Rapid measurement of oxygenin tissues utilijing palce flash photolysis

1.采用红宝石球球阀来改变样品溶液的流动路径，构建了一种绝对厌氧流动闪蒸装置。也可用加压氮气将驱动注射器活塞头接触处的空气氧气吹走，以保证厌氧。这些改进可以跟踪观察池中样品溶液在1毫秒至50分钟的时间范围内的光谱变化，而不会受到空气中氧气污染的干扰。利用该装置，在1 ms ~ 30 min的时间范围内，对细胞色素氧化酶的二亚砜还原过程中的光谱变化进行了跟踪，并在此基础上实现了细胞色素氧化酶中两个血红素α片段的光谱分离。由此导出的绝对吸收光谱可以成功地用于解释还原过程中Soret区域中任何选定波长的多相吸光度变化。这些光谱数据是描述该酶在生物膜上将氧化还原自由能转化为质子驱动力的复杂功能的基础。在细胞色素氧化酶的质子泵送过程中，Cu_A被认为起着至关重要的作用。利用流动闪蒸装置研究了缺乏Cu_A的欧洲亚硝化单胞菌的细胞色素氧化酶与氧的反应。然而，这种缺乏cu_a的酶表现得像正常酶一样；与氧反应的速率以及分子内电子转移的速率甚至比普通酶的速率还要高。这一发现表明Cu_A对电子转移过程并不是必需的，但可以保持和控制酶的三级结构，而三级结构对质子易位的功能至关重要。

1.An ablolutely anaerobic flow flash apparatus was constructed by incorporating ballvalves made of ruby balls to switch the flow path of the sample solutions. Also pressurized nitrogen gas can be used to purge air oxygen from the contact of the piston head of the driving syringe to assure the anaerobiosis. These improvements allow to follow the spectral change of a sample solution in the observation cell in a time range from 1 ms to 50 min without being interfered from contamination of air oxygen. Using this apparatus the spectral changes upon dithionite reduction of cytochrome oxidase were followed in a time range from 1 ms to 30 min, based on which the spectral separation of two heme alpha moieties in this enzyme was achieved. The absolute absorption spectra thus derived can be used successfully to explain multiphasic absorbance changes at any selected wavelengths in the Soret region during the reduction process. These spectral data are fundamental to delineate the intricate function of this enzyme to convert the redox free energy into the proton driving force across the biological membrane.2. In the proton pumping of cytochrome oxidase Cu_A has been assumed to play a crucial role. By using cytochrome oxidase from Nitrosomonas europaea which lacks Cu_A its reaction with oxygen was studied by using a flow flash apparatus. This Cu_A-deficient enzyme, however, behaves like a normal enzyme; the rate for the reaction with oxygen as well as for the intramolecular electron transfer is even higher than that of the normal enzyme. This finding has derived significant implications that Cu_A is not essential to the electron transfer process but assumed to keep and control the tertiary structure of the enzyme which is essential to the function of proton translocation.