Photoreaction of bacteriorhodopsin at various pH levels

Published: 14 April 2022| Version 1 | DOI: 10.17632/5gb59wfr9p.1
Contributor:
Tsutomu Kouyama

Description

The attached excel file "BR-photoreaction-at-various-pHs-in-0MKCl.xlsx" contains raw data of flash-induced absorption changes of bacteriorhodopsin at various pH levels in low-salt membrane suspensions (~ 4 mM pH buffers). The attached excel file "BR-photoreaction-at-various-pHs-2MKCl.xlsx" contains raw data of flash-induced absorption changes of bacteriorhodopsin at various pH levels in high-salt membrane suspensions (2 M KCl). The attached excel file "Temperature Dependence-BR-photoreacton-at-pH5.9-20220413.xlsx" contains raw data of flash-induced absorption changes of bacteriorhodopsin at various temperatures in low-salt membrane auapwnaiona tpH 5.9 (5mM Mes).

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Steps to reproduce

Purple membrane was isolated from Halobacterium salinarum strain JW3 and purified according to established procedure and then stored at -80 ℃ in ~ 40 % sucrose (wt/wt). For the flash experiments, purple membrane was repeatedly washed with distilled water, and its concentration was adjusted to be ~ 0.8 OD at 568 nm in a 5 × 5 × 40-mm quartz cuvette. The medium pH of a membrane suspension was adjusted using a combination of buffer molecules (Mes, Pipes, Hepes, Taps, Ches, bicarbonate, citrate, glycine). The buffer molecules possessing a high buffering capacity and a negatively charged group(s) in the investigated pH region were selectively used for the pH adjustment. The total concentration of pH buffers was – 4 mM for the measurement of absorption kinetics in a low-salt membrane suspension. [In the acidic pH region, the medium pH was adjusted using ~ 2 mM of citrate.] A higher concentration (~ 20 mM) of pH buffers was used for the measurement of absorption kinetics in a high-salt membrane suspension (in 2 M KCl). Transient transmission data of bR at 24 ºC were acquired using a computer-controlled experimental setup with a digital oscilloscope (LeCroy Wavesurfer 422) and a frequency-doubled Nd:YAG laser (Quantel Ultra, 3 ns, 532 nm). The flash intensity (~0.6 mJ/cm2) was adjusted such that the fraction of bR undergoing the proton pumping cycle was <10%. As the measuring light, emission from a 150 W Xe lamp was passed through a monochromator and directed to the sample cell, and the transmitted light was passed through a notch filter and the second monochromator and received by a photomultiplier tube connected to a transimpedance amplifier (Hamamatsu photonics, C13004-01). The notch filter was effective in reducing an undesired effect of the scattered component of light pulses. Flash-induced transmission changes were measured at 30 different wavelengths and at a wavelength interval of 10 nm. At each wavelength, the transmission signal was averaged 300-2000 times. After the systematic noise originating from the pulse-generating system was subtracted, the averaged transmission signal was rescaled to evaluate the time variation of absorbance.

Institutions

Nagoya Daigaku

Categories

Retinal Enzyme, Adsorption Kinetics, Membrane Biophysics

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