Part III: Determining Equilibrium Concentrations 1. To a spectrophotometer cuvet
ID: 946333 • Letter: P
Question
Part III: Determining Equilibrium Concentrations 1. To a spectrophotometer cuvette, add: a 900 uL ethanol (45 mM ethanol in 50 mM Tris buffer, pH 8.17) b. 900 uL NAD* (0.29 mM NAD in 50 mM Tris, pH 8.17) 2. Mix well. When a spectrophotometer is free, place the cuvette in the cell compartment Observe the absorbance reading. What do you expect it to be? 3. Add 100 uL of ADH (enzyme solution containing 120 units/mL), mix well and put back into the spectrophotometer. 4. Record the absorbance every 30 seconds until the system reaches equilibrium and there is no change in the absorbance reading. 5. Perform 3 trials. Do not discard the equilibrium mixtures! You will use them in Part V. Part IV: Changing Initial Conditions 1. Use the volumes determined from pre-lab questions 7 (ethanol) and 8 (NAD) for the second trialHave your TA check your calculations. 2. Add 100 uL of ADH (enzyme solution containing ; 120 units/mL), mix well and put back into the spectrophotometer. 3. Record the absorbance every 1 30 seconds until the system reaches equilibrium and there is no change in the absorbance reading. 4. Perform 3 trials. Do not discard the samples! Part V: Perturbation of Equilibrium 1. To an equilibrium mixture from part III, add 100 uL of ADH. Place the sample in the spectrophotometer and record the absorbance every 30 seconds for 4 minutes. 2. To a second equilibrium mixture, add 200 uL NAD . Place the sample in the spectrophotometer and record the absorbance every 30 seconds for 4 minutes. 3. To a third equilibrium mixture, add 200 WL EtOH. Place the sample in the spectrophotometer and record the absorbance every 30 seconds for 4 minutes.Explanation / Answer
Solution.
2. a) A catalyst speeds up both the forward and the reverse reactions making system to reach the equilibrium state faster.
b) A catalyst does not affect the equilibrium constant as it does not change the equilibrium concentrations of reactants or products.
c) A catalyst does not affect the percent yield of a single reaction. It only affects the time required to obtain some percent yield.
3. Besides the structure, the positive charge of NAD+ affects its ability to attract negative-charged hydride-ion.
When NADH acting as a hydride donor, it loses a hydride-ion, it forms the third double bond in the ring, and the compounds becomes aromatic. Aromatic compounds much more stable due to a resonance, so NAD+ is more stable.