Effects of temperature on the spectral properties of visual pigments were measured in the physiological range (5–28°C) in photoreceptor cells of bullfrog (Rana catesbeiana) and crucian carp (Carassius carassius). Absorbance spectra recorded by microspectrophotometry (MSP) in single cells and sensitivity spectra recorded by electroretinography (ERG) across the isolated retina were combined to yield accurate composite spectra from ca. 400 nm to 800 nm. The four photoreceptor types selected for study allowed three comparisons illuminating the properties of pigments using the dehydroretinal (A2) chromophore: (1) the two members of an A1/A2 pigment pair with the same opsin (porphyropsin vs. rhodopsin in bullfrog “red” rods); (2) two A2 pigments with similar spectra (porphyropsin rods of bullfrog and crucian carp); and (3) two A2 pigments with different spectra (rods vs. long-wavelength-sensitive (L-) cones of crucian carp). Qualitatively, the temperature effects on A2 pigments were similar to those described previously for the A1 pigment of toad “red” rods. Warming caused an increase in relative sensitivities at very long wavelengths but additionally a small shift of λmax toward shorter wavelengths. The former effect was used for estimating the minimum energy required for photoactivation (Ea) of the pigment. Bullfrog rod opsin with A2 chromophore had Ea = 44.2 ± 0.9 kcal/mol, significantly lower (one-tailed P < 0.05) than the value Ea = 46.5 ± 0.8 kcal/mol for the same opsin coupled to A1. The A2 rod pigment of crucian carp had Ea = 42.3 ± 0.6 kcal/mol, which is significantly higher (one-tailed P < 0.01) than that of the L-cones in the same retina (Ea = 38.3 ± 0.4 kcal/mol), whereas the difference compared with the bullfrog A2 rod pigment is not statistically significant (two-tailed P = 0.13). No strict connection between λmax and Ea appears to exist among A2 pigments any more than among A1 pigments. Still, the A1 → A2 chromophore substitution in bullfrog opsin causes three changes correlated as originally hypothesized by Barlow (1957): a red-shift of λmax, a decrease in Ea, and an increase in thermal noise.

Temperature effects on spectral properties of the two types of rod photoreceptors in toad retina, “red” and “green” rods, were studied in the range 0–38°C. Absorbance spectra of the visual pigments were recorded by single-cell microspectrophotometry (MSP) and spectral sensitivities of red rods were measured by electroretinogram (ERG) recording across the isolated retina. The red-rod visual pigment is a usual rhodopsin (λmax = 503.6 nm and 502.3 nm at room temperature (21°C) in, respectively, Bufo marinus and Bufo bufo), that of green rods (λmax = 432.6 nm in Bufo marinus) belongs to the “blue” cone pigment family. In red rods, λmax depended inversely and monotonically on temperature, shifting by −2.3 nm when temperature was raised from 0°C to 38°C. Green-rod λmax showed no measurable dependence on temperature. In red rods, warming caused a relative increase of sensitivity in the long-wavelength range. This effect can be used for estimating the energy needed for photoexcitation, giving Ea = 44.3 ± 0.6 kcal/mol for Bufo marinus rhodopsin and 48.8 ± 0.5 kcal/mol for Bufo bufo rhodopsin. The values are significantly different (P < 0.001), although the two rhodopsins have very similar absorption spectra and thermal isomerization rates. Our recording techniques did not allow measurement of the corresponding effect at long wavelengths in green rods. Although spectral effects of temperature changes in the physiological range are small and of little significance for visual function, they reveal information about the energy states and different spectral tuning mechanisms of the visual pigments.