Supporting information for Falchuk et al. (2002) Proc. Natl. Acad. Sci. USA 99 (1), 251–256. (10.1073/pnas.012616099)

Expanded Experimental Procedures

Emission Spectra of UV Lamps.

The emission spectra of several UV lamps, which are commonly used in the laboratory and described in the pertinent literature, were analyzed with an SX.18MV Emission Photomultiplier (Applied PhotoPhysics, Surrey, U.K.) at a capture time of 50 ms. The manufacturer of the lamps (Ultraviolet Products) categorized the lamps in short (254 nm) and long (366 nm) wavelengths that indicate the presumed predominant emission wavelength and peak. The factory filter attached to the lamps was removed in some measurements. In others, the spectra was obtained after interposing either a polystyrene Petri dish similar to the one used for the irradiation of embryos in this report or a quartz slab similar to that used by other experimenters. The plots represent the relative intensity of the emitted wavelengths. The lamp model is indicated on the side of each graph in Fig. 9. Band peaks are marked in nanometers. The short wavelength/254-nm series lamps emit with a polychromatic spectrum that include bands that peak at 254, 313, and 366 nm. Quartz allows the transmission of all the wavelengths. In some lamps the predominant emission wavelength was not 254 nm but instead longer ones were measured. The long wavelength/366-nm series lamps emit a more monochromatic spectrum with a broad band that peaks at 366 nm. When the factory filter is removed, other longer wavelengths are registered. Polystyrene allows the transmission of the broad band. The range of the emission bands of the lamps is comparable to those provided by the manufacturer, although we found variable relative intensities in different lamps. In the models UV SL 25 and UV SL 58 (a in Fig. 9), the intensity of the band peaking at 254 nm (often assumed to represent the predominant wavelength) is either lower or equivalent to the other bands.

Polystyrene Absorbance and UV Lamp Emission Used.

 Xenopus laevis stage I embryos were placed in polystyrene Petri dishes and exposed to 366-nm UV light by using a UV SL 58 lamp (Ultraviolet Products). The Petri dishes were analyzed for wavelength absorption with a Varian-Cary Bio 50 Spectrophotometer. Air was used as a baseline. The emission spectrum of the ultraviolet lamp is overlaid in Fig. 10. The polystyrene Petri dish has a high absorbance below 300 nm (cut off) and allows full transmission of the UV lamp band that peaks at 366 nm.

The selection of the 366-nm UV lamp was based on several observations: (i) the monochromatic emission spectrum of the UV366 nm lamp, (ii) the teratogenic effect of UV366 nm light on embryos as we demonstrate in this report, (iii) the advantage of using convenient plastic Petri dishes for manipulation and simultaneous irradiation of a large number of embryos, (iv) the wavelength transmission cut-off value of the polystyrene Petri dishes at 300 nm avoids irradiation at shorter wavelengths to other potentially photosensitive molecules, (v) the photosensitivity of biliverdin to 366 nm as we demonstrate in this report, and (vi) the presence of this particular wavelength in the emission spectrum of the 254-nm short wave series UV lamps that has been used by other investigators in teratogenic experiments as indicated in the literature and demonstrated in this report (1, 2).

Purification of a Photo-Sensitive Molecule.

 Procedures involving ovary, oocyte, egg, or embryo manipulation, gradient loading, fraction collection, and extraction with organic solvents were carried out under subdued amber light or light protection with aluminum foil. Solvents were HPLC grade. The cytoplasmic substance was purified from ovaries or spawned eggs of mature frogs. Initially, the ovary of one 9-cm pigmented female frog, or its spawned eggs, was suspended in 1 vol of ice-cold stabilizing buffer (PBS, 5 mg/ml in ascorbic acid and EDTA, adjusted to pH 7.3 with sodium hydroxide) and homogenized with a Dounce tissue grinder. The homogenate was extracted with 2 vol of extraction buffer ethyl acetate/methyl acetate (8:1) with 50 m g/ml butylated hydroxytoluene. The samples were placed on a rotator for 20 min at 4°C and centrifuged at 1,000 ´ g for 10 min, and the organic layer was removed and stored on ice. The extraction procedure was repeated and the organic layers were pooled. The product was dried with a stream of ultra-high purity nitrogen in the dark (procedure A).

The cytoplasmic yolk platelets were found to be the sole location of the UV-sensitive species (M.M., T.S.D., and K.H.F.,unpublished work). Therefore, yolk platelets were used as starting material for an improved method of isolation (procedure B), which yielded sufficient pure material for chemical and biological characterization. The ovaries of four frogs (92 ml) were suspended in 0.75 vol of the above ice-cold stabilizing buffer and the suspension was homogenized at low speed at 0°C for 5 min with a Polytron (Brinkmann). Thirty-five-milliliter aliquots of the mixture were layered onto 5-ml sucrose pellets (1.30 g/ml) in plastic tubes to avoid damage to the platelets during centrifugation at 2,000 ´ g for 10 min. The supernatant was discarded and the yolk platelets were washed twice with deionized water.

The yolk platelet pellet (43 g) was triturated with 100 ml of acetone and stirred further for 15 min at room temperature. The slurry was filtered on a Buchner funnel by using Whatman no. 41 paper and the filter cake was washed with 15 ml of acetone. The filter cake was resuspended in 100 ml of acetone, stirred for 15 min, and filtered as before. The combined filtrates and wash were kept at –20°C for 1 hr, clarified by filtration through Whatman no. 40 filter paper, and concentrated to dryness by flash evaporation and in vacuo over phosphorus pentoxide. An 8-ml aqueous solution of the dry acetone extract was adjusted to pH 8 with saturated sodium bicarbonate and extracted three times with 8 ml ethyl acetate. The yellow ethyl acetate extracts were discarded. The aqueous layer was saturated with sodium chloride and extracted twice with 8 ml 1-butanol. The combined butanol extracts were flash-evaporated and dried over phosphorus pentoxide to yield a green syrup. This product was suspended in 2.5 ml absolute methanol and the supernatant was applied to a 0.9 ´ 17 cm Sephadex LH-20 column prepared from 4 g of solid suspended in absolute methanol. Elution of the column with absolute methanol yielded a yellow-orange contaminant at 0.3–0.5, a yellow contaminant at 0.6–1.0, and the desired dark blue-green product at 1.4 to 1.7 column volumes. The pooled fractions were dried by flash evaporation. The blue-green samples were submitted to fractionation by the HPLC procedure described above. The pertinent fractions were dried, and ammonium acetate was removed in vacuo over phosphorus pentoxide and potassium hydroxide pellets. The sample was stored at –80°C.

A chromatography station (Waters) equipped with an automatic injector, in-line pump, automatic gradient controller, and absorbance detector was used for reversed-phase HPLC. The extracts were dissolved in 1 ml solvent A (20% acetonitrile, 3 mM ammonium acetate, pH 4.5) and 250-ml aliquots were loaded onto a Phenomenex Jupiter 5m C18 300-Å 250 ´ 4.6 mm column. Samples applied at 1.5 ml/min and eluted at the same rate with a linear gradient (solvent B, 100% acetonitrile). Peaks were detected at both 340 and 254 nm. Pooled fractions were dried under UHP nitrogen.

Physical-Chemical Characterization.

TLC sheets were silica gel 60F from Riedel de Han (Seelze, Germany). Absorption spectra were obtained with a Varian Cary Bio 50 Spectrophotometer. Mass spectral analysis was performed in positive ion mode on a ThermoQuest LCQ Classic electrospray ionization/ion trap instrument. Samples, dissolved in 75:25 acetonitrile/water were infused into a 100 mm i.d. capillary with a 10 mm orifice. Aliquots incubated in 99.95 atom % methyl d3 alcohol-d (Aldrich) were similarly analyzed to determine the number of exchangeable protons. Samples for NMR were prepared in the same perdeuterated methanol under dry nitrogen. All NMR experiments were run at 25°C on a Varian Unity Inova 500 spectrometer equipped with a 5-mm triple resonance 1H{13C, 15N} probe head. Unidimensional proton spectra were acquired with a spectral window of 11 ppm, a recycle time of 4 s, and 64 scans.

The free induction decay (FID) contained 16K data points. A total correlation spectroscopy (TOCSY) spectrum was acquired with a spectral window of 8 ppm in t2 and t1. A total of 512 complex FIDs were acquired, each with 2,048 points, and 32 scans. The TOCSY mixing time was 75 ms, and the recycle time was 2 s. A double quantum-filtered COSY experiment was performed with the same parameters as the TOCSY spectrum, except that each FID contained 4,096 points. A 1H-13C distortionless enhancement by polarization transfer-heteronuclear multiple quantum correlation spectrum was acquired with a spectral window of 8 ppm in t2, and 160 ppm in t1. A total of 256 complex FIDs were acquired, each with 1,024 points, and 64 scans. A 1H-13C heteronuclear multiple bond correlation spectrum was acquired with a spectral window of 8 ppm in t2 and 220 ppm in t1. A total of 256 complex FIDs were acquired, each with 2,048 points, and 128 scans. The delay for multiple bond transfer was 55 ms. The spectra were processed on a Silicon Graphics O2 workstation using VNMR software (Varian, version 6.1B). All two-dimensional spectra were zero-filled twice in t1 before Fourier transformation. Proton NMR assignments were confirmed according to published methodology, and by comparison to spectra of commercial biliverdin IXa (3-5). All assignments refer to the numbering scheme in Fig. 6.

The one-dimensional 1H spectrum is consistent with that of biliverdin IXa in terms of chemical shift distribution,and number of protons as determined by integration. The a , b, g, and d isomers of biliverdin are easily identified by differences in chemical shifts using the observations of Bonnett and McDonagh (5). For example, only the a and b isomers have a single resonance above 2.0 ppm, and of these two, only the a isomer has one triplet because of the equivalent b-methylene protons in the two carboxyethyl side chains. This pattern is clearly evident in both the molecule of interest, and in the spectrum of commercial biliverdin IXa. The vinyl protons were identified from analysis of two-dimensional total correlation spectroscopy and double quantum filter correlation spectroscopy spectra.

No attempt was made to assign the NH resonances or to distinguish between the resonances of individual methyl groups. Of significant value is the comparison of spectra from the molecule of interest and commercial biliverdin IXa. The chemical shifts and coupling patterns are identical, providing further evidence that the molecule is biliverdin IXa. Additionally, the coupling between the carbonyl carbon and the a and b methylene protons of the carboxyethyl side chains were verified from the distortionless enhancement by polarization transfer-heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation spectra (data not shown).

Photo-Transformation of Biliverdin by 366 and 254 nm.

 Biliverdin was purified by HPLC as described above. Aliquots of the purified material were placed in quartz cuvettes and irradiated for 30 min with either 366- or 254-nm wavelengths. The source of the monochromatic light was a Varian-Cary Bio 50 Spectrophotometer. The signal read was the absorption at the respective wavelengths and the average integration time for each data point was 10 s. The absorbance units of the two wavelength plots were normalized for an initial ratio value of 1 at t = 0 min. The rate of photo-tranformation expressed by the extinction of the signal or progressive change in the slope of the curve in Fig. 11, indicates that biliverdin is photosensitive to at least the two irradiation wavelengths.

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