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UV–visible spectroscopy

KF Kerstin Fiege
NF Nicole Frankenberg-Dinkel
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UV–vis spectroscopy was performed using an 8453 UV–visible spectrophotometer (Agilent Technologies). Heme spectra were taken at room temperature in 20 mM TES buffer pH 8.0 containing 100 mM KCl and 10% glycerol. Spectra were taken from 350–700 nm under oxidizing conditions. Phytochrome spectra were taken at 25 °C using cell-free lysates (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA). The BphP samples were incubated for 3 min with red light at 690 nm for Pfr spectra and 3 min with far-red light at 750 nm for Pr spectra as described previously [22]. The Pfr spectra were subtracted from the Pr spectra to obtain the red/far-red induced difference spectra. For Cph1, difference spectra were obtained in a similar way, expect that the red and far-red light filters of 630 and 730 nm were used, respectively [23]. To analyze the saturation of Cph1 with its chromophore, difference spectra were measured again after 30 min incubation with 40 µM phycocyanobilin (PCB) for 30 min at room temperature.

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