Absorption spectral properties of purified halorhodopsin

Halorhodopsin in the membrane fragments of Halobacterium halobium Y1 showed an absorption band at 576 nm, the intensity of which decreased on irradiation with red light at 0 degrees C (Ogurusu, T., Maeda, A., Sasaki, N., & Yoshizawa, T. (1981) J. Biochem. 90, 1267-1273). Using this photobleachable property as the basis for an assay of halorhodopsin, we purified halorhodopsin by octyl-Sepharose column chromatography after extracting it from the membrane with Triton X-100. In NaDodSO4-polyacrylamide gel electrophoresis, hR appeared as a major band with an apparent molecular weight of 22,000, but the preparation still showed several other faint bands. The purified halorhodopsin showed a main absorption band at 576 nm and a small band at around 415 nm in 1 M NaCl. The photoreactions of the purified halorhodopsin at 0 degrees C and at -75 degrees C were similar to those of halorhodopsin in membrane fragments. Irradiation of the purified halorhodopsin with red light at 0 degrees C resulted in a decrease of absorbance at around 576 nm with a concomitant increase of absorbance at around 410 nm. A hypsochromic photoproduct was obtained on irradiation with 650 nm light at -75 degrees C. The dependency of the absorption spectrum of halorhodopsin on the concentration of chloride indicates that halorhodopsin has a single chloride binding site, occupation of which is responsible for modifying the spectrum.