Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes.
Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes.
复制标题
光敏色素的初级光过程。
DOI：
10.1021/bi00434a022
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Furuya,M
中科院分区：
文献类型：
--
作者：
Song,PS;Singh,BR;Tamai,N;Yamazaki,T;Yamazaki,I;Tokutomi,S;Furuya,M
材料和方法光敏色素的分离和纯化。从燕麦黄化苗中分离纯化了未降解的124-kDa光敏色素。CV. Garry燕麦，由斯坦福大学种子公司提供，布法罗，纽约）根据Chai et al.（1987年）。分离的光敏色素制剂的分子量和光谱特征通过Chai等人描述的方法确认。（1987年）。根据Song等的方法，从黄化燕麦苗中分离纯化了降解的氨基端截短的118/114-kDa光敏色素。（1981年a）。未降解的，121-kDa的光敏色素从黄化豌豆幼苗（豌豆品种。Alaska）进行分离、纯化，并根据Tokutomi等人的方法进行表征。(1986，1988）。根据Yamamoto和Furuya（1983）和Tokutomi等的方法，从豌豆黄化苗中提取并纯化了114-kDa降解光敏色素。（1988年）。用于皮秒荧光衰减和时间分辨光谱研究的光敏色素制剂具有范围为0.9至1.1的特定吸光度比（SAR= A660/A250）。所用的121-和114-kDa光敏色素的SAR值分别为0.65和0.98。没有明确的依赖性的衰减和光谱性质的光敏色素的纯度被发现以外的实验误差。Pr和Pfr形式的光敏色素通过分别用738-和660-nm光纤源照射样品比色皿1-2分钟而原位制备。除非另有说明，否则如前所述制备具有不同溶剂粘度的光敏色素样品溶液（Song等人，1986年）。溶液的温度控制是通过使用一个专门建造的热电冷却器的细胞室。皮秒荧光衰减测量。这些测量通过使用同步泵浦的腔倒空染料激光器（Spectra Physics 375和344 S）、锁模氩离子激光器（Spectra Physics 171-18）和时间相关单光子计数器来进行，如别处所述（Yamazaki等人，1984年，1985年）。光学设置和条件基本上与我们先前的论文（Song等人，1986年）。激发激光功率在800 kHz下为0.68 mW/cm 2，对于大多数运行，637 nm下的激发脉冲宽度设定为6-7 ps。使用微通道板光电倍增管（R1564 U; Hamamatsu Corp. Ham-amatsu，Japan）进行荧光检测。为了最大限度地减少积累的光敏色素的Pfr物种的样品溶液暴露于重复的637-nm激发laserpulses，脉冲照射区域的溶液进行光化照射的远红光光纤源，而该溶液是磁力搅拌。该转换源由Ushio 500-W的弧光灯、水过滤器、聚焦透镜（f= 50）、中性密度楔形过滤器、738-nm干涉过滤器（半最大带宽= 10.5 nm）和玻璃光纤组成。这种光学布置的示意图先前已经呈现（Song等人，1986年）。在这些条件下，每次数据采集期间产生的Pfr量可以忽略不计，对于2000次计数，每次数据采集持续30-60 s。只有在长时间的数据采集后，激发激光脉冲（光束直径1.5 mm）才能在比色皿中产生可感知的Pfr量，对应于约1.5 mm。从Pr转化10%（Song等人，1986年）。在搅拌速率对测量的衰减数据（寿命及其
Materials and MethodsPhytochrome Isolation and Purification. Undegraded 124-kDa phytochrome was isolated and purifiedfrom etiolated oat seedling shoots (Avena sativa L. cv. Garry oat, supplied by Stanford Seed Co., Buffalo, NY) according to the method of Chai et al.(1987). The molecular weight and spectroscopic characteristics of the isolated phytochrome preparations were confirmed by the procedures described in Chai et al.(1987). The degraded, amino terminus truncated 118/114-kDa phy-tochrome from the etiolated oat shoots was isolated and pu-rified according to the method of Song et al.(1981a). The undegraded, 121-kDa phytochrome from the etiolated pea seedlings (Pisum sativum cv. Alaska) was isolated, purified, and characterized according to the method of Tokutomi et al.(1986, 1988). The 114-kDa degraded pea phytochrome was extracted and purified from the etiolated pea seedlings ac-cording to the procedure of Yamamoto and Furuya (1983) and Tokutomi et al.(1988). The phytochrome preparations used for the picosecond fluorescence decay and time-resolved spectral studies had specific absorbance ratios (SAR= A660/A2so) ranging from 0.9 to 1.1. The 121-and 114-kDa phytochromes used had SAR values of 0.65 and 0.98, re-spectively. No definitive dependence of the decay and spectral properties of the phytochromes on the purity was found outside the experimental errors. The Pr and Pfr forms of phytochrome were prepared in situ by irradiating the sample cuvettewith a 738-and 660-nm optical fiber source for 1-2 min, respec-tively.Unless stated otherwise, the phytochrome sample solutions having different solvent viscosities were prepared as described previously (Song et al., 1986). Temperature control for the solution was achieved by using a specially built thermoelectric cooler for the cell compartment. Picosecond Fluorescence Decay Measurements. These measurements were performed by using a synchronously pumped, cavity-dumped dye laser (Spectra Physics 375 and 344S), a mode-locked argon ion laser (Spectra Physics 171-18), and a time-correlated single photon counter, as described elsewhere (Yamazaki et al., 1984, 1985). Optical setup and conditions were essentially identical with those described in our previous paper (Song et al., 1986). The excitation laser power was 0.68 mW/cm2 at 800 kHz, with the excitation pulse width at 637 nm set at 6-7 ps for most of the runs. Fluorescence detection was achieved by using a microchan-nel-plate photomultiplier (R1564U; Hamamatsu Corp. Ham-amatsu, Japan). To minimize the buildup of the Pfr species of phytochrome resulting from the exposure of the sample solution to the repeated 637-nm excitation laserpulses, the pulse-irradiated area of the solution was actinically irradiated with a far-red light optical fiber source while the solution was magnetically stirred. This conversion source consisted of an Ushio 500-W Xe arc lamp, a water filter, a focal lens (f= 50), a neutral density wedge filter, a 738-nm interference filter (half-maximum bandwidth= 10.5 nm), and a glass optical fiber. The schematics of this optical arrangement have been presented previously (Song et al., 1986). Under these con-ditions, negligible amounts of Pfr were produced during each data acquisition, which lasts 30-60 s for 2000 counts. Only after an extended period of data acquisitiondid the excitation laser pulses (beam diameter 1.5 mm) produce appreciable amounts of Pfr in the cuvette, corresponding to ca. 10% con-version from Pr (Song et al., 1986). After the effect of the stirring rate on the measured decay data (lifetimes and their