OTHER METHODS OF STUDYING VISUAL PIGMENTS 



and 20°C respectively. After 24 hr at the higher temperature the 

 thermal breakdown of the initial photoproduct (very noticeable in 

 fresh suspensions) was no longer apparent. In suspensions stored for 

 3 days at 20°C the initial photoproduct was quite stable and the 

 negative and positive Umbs of the difference spectrum were then of 

 nearly equal heights. 



To investigate the carotenoids present in the suspensions, arden 

 (1954b) exposed a suspension to light, diluted it with Ringer-Locke 

 and centrifuged it. After removing the supernatant, the precipitated 

 outer segments were freeze-dried and then extracted with chloroform. 

 The extract had an absorption band Amax at 390 m/i (retinenci). The 

 same experiment was then carried out on another suspension except 

 that the procedures subsequent to the bleaching were delayed for an 

 hour. The chloroform extract in this case had an absorption band at 

 330 m/x which is characteristic of vitamin A^. 



These results strongly suggest that the initial photoproduct formed 

 when suspensions are exposed to light is retinenci (or indicator 

 yellow). In fresh suspensions this is then reduced to vitamin A^, but 

 during storage of suspensions the mechanism responsible for the 

 reduction becomes inactive. 



IS there an extra pigment in the suspensions? 



The density maximum of pure (frog) visual purple is at 502 m^. 

 Difference spectra have Amax displaced to slightly longer wavelengths. 

 This arises from the 'overlap' between the absorption bands of pig- 

 ment and photoproduct. In solution, the amount of displacement 

 depends on the pH, for this determines the spectral location of the 

 photoproduct absorption band. In acid solution the displacement 

 is maximal and the Amax is then at c. 510 m^. 



Now the Amax of the difference spectra of suspensions was also at 

 c. 510 m/Li. arden found that the pH of the suspending medium had 

 no effect on the difference spectrum. This, no doubt, was due to the 

 fact that the external pH did not alter conditions inside the suspended 

 visual cells. Thus no direct evidence of the effect of pH could be 

 obtained. 



arden considered that the high Amax of suspension difference 

 spectra could not be attributed entirely to a photoproduct artifact. 

 Even in those cases where the initial photoproduct had been reduced 

 to the colourless vitamin A (see Fig. 7.1, curve HIJ) the X 

 (505-510 m^) was still higher than that for visual purple. 



185 



