Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin

Abstract

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR568 and bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with HCl showed the transition to a form absorbing at 605 nm .**GRAPHIC**. with pK = 2.9 and to a 2nd form absorbing at 565 nm .**GRAPHIC**. with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a 3rd isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, .**GRAPHIC**. and .**GRAPHIC**. all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-.mu.s time resolution) of .**GRAPHIC**. and .**GRAPHIC**. showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for .**GRAPHIC**. Chromophore extraction from membrane suspensions yielded all-trans-retinal for .**GRAPHIC**. and a mixture of 13-cis and trans isomers for .**GRAPHIC**.