INTRODUCTION AND METHODS 



bottom of the tube. These, after washing with pH 4-6 buffer, are 

 treated with digitonin or other extractant. 



A modified form of saito's flotation technique has been used by 

 COLLINS, LOVE and MORTON (1952). The retinae are shaken with 

 saline solution and the mixture filtered through 60-mesh gauze. The 

 filtrate, which contains tissue fragments, blood, melanin, and rod 

 outer segments is transferred to a centrifuge tube. Saturated sucrose 

 solution is then carefully poured down the side of the tube to form a 

 lower layer. A flat-ended glass rod is lowered into the tube and the 

 sahne-sucrose interface gently stirred so that a concentration gradient 

 is set up. On then centrifuging the tube, red blood cells, melanin 

 granules, and rod outer segments find their separate density levels 

 and may be cleanly pipetted off". The reader is referred to the paper 

 for practical details and for other useful information on the prepara- 

 tion of visual pigment solutions, kimura (1952) has also used a 

 basically similar modification of the flotation technique. 



Finally, if the separated outer segments are dried and then treated 

 with fight petroleum ether (B.P. 40-60°C) a small amount of yellow 

 pigment (rod hpid) is removed without affecting the visual purple 

 (lythgoe, 1937), which can then be extracted in the usual manner. 



Visual pigment solutions, adequate for most purposes, however, 

 can be obtained simply by extracting whole retinae after they have 

 been washed in three or four changes of pH 4-6 buffer solution. The 

 treatment with acid buffer removes all blood pigments and renders 

 insoluble a good deal of protein material. Also, the trace of buffer 

 which remains after removal of the washings, ensures that the sub- 

 sequent extraction is carried out in acid conditions. Extracts made 

 in this way are hardly inferior to those made by saito's sugar 

 flotation process. The small amounts of yellow impurities which 

 they contain are quite stable, both thermally and photochemically. 



Digitonin extracts of visual purple, buffered at pH 6-10, keep well 

 if stored in darkness in a refrigerator. An extract of frog's retinae, 

 buffered at pH 7-7, was kept by the author for 14 months at 2-3°C 

 without measurable loss of photosensitive pigment. 



CHARACTERIZATION OF VISUAL PIGMENTS 



ABSORPTION AND DENSITY SPECTRA 



The only known way of characterizing the visual pigments is to 

 measure their fight-absorbing properties. To do this a sample of an 



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