Rapid measurement of oxygenin tissues utilijing palce flash photolysis

1.通过采用红宝石球制成的球阀来切换样品溶液的流动路径，构建了一种完全无氧流动闪蒸装置。加压氮气还可以用来从驱动注射器的活塞头接触处清除空气中的氧气，以确保厌氧。这些改进允许在1ms到50min的时间范围内跟踪观察池中样品溶液的光谱变化，而不受空气中氧气污染的干扰。利用该装置在1ms~30min的时间范围内跟踪了二亚硫酸盐还原细胞色素氧化酶时的光谱变化，从而实现了该酶中两个血红素α基团的光谱分离。由此得到的绝对吸收光谱可以成功地解释还原过程中Soret区任何选定波长的多相吸光度变化。这些光谱数据是描述这种酶将氧化还原自由能转换为跨越生物膜的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过程中，CuA被认为起着至关重要的作用。用流动闪蒸装置研究了缺乏铜A的亚硝酸单胞菌的细胞色素氧化酶与氧的反应。然而，这种缺乏铜A的酶的行为与正常酶相似，与氧反应的速度以及分子内电子转移的速度甚至高于正常酶。这一发现表明，CuA对电子转移过程不是必需的，而是假定保持和控制酶的三级结构，而三级结构是质子转移功能所必需的。

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.