Supplementary information
Contents: supplementary figures, equations and methods.
Supplementary figures
Figure S1. Chlorin absorbance in salamander rods by microspectrophotometry. a,
Absorption spectra of outer (solid line) and inner (dashed line) segments of a rod exposed
to 200 M chlorin for 3 hrs. Spectra were divided by the absorbance values of their
protein peak at 278 nm. Inset, diagram of an isolated rod. b, Linear dichroism of rods
incubated in chlorin. Averaged spectra determined with the measuring beam polarized
perpendicular (continuous line) or parallel (dashed line) to the rod axis. The former
spectrum was normalized to one at 278 nm and the latter was scaled by the same factor.
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c, Spectral shifts in chlorin absorption as a function of concentration in solution. Dashed
line, 1 M; solid line, 400 M. d, Changes in the chlorin spectrum upon binding to rods.
The difference spectrum (dashed line), obtained from a rod with high chlorin uptake and
the average of untreated rods, is shown superimposed on the spectrum of 400 M chlorin
in solution (solid line). Both spectra were normalized to one at the short wavelength peak.
The ratio between chlorin absorption in rods at 402 nm and 668 nm is 3.7.
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Figure S2. a, Increased rate of rhodopsin bleaching in rod outer segments with high
chlorin content. With 668 nm light, t’, the time required to bleach 50% of rhodopsin,
was 14 s with chlorin (open symbols) and 2,150 s in its absence (filled symbols) for this
pair of rod outer segments. For clarity, only eight of ten measurements on the untreated
outer segment are shown. Exposure times to 668 nm for the untreated outer segments
were recalculated for a standardized bleaching intensity (see Methods). Inset, mean t’
values ± SEM, n. Chlorin decreased t’ by 180-fold at 668 nm (p<0.017; ANOVA
followed by a Scheffe test), without changing it at 560 nm (p<0.081). b, Regeneration of
a photosensitive pigment after bleaching with red light. The absorbance of a rod treated
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with chlorin was measured before (thick black trace) and after a bleach with 675 nm light
(thin black trace). Subsequent incubation in 9-cis retinal yielded a blue-shifted visual
pigment with a max = 484 nm (gray trace). A rod treated with chlorin exhibited a
prominent pigment peak (520 nm) before bleaching (thick black trace), which was
reduced markedly after exposure to 675 nm light (thin black trace). Subsequent
incubation with a retinal analogue, 9-cis retinal, yielded a visual pigment whose max =
484 nm (grey trace) was blue-shifted relative to that of rhodopsin with its native
chromophore1.
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Supplementary equations
Calculation of chlorin content: We estimated the amount of chlorin relative to
rhodopsin according to:
chlorin/rhodopsin = (OD402fRR)/(OD520C)  0.34(OD402)/OD520
(1)
where OD402 was found from the difference spectrum of treated and untreated rods (e.g.
Fig.S1), OD520 was the absorbance at 520 nm, C = 134,000 M-1cm-1 determined for
chlorin at 402 nm, R = 30,000 M-1cm-1 for rhodopsin2, and fR = 1.5 corrected for the
molecular orientation of rhodopsin in the rod. Although absorbance measurements at 668
nm indicated that chlorin was aligned in the outer segment, no correction was made for
its orientation because linear dichroism at 402 nm was minor (Fig. S1b).
Calculation of quantum efficiency: For light absorbed by rhodopsin, the quantum
efficiency (E), given as the ratio of the concentration of bleached molecules [R] to the
concentration of rhodopsins absorbing a photon [R],
E = [R]/[R] = 0.67
(2)
is constant across the visible spectrum3. To find the relative efficiency of rhodopsin
bleaching with light absorbed by chlorin, we compared values for [R’] and [C’], the
concentration of rhodopsin and chlorin that absorbed a photon and yielded 50% fractional
bleaches:
[C’]  C668[chlorin]I668t’668
(3a)
[R’]  fRR560[rhodopsin]I560t’560.
(3b)
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Here [chlorin] and [rhodopsin] are the respective concentrations of chlorin and rhodopsin
in a dark adapted rod, I is the light intensity in photons m-2 s-1, t’ is the duration of
light exposure in s, fR = 1.5 is the polarization factor from eq. (1), and C668 and R560 are
the molecular absorbance coefficients for chlorin at 668 nm and for rhodopsin at 560 nm,
respectively. Then E668/E560 = [R’]/[C’]. The light intensities were equivalent at the
two wavelengths (I560 = I668). Setting R560 /C668 equal to the ratio of the corresponding
molar extinction coefficients = 21,720 M-1cm-1 / 36,216 M-1 cm-1 on the assumption that
for chlorin in rods (from Fig.S1d) 668 = (402)/(402/668) = (134,000 M-1cm-1)/(3.7), t’560
= 14 s and t’668= 18 s (from Fig.S2):
E668/E560 = (fRR560[rhodopsin]t’560)/( C668[chlorin]t’668)
 0.7([rhodopsin]/[chlorin]).
(4)
In our bleaching experiments, [rhodopsin]/[chlorin] = 1.3, therefore the efficiency of
chlorin-assisted bleaching was 60% (0.7 x 1.3 x 0.67 = 0.6), nearly as efficient as direct
photoisomerization of the native chromophore (67%)3. A combined estimate
encompassing the results from the bleaching and photoresponse experiments suggests a
quantal efficiency of rhodopsin isomerization by the red light absorbed by chlorin of
about 0.4.
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Supplementary methods
Animals: Care and use of all animals were in strict agreement with NIH and institutional
guidelines. Larval tiger salamanders (Ambystoma tigrinum, Charles D. Sullivan, Inc.,
Nashville, TN) were dark-adapted overnight before use. Retinas were isolated under
infrared illumination and stored in Ringer’s consisting of (mM): 108 NaCl, 2.5 KCl, 1.0
MgCl2, 10 HEPES, 1.5 CaCl2, 0.02 EDTA, 10 glucose, 7.5 x 10-4 bovine serum albumin
(Fraction V, -globulin-free; Sigma), pH 7.6 at 4°C. Pieces of retina were incubated in
100 to 400 M chlorin e6 (Frontier Scientific, Inc., Logan, UT) in Ringer’s without
bovine serum albumin for 3 to 18 hr at 4C. The final concentration of chlorin was
determined spectroscopically for each experiment.
Measurement of single cell absorption: A piece of retina was mechanically dissociated,
transferred to a chamber formed by two 0.2 mm thick quartz coverslips separated by 0.1
mm, and placed on the stage of the Williams-Webbers microspectrophotometer4. The
probe beam had a nominal size of 1 m by 1 m at the level of the chamber. The
measuring beam was polarized with its electric vector perpendicular to the long axis of
the rod for optimal rhodopsin absorption for all scans except those used to determine
linear dichroism. Scans were taken from 700 to 250 nm in 2 nm bins in a cell free area
and averaged to obtain a baseline spectrum. Measurements were then taken with the
beam positioned on a rod. Absorbance was calculated online and stored on a computer.
Pigment bleaching by the probe beam was negligible.
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For bleaching experiments, the beam was resized to cover the cell width, typically 7 m
by 10 m. Bleaching wavelength was set to either 560 nm or 668 nm, and the exposure
was controlled manually. After bleaching, the beam size was reduced for absorbance
measurement. For bleaching with 668 nm light in the absence of chlorin, the light
intensity was increased to reduce exposure duration. To simplify the analysis, the brighter
bleaching light was equated with a proportional increase in exposure time to the lower
intensity. After bleaching, a 0.05% ethanolic solution of ~30 M 9-cis retinal in Ringer’s
was introduced into the chamber to regenerate the visual pigment.
Recording of the rod’s electrical response to light: Tissue was mechanically
dissociated, placed in the recording chamber and perfused continuously with Ringer’s
with bovine serum albumin at 20-22ºC. Photocurrents were recorded from the inner
segment of a single rod with a suction electrode5 and a patch clamp amplifier (Axopatch
200A, Axon Instruments, Foster City, CA), low-pass filtered with an 8-pole Bessel (30
Hz, -3 dB) and digitized at 400 Hz. Cells were stimulated with light from a xenon arc
lamp that passed through a six-cavity interference filter (10 nm bandwidth at halfmaximal transmission, Omega Optical, Brattleboro, VT) and an electronic shutter. A
nominal duration of 22 ms was used for flashes. Light was calibrated with a photometer
(UDT 350, Graseby, Orlando, FL) through a 200 m diameter pinhole (Melles Griot,
Carlsbad, CA) placed at the level of the recording chamber.
References:
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1. Kefalov, V.J., Estevez, M.E., Kono, M., Goletz, P.W., Crouch, R.K., Cornwall, M.C.,
& Yau, K.-W. (2005) Neuron 46, 879-890.
2. Harosi, F.I. Absorption spectra and linear dichroism of some amphibian
photoreceptors. J. Gen. Physiol. 66, 357-382 (1975).
3. Dartnall, H.J.A. The photosensitivities of visual pigments in the presence of
hydroxylamine. Vision Res. 8, 339-358 (1972).
4. Makino, C.L., Howard, L.N., & Williams, T.P. Axial gradients of rhodopsin in lightexposed retinal rods of the toad. J. Gen. Physiol. 96, 1199-1220 (1990).
5. Makino, C.L., Groesbeek, M., Lugtenburg, J., & Baylor, D.A. Spectral tuning in
salamander visual pigments studied with dihydroretinal chromophores. Biophys. J.
77, 1024-1035 (1999).
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