Cytochrome b_559 in photosystem Ⅱ reaction center was purified from spinach ( Spinacia oleracea L.) and rice ( Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emission and e...Cytochrome b_559 in photosystem Ⅱ reaction center was purified from spinach ( Spinacia oleracea L.) and rice ( Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emission and excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The author's purification methods, which enhanced the yield of pure protein and shorted the time for isolation, have several advantages: 1. use of oxygen_evolving PSⅡ core complexes as the starting material in order to avoid disturbing from other cytochromes; 2. isocratic elution of cytochrome b_559 from a DEAE_Sephacel column for eliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal of excess Triton X_100. Purified cytochromes b_559 from these species have similar optical spectra and mobility during 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 molecular weight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for the purified cytochrome b_559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at 563 nm and 668 nm. This is the first indication that Cyt b_559 is able to emit fluorescence and also transfer excited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the first time that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b_559.展开更多
Mossbauer spectrum measured for the iron components of photosystem Ⅱ (PS Ⅱ ) particles of spinach is a superposition of 4 doublets. Quadrupole splitting and chemical shifting of doublets I -IV are characteris-tics o...Mossbauer spectrum measured for the iron components of photosystem Ⅱ (PS Ⅱ ) particles of spinach is a superposition of 4 doublets. Quadrupole splitting and chemical shifting of doublets I -IV are characteris-tics of proteins with oxidized cytochrome b-559, reduced cytochrome b-559, Fe3 + -Q complex and Fe2+ -Q complex re-spectively . After the PS II particles are treated with La3+ , two doublets of Fe2+ disappear and Fe2+ is converted into Fe3+ , indicating that the reduced cytochrome b-559 has been converted into the oxidized cytochrome b-559, and Fe2+-Q complex into Fe3+-Q complex. The Mossbauer spectrum of PS II particles treated with La3+ and Ca2+ shows that Ca2+ can weaken the inhibitory effect of La3+ in part, and a portion of the reduced cytochrome b-559 and Fe-Q complex still exist.展开更多
文摘Cytochrome b_559 in photosystem Ⅱ reaction center was purified from spinach ( Spinacia oleracea L.) and rice ( Oryza sativa L.) by a rapid and simple procedure. Their low temperature fluorescence emission and excitation spectra, ultraviolet fluorescence spectra and absolute absorption spectra were presented. The author's purification methods, which enhanced the yield of pure protein and shorted the time for isolation, have several advantages: 1. use of oxygen_evolving PSⅡ core complexes as the starting material in order to avoid disturbing from other cytochromes; 2. isocratic elution of cytochrome b_559 from a DEAE_Sephacel column for eliminating the impurity and yielding the protein in pure state; 3. a simple column procedure for removal of excess Triton X_100. Purified cytochromes b_559 from these species have similar optical spectra and mobility during 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 molecular weight 9 kD and 4 kD, respectively). By measuring of 77 K fluorescence spectra, it was shown that for the purified cytochrome b_559 there were two excitation peaks at 439 nm and 413 nm, and two emission peaks at 563 nm and 668 nm. This is the first indication that Cyt b_559 is able to emit fluorescence and also transfer excited electrons to chlorophyll. By the use of ultraviolet fluorescence spectra, it was demonstrated for the first time that the location of Trp residue could be in the hydrophobic transmembrane region of cytochrome b_559.
基金Project supported by the National Natural Science Foundation of China and the Natural Science Foundation of the Chinese Educational Commission.
文摘Mossbauer spectrum measured for the iron components of photosystem Ⅱ (PS Ⅱ ) particles of spinach is a superposition of 4 doublets. Quadrupole splitting and chemical shifting of doublets I -IV are characteris-tics of proteins with oxidized cytochrome b-559, reduced cytochrome b-559, Fe3 + -Q complex and Fe2+ -Q complex re-spectively . After the PS II particles are treated with La3+ , two doublets of Fe2+ disappear and Fe2+ is converted into Fe3+ , indicating that the reduced cytochrome b-559 has been converted into the oxidized cytochrome b-559, and Fe2+-Q complex into Fe3+-Q complex. The Mossbauer spectrum of PS II particles treated with La3+ and Ca2+ shows that Ca2+ can weaken the inhibitory effect of La3+ in part, and a portion of the reduced cytochrome b-559 and Fe-Q complex still exist.
