1. VISUAL PIGMENTS
19
chloride reagent is added to a chloroform extract containing retinene. The
blue color quickly fades, necessitating a highly standardized procedure
for reproducible results. Water and some other substances can cause
turbidity, upsetting the analyses; sometimes acetic anhydride is added
to reduce this turbidity. Finally, the spectra of the absorbance bands
seem to be variable, A, , , reported for the retinene, band varying from
661 to 664 nm, and for retinene, from 705 to 741 nm (Grangaud et al.,
1962; Naito and Wilt, 1962; Plack, 1961; Wald, 1939a; Wilt, 1959). The
second method uses the results of partial bleaching experiments. The
difference spectrum of the product of bleaching (Figs. 6 and 7) can be
used to characterize retinene, and retinenez (Crescitelli, 1958). The A, , ,
values are less variable than in the antimony trichloride reaction; and the
presence of stable, light-absorbing impurities is unimportant. Both methods appear to be reliable in the hands of experienced workers.
3. VISUAL PIGMENTS IN PHOTORECEPTORS
The light absorption of visual pigments is the same, whether they are
solubilized in retinal extracts or are in situ in suspensions of the visualcell outer segments (Dartnall, 1961, 1962). When the outer segments
are oriented, however, as in the retina, light absorption is quantitatively
greater than in randomly oriented suspensions or in extracts (Denton and
Wyllie, 1955). A method devised by Denton (Denton and Warren, 1957;
Denton and Walker, 1958; Denton, 1959; Denton and Nicol, 1964) uses
the intact retina. The light absorption is measured at several wavelengths
before and after bleaching the visual pigment. Results are similar to the
more precise measurements obtained from retinal extracts but generally
lack the essential qualification that homogeneity must be tested by partial
bleaching. Nevertheless, they have the advantage of giving information
on how much of the light incident on the retina is absorbed there. To
anticipate slightly, deep-sea fish retinas can capture a very large fraction
of the incident light; at A, , ,
of the visual pigment, this may be more
than 90% (Denton and Warren, 1957; Denton, 1959). The intact retina
is also used in the method of microspectrophotometry (Section 11, E ) .
C. A Choice of Retinenes: Rhodopsin and Porphyropsin
Wald (1936, 1939b) showed that certain freshwater fishes have a
different visual pigment from marine fishes. Finding mixtures of both
visual systems in some euryhaline species ( Wald, 1941), he proposed an
elegant generalization (Wald, 1947, 1958, 1959, 1960) that is widely
accepted. According to this view, marine fishes have rhodopsin, the visual
pigment based on retinene,. Freshwater fishes have a different prosthetic
19
chloride reagent is added to a chloroform extract containing retinene. The
blue color quickly fades, necessitating a highly standardized procedure
for reproducible results. Water and some other substances can cause
turbidity, upsetting the analyses; sometimes acetic anhydride is added
to reduce this turbidity. Finally, the spectra of the absorbance bands
seem to be variable, A, , , reported for the retinene, band varying from
661 to 664 nm, and for retinene, from 705 to 741 nm (Grangaud et al.,
1962; Naito and Wilt, 1962; Plack, 1961; Wald, 1939a; Wilt, 1959). The
second method uses the results of partial bleaching experiments. The
difference spectrum of the product of bleaching (Figs. 6 and 7) can be
used to characterize retinene, and retinenez (Crescitelli, 1958). The A, , ,
values are less variable than in the antimony trichloride reaction; and the
presence of stable, light-absorbing impurities is unimportant. Both methods appear to be reliable in the hands of experienced workers.
3. VISUAL PIGMENTS IN PHOTORECEPTORS
The light absorption of visual pigments is the same, whether they are
solubilized in retinal extracts or are in situ in suspensions of the visualcell outer segments (Dartnall, 1961, 1962). When the outer segments
are oriented, however, as in the retina, light absorption is quantitatively
greater than in randomly oriented suspensions or in extracts (Denton and
Wyllie, 1955). A method devised by Denton (Denton and Warren, 1957;
Denton and Walker, 1958; Denton, 1959; Denton and Nicol, 1964) uses
the intact retina. The light absorption is measured at several wavelengths
before and after bleaching the visual pigment. Results are similar to the
more precise measurements obtained from retinal extracts but generally
lack the essential qualification that homogeneity must be tested by partial
bleaching. Nevertheless, they have the advantage of giving information
on how much of the light incident on the retina is absorbed there. To
anticipate slightly, deep-sea fish retinas can capture a very large fraction
of the incident light; at A, , ,
of the visual pigment, this may be more
than 90% (Denton and Warren, 1957; Denton, 1959). The intact retina
is also used in the method of microspectrophotometry (Section 11, E ) .
C. A Choice of Retinenes: Rhodopsin and Porphyropsin
Wald (1936, 1939b) showed that certain freshwater fishes have a
different visual pigment from marine fishes. Finding mixtures of both
visual systems in some euryhaline species ( Wald, 1941), he proposed an
elegant generalization (Wald, 1947, 1958, 1959, 1960) that is widely
accepted. According to this view, marine fishes have rhodopsin, the visual
pigment based on retinene,. Freshwater fishes have a different prosthetic