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Laboratory Exercise: Activation Energy
Introduction
The hydrolysis ('inversion') of sucrose, completely or partially, to glucose and fructose provides sweet syrups that are more stable (i.e. less likely to crystallize) than pure sucrose syrups. This hydrolysis reaction has historically been performed using acid hydrolysis (e.g. Lyle's Golden Syrup). However, with the growth in the market for colorless invert syrups, which are costly to produce using acid due to the downstream purification needed, the use of the yeast enzyme invertase has become more popular. Sucrose is α-D-glucopyranosyl (1-2)-β-D-fructofuranoside and can therefore be hydrolyzed both by α-glucosidases and β-fructofuranosidases. Invertase is a β-fructofuranosidase that hydrolyses sucrose as well as other β-fructans such as raffinose.
Reagents
- Sucrose: 100 mM in 0.5 M acetate buffer, pH 4.8
- Invertase enzyme solution
- 3,5-dinitrosalicylic acid reagent: 1 g 3,5-dinitrosalicylic acid (DNSA) + 30 g sodium potassium tartrate + 20 mL 2 N NaOH, diluted to a final volume of 100 mL
Equipment
- 10 mL graduated cylinder
- Dessicators
- Microwave oven
- Vacuum oven
- Small plastic cups
- Aluminum weigh cups
- Weighing pads for use in microwave
- Water activity meter
- Water activity sample containers
Procedure
You will be measuring the effects of temperature on enzyme activity. A number of water baths ranging from 4°C to 40°C will be found in the room. Each group is to perform analysis at room temperature (RT) and at two additional temperatures. These temperatures will be assigned by the instructor. For each temperature, place a tube containing 20 mL of substrate solution and another containing 1.0 mL of enzyme solution in the water bath and allow them to equilibrate for 5 minutes. Place 2 mL of DNSA solution in each of 5 test tubes. Place 0.2 mL of enzyme solution in the tube containing the substrate and mix well by inverting the tube twice. Remove 1.0 mL of this solution and add it to a tube containing 2.0 mL of DNSA. This sample will be counted as the 0 time sample. Remove additional samples at 2, 4, 6 and 8 minutes. Place the tubes in a boiling water bath for exactly 5 minutes. Cool the tubes for 5 minutes, then record the absorbance of the solution at 540 nm.
Results
Present all of your data. Plot the reaction rate data obtained for the three temperatures you performed. Data obtained by other groups at other temperatures will be given to you by the instructor. Use this information to obtain an energy of activation for the inversion of sucrose by the enzyme.
Questions
- What is the Ea you obtained for the reaction? How does this compare to literature values?
- The reported Ea for the acid is 25.2 Kcal/mole. What would be the relative rates of these two reactions at 50°C?
- At what temperature does the rate of the inversion of sucrose by acid equal the rate obtained by the enzyme at 37°C?
- Given the information obtained in Question 1 if you measured the rate of invertase at 30°C but later found that your thermometer was not properly calibrated and the reaction was actually measured at 33°C, how large would your error be?
- An enzyme catalyzed reaction was found to be 3 times faster at 37°C than at 12°C. What was the energy of activation of the enzyme catalyzed reaction? What can you say about DG of the reaction?