Analyze The Data

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 centrifuga-tion and then resuspended in nonfluorescent medium.

450 500 550 600 650 Wavelength (nm)

a. When these vesicles were incubated in a physiological buffer containing NADH, ADP, P(, and O2, the fluorescence of BCECF trapped inside gradually decreased in intensity. What does this decrease in fluorescent intensity suggest about this vesicular preparation?

b. How would you expect the concentrations of ADP, P(, and O2 to change during the course of the experiment described in part a? Why?

c. After the vesicles were incubated in buffer containing ADP, Pj, and O2 for a period of time, addition of dinitrophe-nol caused an increase in BCECF fluorescence. In contrast, addition of valinomycin produced only a small transient effect. Explain these findings.

d. Predict the outcome of an experiment performed as described in part a if brown-fat tissue was used as a source of unsealed, isolated inner mitochondrial membranes. Explain your answer.

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