Chemical modification of bacteriorhodopsin with N-bromosuccinimide

Received 20 May 1977 1. Introduction Bacteriorhodopsin in the purple membrane of halophilic bacteria functions as a light-driven proton pump. Proton translocation is driven by light absorp- tion by the retinal chromophore [1 ], but the way in which the chromophore excitation interacts with protein, and the process of vectorial proton transloca- tion which involve the apo-protein are not yet well characterized. There is evidence that deprotonation and reprotonation of the Schiff base at the site of attachment of retinal to the protein is a primary step in proton translocation. The deprotonated intermediate state is detected by an absorption band at 412 nm during the photoreaction cycle [2-5]. The amino acid residues involved in this reaction and in the movement of the proton through the protein molecule are unclarified. Chemical modification of bacteriorhodopsin in purple membrane preparations is a good approach to study such questions because this protein is the only one in the preparation, and because it exhibits considerable stability [1,6]. In a previous study, we demonstrated that modification of amino groups using certain lysine specific reagents (imidoesters) and glutaraldehyde inhibited proton pump activity concommitant with a loss of intrinsic tryptophan fluorescence [7]. Oesterhelt and Hess [9] found a decrease of tryptophan fluorescence during illumination of purple membranes in ether saturated salt solutions. Furthermore Bensasson et al. [8] have proposed that in mammalian rhodopsin, tryptophan residues interact with the retinal Schiff base to form a charge transfer complex resulting in a large red shift of chromophore absorption. A similar situation may occur in the case of bacteriorhodopsin. In the present study the effect of a tryptophan specific reagent, N-bromosuccinimide, on bacteriorhodopsin has been investigated. Correlation of chemical modification tryptophan residues by this reagent with measurements of the formation the 412 nm photoreaction cycle intermediate and chromophore absorption, reveal that tryptophan residues are essential for proton pump activity. 2. Materials and methods Purple membrane preparations kindly provided by Dr J. Lanyi (NASA-Ames Research Center) containing 4-8 gM of bacteriorhodopsin, as calculated from 570 nm absorption using the molar extinction coefficient of 63 000 M -t cm-1, [9] were employed in experiments. Absorption spectra were recorded in a Cary 14 spectrophotometer and difference spectra were measured in the Aminco Chance DW2 dual wavelength spectrophotometer. The height of the 412 nm peak induced by a single flash is a direct indication of the number of bacteriorhodopsin molecules photocycling, and therefore provides an assessment of proton pump activity. The rate of decay the 412 nm intermediate is proportional to the kinetics of proton pump activity. Flash photometry was carried out in an apparatus designed for these measurements in our laboratory which contained a photoflash unit (Vivitar 283) 1 ms maximum flashes from a xenon lamp. A pair of narrow band interference filters with a peak transmission of 410 nm (Baird Atomic series) were used to isolate the