Aplysia calif ornica — W inkler 
359 
Fig. 1. The spectral curves of the aplysia purple 
taken from (A) a Plocamium-ieeding adult, and (B) a 
young specimen showing no signs of feeding on 
Plocamium. The pigment was diluted in 10 per cent 
hydrochloric acid, 
tions. The absorption spectrum (Fig. 2 ) 
showed a far sharper peak but a much less 
acute slope toward the red end of the spec- 
trum than was noted earlier in the lavender 
collections. This suggested that it constituted 
a separate component. Paper chromatography 
was then tried as a means of separation, and 
by this it was possible to prove the existence 
of a third component of the aplysia ink of 
A. calif ornica. On the dried paper a rhodonite 
pink component (Ridgway, 1912 ) left a wide 
band in the center of the paper. The second, 
a deep lavender, lagged behind at one-quarter 
the distance above the starting line. Scarcely 
clearing the initial line was a calamine blue 
area. Elution produced weak concentrations 
of the pigments, but these were adequate to 
produce curves on the Beckman Model DU 
spectrophotometer. However, these papers 
were dried and stored before elution, and this 
treatment gave opportunity for oxidative 
changes (Fig. 2 ). 
DISCUSSION AND CONCLUSIONS 
Though a component was isolated which 
gave a virtually pure curve at 496 m/x (Fig. 3), 
the writer has never succeeded in isolating a 
fractional component with a peak in the high 
500 region which did not also have a weak 
peak at 499.5 m/x (not 496). As this material 
having two peaks (which will be shown to be 
composed of more than one, separable, but 
similar components) stands under refrigera- 
tion over a period of time, the relative heights 
of the two peaks change. The D band peak 
decreases and the F band peak increases in 
height, with a gradual shift of the F band 
from 499.5 to 497 and finally to 496 m/x, 
eventually giving approximately the same 
spectrum and gross color as the original 
aplysia purple from which it was separated 
(Fig. 3). 
Aplysia purple is believed to be a bilin 
(Fox, 1953), a linear chain of pyrrole mole- 
cules connected by -CH= or -CH2- linkages, 
as it gives a positive Gmelin reaction. Erode 
(1955), by using commercial dyes, showed 
that the spectrophotometric effect of these 
linkages is insulating for -CH2- and coupling 
for the -CH= configaration. In Aplysia the 
pyrrole molecules of the bilins seem to be 
joined by the insulating linkages when in the 
blue state, which thus inhibits the normal F 
band absorption characteristic of the chain of 
pyrrole molecules absorbing together. In 
time, these insulating linkages gradually 
change to coupling linkages, perhaps due to 
enzymatic activity or other causes as yet un- 
known. As Erode (1955) puts it, ’Tf the 
linkage is changed to a conjugating (=cou- 
pling) connection . . . there is a marked 
change in the spectrum with the production 
of a single resonance structure involving both 
