Rapid-flow resonance Raman spectroscopy of photolabile molecules: rhodopsin and isorhodopsin.

We have devised a method for obtaining the resonance Raman spectrum of a photolabile molecule before it is modified by light. The essence of this technique is that the sample is flowed through the light beam at a sufficiently high velocity so that the fraction of photoisomerized (or photodestroyed) molecules in the illuminated volume is very low. This rapid-flow technique has enabled us to measure the resonance Raman spectrum of unphotolyzed bovine rhodopsin in Ammonyx LO detergent solution and in sonicated retinal disc membranes. The major features of these spectra, which are very similar to one another, are the protonated Schiff base line near 1660 cm-1, the ethylenic line at 1545 cm-1, lines due to skeletal modes at 1216, 1240, and 1270 cm-1, and a line due to C-H bending at 971 cm-1. The resonance Raman spectrum of unphotolyzed isorhodopsin formed by the addition of 9-cis-retinal to opsin was also measured. The spectrum of isorhodopsin is more complex and differs markedly from that of rhodopsin. In isorhodopsin, the ethylenic line is shifted to 1550 cm-1, and there are six lines between 1153 and 1318 cm-1. The rapid-flow technique described here makes it feasible to control the extent of interaction between light and any photolabile molecule. We present a theory for predicting the effective sample composition in the illuminated volume as a function of the flow rate, light intensity, and spectral characteristics of the photolabile species.