CAJ | 학술논문

采用一种快速简捷的方法从菠菜(Spinacia oleracea L.)和水稻(Oryza sativa L.)中分离纯化了光系统Ⅱ反应中心内的细胞色素b-559,并且研究了其低温可见光区荧光光谱、室温紫外区荧光光谱、吸收光谱以及电泳特性.该方法的主要特点:1.以放氧核心复合物为起始材料以避免其他细胞色素的干扰;2.选用DEAE-Sephacel为层析介质,用等度洗脱除去杂蛋白和叶绿素;3.用同一介质不同条件去除过量的去垢剂.从两种植物中分离纯化的Cyt b-559具有相似的吸收光谱,在非变性电泳中有相同的泳动特征.用修订的适用于分析小蛋白的Tricine-SDS-PAGE证明,从两种植物中分离得到的Cyt b-559都是由两个多肽亚基组成,它们的表观分子量分别为9 kD和4 kD.低温荧光光谱的结果表明,Crt b-559的荧光激发峰位为413nm和439 nm,荧光发射峰位在563 nm和668 nm,首次证明Cyt b-559可以发出荧光并将电子传递给叶绿素.首次通过Cyt b-559的紫外荧光光谱证明Trp残基位于该蛋白的疏水跨膜区内.
채용일충쾌속간첩적방법종파채(Spinacia oleracea L.)화수도(Oryza sativa L.)중분리순화료광계통Ⅱ반응중심내적세포색소b-559,병차연구료기저온가견광구형광광보、실온자외구형광광보、흡수광보이급전영특성.해방법적주요특점:1.이방양핵심복합물위기시재료이피면기타세포색소적간우;2.선용DEAE-Sephacel위층석개질,용등도세탈제거잡단백화협록소;3.용동일개질불동조건거제과량적거구제.종량충식물중분리순화적Cyt b-559구유상사적흡수광보,재비변성전영중유상동적영동특정.용수정적괄용우분석소단백적Tricine-SDS-PAGE증명,종량충식물중분리득도적Cyt b-559도시유량개다태아기조성,타문적표관분자량분별위9 kD화4 kD.저온형광광보적결과표명,Crt b-559적형광격발봉위위413nm화439 nm,형광발사봉위재563 nm화668 nm,수차증명Cyt b-559가이발출형광병장전자전체급협록소.수차통과Cyt b-559적자외형광광보증명Trp잔기위우해단백적소수과막구내.
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.