimental Condition Result (Light/No Light)
9. Hexokinase/L-Glucose/ATP
10. Hexokinase/Sucrose/ATP
11. Hexokinase/Mannose/ATP
12. Hexokinase/Fructose/ATP
13. Hexokinase/Arabinose/ATP
14. Hexokinase/Maltose/ATP
15. Hexokinase/Ribose/ATP
16. Hexokinase/Lactose/ATP
17. Hexokinase/Erythrose/ATP
18. Hexokinase/Galactose/ATP
19. Hex/D-Glu/PhCr/CrPhK in Buffer (no ATP)
Answer the following questions in the separate worksheet:
Thinking about assay experiments 1 through 5, answer the following:
What is the hypothesis that these experiments (1-5) are designed to test? Did they support or reject that hypothesis?
Which experiments/cuvettes (1-5) are controls? What parameters does each cuvette control for in the overall experiment? In other words, for each cuvette listed as a control, what part of the overall hypothesis test would you be unsure of if you hadn't run it?
Thinking about assay experiments 6 through 8, answer the following:
What hypothesis do you think these experiments (6-8) are designed to test? Did they support or reject that hypothesis?
Which experiments/cuvettes (6-8) are controls? What parameters does each cuvette control for in the overall experiment? In other words, for each cuvette listed as a control, what part of the overall hypothesis test would you be unsure of if you had not run it?
Thinking about the lab overall and experiments 9-18, answer the following questions:
Why do you think enzyme concentrations are often listed in activity units (i.e., concentration of substrate consumed per minute/ml) rather than absolute concentration (mg/ml or molarity)?
When Hexokinase/Glucose and CreatPhosKin/Phosphocreatine reactions are running concurrently, what will determine which set of reactions will predominate or react the longest? We ran this experiment in cuvette 8. Which reaction ran the longest? Explain how you determined this and explain specifically why that reaction predominated.
Looking at the results of your group and your classmates with regards to tests 9 - 19, what hypothesis can you draw from the results with respect to hexokinase's substrate specificity? Tip: You might want to organize your hexokinase-sugar data by grouping together data from sugars with equivalent numbers of carbons, then looking to see whether all 4 carbon sugars react similarly, 5 carbon sugars, disaccharides etc. Note that to do this you will have to look up the number of carbons of each sugar. For those sugars which are disaccharides, you may want to look at which two monosaccharides they are composed of.