LOW TEMPERATURE ABSORBANCE AND FLUORESCENCE SPECTROSCOPY OF THE PHOTOACTIVE YELLOW PROTEIN FROM Ectothiorhodospira halophila

Autor: Klaas J. Hellingwerf, Wouter D. Hoff, Stefan L.S. Kwa, R. van Grondelle
Rok vydání: 1992
Předmět:
Zdroj: Photochemistry and Photobiology. 56:529-539
ISSN: 1751-1097
0031-8655
DOI: 10.1111/j.1751-1097.1992.tb02197.x
Popis: — A simplified procedure was developed to purify the photoactive yellow protein (PYP) from Ecrorhiorhodospira halophila. Specific antibodies were used to follow the distribution of PYP through the separate purification steps. Low temperature absorbance and fluorescence characteristics of this photoactive protein were investigated. The absorbance spectrum of PYP in 67% (vol/vol) glycerol peaked at 449 and 447 nm, at room-and liquid nitrogen temperatures, respectively. It sharpened significantly upon cooling to 77 K and displayed fine-structure on the blue side of its absorbance maximum, with a spacing of 25 nm. At room temperature PYP fluoresced with a quantum yield of approximately 3.5 times 10−-3 an emission maximum of 495 nm. Maximal excitation occurred at 457 nm, 10 nm red-shifted with respect to the absorbance maximum. At -low temperature the excitation maximum remained unaltered but maximal emission shifted significantly to the blue (to 482 nm). The quantum yield of fluorescence increased to 0.07 at this temperature. Illumination of PYP at low temperature with light from the visible part of the spectrum of electromagnetic radiation induced pronounced changes in its absorbance and fluorescence characteristics. At least two new stable intermediates were formed: one highly fluorescent, with an excitation maximum at 430 nm; additionally, a non-fluorescent red-shifted intermediate with an absorbance maximum at 490 nm. The amount formed of these two intermediates depended strongly on the wavelength of actinic illumination. In combination, these data underline the spectroscopic similarities between PYP and the retinal-containing chromoproteins that are present in Halobacterium halobium.
Databáze: OpenAIRE
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