Measuring the redox state inside of cells is challenging. Recently, scientists have developed a redox sensitive GFP (roGFP) that contains a pair of redox sensitive cysteines. When reduced, roGFP fluoresces differently than when the disulfide is formed (i.e. oxidized). a. Suppose you wanted to characterize roGFP in vitro in order to better understand in vivo measurements in the following manner: You mix a small amount of roGFP with an excess of the reducing agent DTT at 300K. The DTT reaction is: DTT(ox) + 2H+ + 2e - → DTT (red) and E' = -323 V By following fluorescence of roGFP, you find that when the reaction reaches equilibrium, you measure the concentrations of the individual components and find that the reaction quotient is 5.4. Calculate the biochemical standard reduction potential for roGFP. Question 3. continued. b. For more characterization, you decide to investigate how free cysteine in solution behaves. It turns out that the reduction potential for converting a disulfide linked dimer of cysteine to two molecules of cysteine has been measured in solution as Eº'= - 230V. Calculate the AG' for the case of both the protein (i.e. in roGFP) or the free cysteines being reduced by DTT under standard biochemical conditions. c. These calculations suggest contrasting behavior between the solution and protein cysteine pair found in roGFP and that one species favors the oxidized state more than the other. Comment on and explain the difference.
