Rapid and sensitive measurement of dissolved gases by permeable membrane mass-spectrometry.

采用透膜质谱仪对闪蒸析氧进行了测量。释放出的气体通过透气膜（0.024 mm厚，硅橡胶，通用电气，美国）进入四极杆质量分析仪（MSQ 15OA， ULVAC，日本）。将冻干后的PS II膜的纸盘轻轻浸入0.3-1.5 ml的50 mM Hepes/NaOH （pH 6.8）中，并用Ar气冲洗。反应室（直径1厘米）用气密的丙烯酸活塞盖关闭。在25ºC条件下，暗孵育5 ~ 10 min后，在32 m/e的离子峰下，在黑暗间隔20 ~ 30 s的Xe闪蒸（30 ~ 50 J, t_<1/2> = 0.03 ms; Sugawara Kenkyusho, Japan）下测量析氧。当样品溶液厚度为0.1 mm时，闪蒸析氧信号的半衰减时间约为2 s。在连续光照条件下，重悬冻干PS II膜的出氧活性为0.27 mmol (mg Chl)^<-1>h^<-1>，为对照PS II膜的70%。我们研究了冻干对闪光灯下析氧振荡模式的影响。对照PS II膜的析氧在第3闪时达到最大值，振荡周期为4闪。而浸泡在Hepes缓冲液中的冻干后的PS II膜，在第6闪时氧的析出量最大，说明冻干后水氧化酶明显降低到“S_<-2>状态”。预光照后，冻干膜的析氧过程与对照膜一样振荡。冻干和预照射后的PS II膜的析氧模式的脱靶概率和二次脱靶概率以及S_1/(S_0+S_1)与对照膜相似。冻干降低了水氧化酶的活性，只有当水与酶的高亲和位点结合时，两个光子才使酶回到S_0状态。少

Flash induced oxygen evolution was measured using a permeable membrane-mass spectrometer. Evolved gases were introduced through a gas-permeable membrane (0.024 mm thick, silicone rubber; General Electric, USA) to a quadrupole mass analyzer (MSQ 15OA; ULVAC, Japan). The paper disk of the lyophillized PS II membranes was gently immersed in 0.3-1.5 ml of 50 mM Hepes/NaOH (pH 6.8) which had been flushed with Ar gas. The reaction chamber (1 cm in diameter) was closed with a gas-tight, acrylic piston lid. After incubation in the dark for 5 to 10 min at 25ﾟC, the oxygen evolution was measured at the ion peak of 32 m/e under a Xe flash (30-50 J, t_<1/2> = 0.03 ms; Sugawara Kenkyusho, Japan) at dark intervals of 20-30 s. The half-decay time of the signal of the flash induced oxygen evolution was about 2 s when the thickness of the sample solution was 0.1 mm. The oxygen-evolving activity of the resuspended lyophillized PS II membranes was 0.27 mmol of oxygen (mg Chl)^<-1>h^<-1> under continuous … More light which was 70% that of the control PS II membranes. We examined the effect of lyophillization on the oscillation pattern of oxygen evolution by flash illuminations. The oxygen evolution of the control PS II membranes was maximal at the third flash and oscillated with a period of 4 flashes. The lyophillized PS II membranes which were immersed in Hepes buffer, however, showed the maximal evolution of oxygen at the sixth flash, indicating the water-oxidizing enzyme was apparently reduced to "S_<-2> state" by lyophillization. After preillumination the oxygen evolution of the lyophillized membranes oscillated as the control membranes did. Moreover, the miss-hit and double-hit probabilities and S_1/(S_0+S_1) for the oxygen evolution pattern of the lyophillized and preilluminated PS II membranes were similar to those of the control membranes. The lyophillization reduces the water-oxidizing enzyme, and the two photons return the enzyme to the S_0 state only in the presence of the water bound the the high-affinity sites in the enzyme. Less