Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: determination of number of binding sites and dissociation constants by equilibrium dialysis.

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: determination of number of binding sites and dissociation constants by equilibrium dialysis.
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细胞色素 c 与球形红假单胞菌 R-26 反应中心的相互作用:通过平衡透析测定结合位点数量和解离常数。

DOI:
10.1021/bi00566a004
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发表时间:
1980
期刊:
影响因子:
2.9
通讯作者:
Feher,G
Feher,G
中科院分区:
生物学3区
文献类型:
--
作者:
Rosen,D;Okamura,MY;Feher,G

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材料与方法平衡透析。使用五室旋转平衡透析组件(Spectrum Medical Instruments,Los Angeles,CA);每个隔室的体积为1 mL。膜为再生纤维素系列(Schleicher & Scheull,Keene,NH,RC-52和RC-53)。将它们在10 mM Tris-HCl,pH 8.0,0.1% Triton X-100(TT缓冲液)和1 mM EDTA中洗涤5次,持续数小时,然后在使用前用TT缓冲液冲洗。通过0.22-µ Millipore过滤器过滤所有缓冲液,以去除可能堵塞膜的颗粒。除非另有说明,否则在4 ℃下在黑暗中进行透析48小时。反应中心。根据公开的程序制备RC(Feher和Okamura,1978)。发现增溶洗涤剂月桂基二甲基氧化胺(LDAO)改变了cyt的氧化态,因此用0.1%Triton X-100代替LDAO,通过在DEAE柱上吸附RC,用TT缓冲液洗涤,并用1
Materials and MethodsEquilibrium Dialysis. A five-cell rotating equilibrium dialysis assembly (Spectrum Medical Instruments, Los An-geles, CA) was used; the volume of each compartment was 1 mL. Themembranes were of the regenerated cellulose series (Schleicher & Scheull, Keene, NH, RC-52 and RC-53). They were washed for several hours with five changes in 10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100 (TT buffer) and 1 mM EDTA and then rinsed with TT buffer before use. All buffers were filtered through 0.22-µ Millipore filters to remove particles that might clog the membrane. Dialyses were performed at 4 C in the dark for 48 h unless otherwise specified. Reaction Centers. RCs were prepared according to pub-lished procedures (Feher & Okamura, 1978). The solubilizing detergent, lauryldimethylamine oxide (LDAO), was found to change the oxidation state of cyt; it was therefore replaced with 0.1% Triton X-100by absorbing RCs on a DEAE column, washing with TT buffer, and eluting with 1
DOI: 10.1021/bi00704a014
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