Photosensitivity spectrum of crayfish rhodopsin measured using fluorescence of metarhodopsin.

Abstract

Discrepancies exist among spectral measurements of sensitivity of crayfish [Orconectes rusticus and Procambarus clarkii] photoreceptors, their absorption in situ, and the number and absorption spectra of crayfish photopigments that are extracted by digitonin solutions. The photosensitivity spectrum of crayfish rhodopsin was determined in isolated rhabdoms using long wavelength fluorescence emission from crayfish metarhodopsin as an intrinsic probe. There is no measurable metarhodospin in the dark-adapted receptor, so changes in the emission level are directly proportional to metarhodospin concentration. Changes in metarhodospin fluorescence were used to construct relaxation and saturation (photoequilibrium) spectra, from which the photosensitivity spectrum of crayfish rhodopsin was calculated. This spectrum peaks at .apprx. 530 nm and closely resembles the previously measured difference spectum for total bleaches of dark-adapted rhabdoms. Measurements of the kinetics of changes in rhabdom fluorescence and in transmittance at 580 nm were compared with predictions derived from several model systems containing 1 or 2 photopigments. Only a single rhodopsin and its metarhodopsin are present in the main rhabdom of crayfish. Other explanations must be sought for the multiple pigments seen in digitonin solution. There is no detectable formation of isorhodopsin in the rhabdom.