To be, or not be (a chemical equilibrium), that is the question:

Transcription

1 To be, or not be (a chemical equilibrium), that is the question: Enzymes are catalysts and cannot deviate from the laws of thermodynamics. nce the Gibbs free energy change ( G) for the overall reaction that an enzyme catalyzes is zero, the reaction is at equilibrium and no additional product accumulates. This might seem to be a problem for a cell in that enzymes will bring everything to equilibrium and then the flux stops. Fortunately, chemical reactions within a cell have evolved to utilize one another s products. In this way, flux through any particular reaction can be continuous since the products of the reaction are being utilized in other reaction and equilibrium is often not reached. In this experiment, you are going to examine a redox reaction that does not go to completion. You will determine the equilibrium position of the reaction. Finally, you are going to observe what happens when a reaction at equilibrium is coupled to a second reaction, which depletes a product (as in metabolism). Additionally, you will determine the [alcohol] in an unknown sample. ur reaction of interest is the oxidation of ethanol to acetaldehyde as catalyzed by alcohol dehydrogenase (AD):

2 To observe this reaction, we are going to utilize the UV absorbance properties of AD (ε 340nm = mm 1 cm 1 ). AD does not absorb light at 340 nm. You will utilize a pyrophosphate buffer at p 8.0. Some of your reactions will contain semicarbazide, which will react with aldehydes to form a Schiff s base: Semicarbazide Ethanol Yeast Alcohol Dehydrogenase 2 AD 2 2 Semicarbazide Acetaldehyde 2 2 AD on-enzymatic Reaction 2 2 Acetaldehyde Semicarbazone 2 2 AD

3 Reactions that contain semicarbazide will go to completion since the acetaldehyde will be reacted any. Reactions without semicarbazide will go to equilibrium containing ethanol, AD, AD, and acetaldehyde. Before Lab: 1. Make a table that summarizes the starting concentration of semicarbazide, AD, and AD in each tube. 2. If all of the AD in a tube were converted to AD, what would be the absorbance at 340 nm? 3. Rank each of the following from lowest to highest expected absorbance at 340 nm: a. Tube 1; Tube 3; and Tube 4 b. Tube 2; Tube 5; and Tube 6 In lab: Make the following solutions very carefully [add everything accept the AD, mix well, then add the AD]: Tube Buffer (without semicarbazide) Buffer (with 75 mm semicarbazide) 16 mm AD 1200 units/ml AD Et test solution Unknown solution d 2 1 2,625 ul ul 75 ul ul 2 2,625 ul 150 ul 75 ul ul 3 2,625 ul ul 75 ul 150 ul ,625 ul 150 ul 75 ul 150 ul ,625 ul ul 75 ul ul - 6-2,625 ul 150 ul 75 ul ul - Incubate the solutions at room temperature until equilibrium is reached (~20 minutes). Record the absorbance of each solution at 340 nm using d2 as a blank. Subtract the absorbance value of Tube 1 from the absorbance values of Tubes 3 and 5. Likewise, subtract the absorbance value of Tube 2 from the absorbance values of Tubes 4 and 6.

4 1.) Determine each of the following concentrations for tube 3 in units of mm (show your work): [AD]eq [AD ]eq [C3C2]eq [C3C]eq [ ]eq [AD]eq Use Beer s Law to convert the absorbance value at 340 nm into [AD]eq: A = εlc c = *, [C3C]eq The 1:1 ratio of stoichiometric coefficients within equation 1 require: [AD]eq=[C3C]eq. [AD ]eq Determine the initial concentration of AD that you added to tube 3. The 1:1 ratio of stoichiometric coefficients within equation 1 require: [AD ]eq=[ad ]initial-[ad]eq [ ]eq p=-log[ ] (this is M, and you need mm) [C3C2]eq Determine the initial concentration of ethanol that you added to tube 3. The 1:1 ratio of stoichiometric coefficients within equation 1 require: [C3C2]eq=[C3C2]initial-[AD]eq 2.) Determine K obs = [AD] eq[c3c] eq [<] eq [AD<] eq [C3C2] eq. 3.) Use the calculated equilibrium constant (Kobs from #2) to determine the [C3C2] in the unknown solution of tube 5. You will need to determine: [AD]eq [AD ]eq [C3C]eq [ ]eq You cannot determine [C3C2]eq [AD] EF [C I C] EF [ < ] EF K?@A = [AD < ] EF [C I C ] QR, [AD] EF If the concentration of ethanol in the unknown is low, the equilibrium position in tube 5 will tend towards the reactants side. Since you are observing the absorbance of a product, the further the equilibrium tends toward the reactants, the lower the signal and greater the background noise.

5 4.) ow did the presence of semicarbazide affect the measured concentration of AD in samples containing Et? 5.) Does this support the conclusion that semicarbazide pulls the alcohol dehydrogenase reaction to completion? Since AD remains after acetaldehyde reacts with semicarbazide and one AD molecule is produced per ethanol molecule that is oxidized to acetaldehyde, the concentration of AD in Tube 6 should be equal to the concentration of ethanol in the unknown (after taking into account any dilutions). 6.) Determine the concentration of ethanol in the unknown from the absorbance data of Tube 6. 7.) ow does your determination of the [Et] of the unknown with and without semicarbazide compare? 8.) Staple in the following table to summarize your results.