Analyze the Data 12-1: The Mitochondrial pH Gradient
A proton gradient can be analyzed with fluorescent dyes whose emission-intensity profiles depend on pH. One of the most useful dyes for measuring the pH gradient across mitochondrial membranes is the membrane-impermeant, water-soluble fluorophore 2′,7′-bis-(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of pH on the emission intensity of BCECF, excited at 505 nm, is shown in the accompanying figure. In one study, sealed vesicles containing this compound were prepared by mixing unsealed, isolated inner mitochondrial membranes with BCECF; after resealing of the membranes, the vesicles were collected by centrifugation and then resuspended in nonfluorescent medium.
a. When these vesicles were incubated in a physiological buffer containing NADH, ADP, Pi, and O2, the fluorescence of BCECF trapped inside gradually decreased in intensity. What does this decrease in fluorescence intensity suggest about this vesicular preparation?
_feedback: The electron-transport system normally pumps protons out of the mitochondrial matrix, increasing the pH of the matrix; thus, the fluorescence of matrix-trapped BCECF would increase in intensity. The observed decrease in intensity of BCECF trapped inside the vesicles suggests that the vesicles have an inverted (inside-out) orientation, so that protons were pumped from the outside to the inside of the vesicles.
b. How would you expect the concentrations of ADP, Pi, and O2 to change during the course of the experiment described in part a? Why?
_feedback: The concentrations of ADP, Pi, and oxygen should decrease over time as the process of oxidative phosphorylation utilizes oxygen as an electron acceptor and uses ADP and Pi to synthesize ATP.
c. After the vesicles were incubated in a buffer containing ADP, Pi, and O2 for a period of time, addition of dinitrophenol caused an increase in BCECF fluorescence. In contrast, addition of valinomycin produced only a small transient effect. Explain these findings.
_feedback: Dinitrophenol compromises the pH gradient and the resulting equilibration of protons leads to an increase in the intravesicular pH and corresponding increase in emission intensity. Valinomycin, a potassium ionophore, affects the electric potential more than the pH gradient. Since BCECF fluorescence reflects the pH of the milieu, it is largely unaffected by valinomycin-induced changes in the transmembrane electric potential.
d. What result would you expect to see if the source of the mitochondrial membranes were brown-fat mitochondria? Explain.
_feedback: The fluorescence intensity inside the vesicles should remain constant over time since inner mitochondrial membranes from brown fat tissue would likely contain thermogenin, a protein that functions as an uncoupler of oxidative phosphorylation. Since thermogenin is a proton transporter, its presence would prevent the generation of a proton gradient and thus no fluorescence change would be expected.
e. Chloroplasts could also be used as a source of membranes in a similar experiment (as in part a) involving BCECF. In this case, the BCECF would be surrounded by what membrane? How would the fluorescence change upon addition of light, ADP, and Pi?
_feedback: To examine proton movement in chloroplast you would use the thylakoid membranes. In this case, the proton concentration is high in the thylakoid lumen and low outside, so the pH inside the thylakoid would increase as time passed, thus producing an increase in BCECF fluorescence.
