Absorption Spectral Properties of Purified Halorhodopsin1

Abstract

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°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°C and at −75°C were similar to those of halorhodopsin in membrane fragments. Irradiation of the purified halorhodopsin with red light at 0°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°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.