ROLE OF PALMITIC ACID ON THE ISOLATION AND PROPERTIES OF HALORHODOPSIN

Purified halorhodopsin was isolated from Halobacterium halobium as pre viously described (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016- 17022). Two purple bands were eluted from phenyl-Sepharose column, ind icating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) am ong lipids associated with purple fractions. Typically the palmitate c ontent of the first eluted band was higher than that of the second, in dicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no pa lmitate was released from denaturated halorhodopsin. By adding palmita te to buffers used in the phenyl-Sepharose chromatography, only one sh arp purple band was collected, corresponding to the less retained halo rhodopsin fraction. Pentadecanoic fatty acid could also affect the hal orhodopsin chromatography. Chromatography of halorhodopsin in the pres ence of beta-mercaptoethanol showed only one band, corresponding to th e more retained halorhodopsin form. The two halorhodopsin fractions ha d different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the ab sorbance at 570 nm upon illumination. By following the decay of the ab sorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pK(a) values of deproton ation.