Purification and Spectral Characteristics of Cytochrome b-559 from Oxygen-evolving Photosystem Ⅱ Core Complexes of Spinach and Rice
Author(s): XIN Yueyong, YU Fei, TANG Chongqin, LI Liangzuo, KUANG Tingyun
Pages: 1225-1230
Year: 2000 Issue: 12
Journal: ACTA BOTANICA SINICA
Keyword: cytochrome b-559; oxygen evolution PS Ⅱ core complexes; Tricine-SDS-PAGE; optical spectra;
Abstract: Cytochrome b-559 in photosystem Ⅱ reaction center was purified from spinach (Spinacia oleraceaL.) and rice (Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emissionand excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The au-thor' s purification methods, which enhanced the yield of pure protein and shorted the time for isolation, haveseveral advantages: 1. use of oxygen-evolving PS Ⅱ core complexes as the starting material in order to avoiddisturbing from other cytochromes; 2. isocratic elution of cytochrome b-559 from a DEAE-Sephacel column foreliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal ofexcess Triton X-100. Purified cytochromes b-559 from these species have similar optical spectra and mobilityduring gel electrophoresis under native conditions. From the results of novel electrophoresis (Tricine-SDS-PAGE), cytochrome b-559 from both spinach and rice reveal two polypeptide bands (apparent molecularweight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for thepurified cytochrome b-559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at563 nm and 668 nm. This is the first indication that Cyt b-559 is able to emit fluorescence and also transferexcited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the firsttime that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b-559.
Pages: 1225-1230
Year: 2000 Issue: 12
Journal: ACTA BOTANICA SINICA
Keyword: cytochrome b-559; oxygen evolution PS Ⅱ core complexes; Tricine-SDS-PAGE; optical spectra;
Abstract: Cytochrome b-559 in photosystem Ⅱ reaction center was purified from spinach (Spinacia oleraceaL.) and rice (Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emissionand excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The au-thor' s purification methods, which enhanced the yield of pure protein and shorted the time for isolation, haveseveral advantages: 1. use of oxygen-evolving PS Ⅱ core complexes as the starting material in order to avoiddisturbing from other cytochromes; 2. isocratic elution of cytochrome b-559 from a DEAE-Sephacel column foreliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal ofexcess Triton X-100. Purified cytochromes b-559 from these species have similar optical spectra and mobilityduring gel electrophoresis under native conditions. From the results of novel electrophoresis (Tricine-SDS-PAGE), cytochrome b-559 from both spinach and rice reveal two polypeptide bands (apparent molecularweight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for thepurified cytochrome b-559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at563 nm and 668 nm. This is the first indication that Cyt b-559 is able to emit fluorescence and also transferexcited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the firsttime that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b-559.
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