PNAS 1995-01-17

Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk.

U Alexiev, R Mollaaghababa, P Scherrer, H G Khorana, M P Heyn

Index: Proc. Natl. Acad. Sci. U. S. A. 92(2) , 372-6, (1995)

Full Text: HTML

Abstract

The pH-indicator dye fluorescein was covalently bound to the surface of the purple membrane at position 72 on the extracellular side of bacteriorhopsin and at positions 101, 105, 160, or 231 on the cytoplasmic side by reacting bromomethylfluorescein with the sulfhydryl groups of cysteines introduced by site-directed mutagenesis. At position 72, on the extracellular surface, the light-induced proton release was detected 71 +/- 4 microseconds after the flash (conditions: pH 7.3, 22 degrees C, and 150 mM KCl). On the cytoplasmic side with the dye at positions 101, 105, and 160, the corresponding values were 77, 76, and 74 +/- 5 microseconds, respectively. Under the same conditions, the proton release time in the bulk medium as detected by pyranine was around 880 microseconds--i.e., slower by a factor of more than 10. The fact that the proton that is released on the extracellular side is detected much faster on the cytoplasmic surface than in the aqueous bulk phase demonstrates that it is retained on the surface and migrates along the purple membrane to the other side. These findings have interesting implications for bioenergetics and support models of local proton coupling. From the small difference between the proton detection times by labels on opposite sides of the membrane, we estimate that at 22 degrees C the proton surface diffusion constant is greater than 3 x 10(-5) cm2/s. At 5 degrees C, the proton release detection time at position 72 equals the faster of the two main rise times of the M intermediate (deprotonation of the Schiff base). At higher temperatures this correlation is gradually lost, but the curved Arrhenius plot for the proton release time is tangential to the linear Arrhenius plot for the rise of M at low temperatures. These observations are compatible with kinetic coupling between Schiff base deprotonation and proton release.


Related Compounds

Related Articles:

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein.

1993-06-15

[Biochemistry 32 , 6095, (1993)]

Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

2013-01-01

[Int. J. Environ. Res. Public Health 10 , 47-71, (2013)]

5-Bromomethyl fluorescein (5-BMF) for derivatization of carboxyl containing analytes for use with laser-induced fluorescence detection.

1995-06-01

[Pharm. Res. 12(6) , 930-6, (1995)]

Measurement of thiol-containing amino acids and phytochelatin (PC2) via capillary electrophoresis with laser-induced fluorescence detection.

2002-01-01

[Electrophoresis 23(1) , 81-7, (2002)]

Analysis of gamma-(cholesteryloxy)butyric acid in biologic samples by derivatization with 5-(bromomethyl)fluorescein followed by high-performance liquid chromatography with laser-induced fluorescence detection.

1996-01-15

[Anal. Chem. 68(2) , 327-32, (1996)]

More Articles...