Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study.
Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study.
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铁氧还蛋白-铁氧还蛋白-NADP 还原酶复合物的还原动力学：激光闪光光解研究。
DOI：
10.1021/bi00364a030
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Tollin,G
中科院分区：
文献类型：
--
作者：
Bhattachryya,AK;Meyer,TE;Tollin,G
材料与方法根据Zanetti和Curti（1980）的方法纯化菠菜FNR和Fd。用E.恶心在458和420 nm处的消光系数为10.3 X 103和9.7 X 103 M-1cm-1（Foust等人，1969）分别用于测定氧化的FNR和Fd的浓度。所有缓冲液组分均为ACS试剂级。在pH 6.0和7.0下用于低离子强度实验的缓冲液为4 mM磷酸钾-0.5 mM乙二胺四乙酸（EDTA）和约100 µ 5-dRf（i = 10 mM）。在pH 8.0下，使用10 mM Bistris propane-0.5 mM EDTA缓冲液。氯化钾用于将缓冲溶液调节至适当的离子强度。所有动力学实验均在伪一级条件下进行，其中氧化蛋白的浓度远远超过每次闪蒸产生的5-dRf* 的量（< 0.7 μ）。除非需要定量（例如，闪光诱导的差异），否则每个动力学迹线的闪光次数不同。此外，在氧化蛋白量增加的情况下，由于激发激光的吸收，每次闪光产生的5-dRf* 的量减少。通过拟合指数曲线手动分析所有动力学迹线。图1：动力学迹线表示以下：（a）在不存在Fdg和FNR * 的情况下，在I= 10 mM，pH 7下，5-dRf* 快速形成和较慢分解。(b)在500 nm处监测FDox的还原。缓冲液条件如（a）中。蛋白质浓度为9 µ。插图显示了在（）10、（）310和（）460 mM离子强度下fcobsd对FDox浓度的二阶图。（c）在590 nm处监测的FNR* 的形成。FNR 0X浓度为11 µ。初始快速上升（虚线）对应于散射伪影。然而，迹线的指数部分外推到零时刻的预闪基线。插图显示了在（0）10和（0）310 mM的离子强度下fcohd相对于FNR 0X浓度的二阶图，以及（0）1：1 Fd 0X-FNR 0X混合物在f = 10 mM下的二阶图。（B）和（c）中的实线是通过数据绘制的单指数曲线。
Materials and MethodsSpinach FNR and Fd were purified according to the method of Zanetti and Curti (1980). A few experiments were per-formed with FNR that was a kind gift from Dr. E. Gross. Extinction coefficients of 10.3 X 103 and 9.7 X 103 M'1 cm" 1 at 458 and 420 nm (Foust et al., 1969) were used todetermine the concentrations of oxidized FNR and Fd, respectively. All buffer components were ACS-reagent grade. The buffer used for the low ionic strength experiments at pH 6.0 and 7.0 was 4 mM potassium phosphate-0.5 mM ethylenediaminetetra-acetic acid (EDTA) and~ 100 µ 5-dRf (/= 10 mM). At pH 8.0, 10 mM Bistrispropane-0.5 mM EDTA buffer was used. Potassium chloride was used to adjust buffer solutions to the appropriate ionic strengths. All kinetic experiments were performed under pseudo-first-order conditions, in which the concentration of oxidized protein was in large excess over the amount of 5-dRf* produced per flash (< 0.7 µ). Unless quantitation was required (eg, for flash-induced differencespectra) the number of flashes per kinetic trace varied. Furthermore, in the presence of increasing amounts of oxidized protein, due to absorbance of the exciting laser light, the amount of 5-dRf* produced per flash was di-minished. All kinetic traces were analyzed by hand, by fitting to an exponential curve. All kinetic experiments were carried figure 1: Kinetic traces representing the following:(a) Rapid formation and slower disproportionationof 5-dRf* in the absence ofFdg, and FNR^ at I= 10 mM, pH 7.(b) Reduction of Fdox monitored at 500 nm. Buffer conditions as in (a). The protein concentration was 9 µ. The inset shows second-order plots of fcobsd vs. Fdox concentration at () 10,(O) 310, and () 460 mM ionic strengths,(c) Formation of FNR* monitored at 590 nm. FNR0X concentration was 11 µ. The initial rapid rise (dashed line) correspondsto a scattering artifact. However, the exponential portion of the trace extrapolates to the preflash base line at zero time. The inset shows second-order plots of fcohd vs. FNR0X concentration at ionic strengths of (O) 10 and () 310 mM and () a 1: 1 Fd0X-FNR0X mixture at/= 10 mM. Solidlines in (b) and (c) are single-exponential curves drawn through the data.