Development of a method to examine the sensitivity of tissues and bacteria for drugs on the basis of direct and sensitive measurement of the concentration of oxygen

1.我们开发了一种系统，可以提供含氧小于1 μM的缓冲液。在这个系统中，注射器中含有的缓冲液中的氧分子在几秒钟内被儿茶酚2,3-双加氧酶（Mpc）催化氧化成4-氯儿茶酚（4-CC），然后缓冲液通过一个由C18和强阴离子交换凝胶填充的柱，将剩余的4-CC和酶促产物除去，然后供应到反应容器中。已经生产出了几个月来保持高酶活性的mpc固定凝胶。在2-羟吡啶n -氧化物（一种有效的竞争性抑制剂）存在下，Mpc与TSKgel aft - tresyl Toyopearl凝胶反应，得到Mpc固定凝胶：填充凝胶的微柱（1.0 mm×30 mm）显示出足够的活性，几乎可以在0.1 ml/min的流速下完成O_2与4-CC的反应。我们开发了一种系统来提供含有0-250 μM O_2的缓冲液。使用两个注射泵，将空气饱和缓冲液和不含氧的缓冲液（见1）用T形接头按规定的比例混合。用氧电极测定了系统洗脱液中O_2的浓度。使用仅含有阴离子交换剂的捕集柱，通过微Mpc柱后，用紫外-可见监测器成功地监测了O_2的浓度（见2）。用100 μ l的蛋白质-配体混合物成功地进行了正面凝胶层析（FGC）。FGC允许我们获得蛋白质与配体相互作用的结合曲线。因此，目前的微尺度FGC为其在后基因组时代的功能表征研究开辟了道路。对Mpc-2-HPNO配合物进行了结晶和初步的x射线衍射研究。

1.We have developed a system to supply a buffer containing oxygen less than 1 μM. In this system, the oxygen molecules in the buffer contained in a syringe are consumed completely within several seconds by the oxygenation to 4-chlorocatechol (4-CC) catalyzed by catechol 2,3-dioxygenase (Mpc), then the buffer is supplied to a reaction vessel after passing through a column packed with both C18 and strong anion exchange gels which remove the remaining 4-CC and the enzymatic products.2.Mpc-fixed gels maintaining a high enzyme activity over months have been produced. In the presence of 2-hydroxypyridine N-oxide, a potent competitive inhibitor, Mpc was reacted with TSKgel AF-Tresyl Toyopearl gels to give Mpc-fixed gels : a micro column (1.0 mm×30 mm) packed with the gel showed a sufficient activity to nearly complete the reaction of O_2 with 4-CC at a flow rate of 0.1 ml/min.3.We have developed a system to supply a buffer containing 0-250 μM O_2. Using two syringe pumps, the air-saturated buffer and the buffer containing no oxygen (see 1.) were mixed in a defined ratio with a T joint. The concentration of O_2 in the buffer eluted from the system was confirmed by an oxygen electrode. Using the trap column only containing anion exchanger, the concentration of O_2 was successfully monitored by a UV-Vis monitor after passing through the micro Mpc column (see 2.).4.Frontal gel chromatography(FGC) using a 100-μL amount of a protein-ligand mixture has been successfully performed. FGC allows us to obtain the binding curve for the interaction between a protein and ligands. Therefore, the present micro scale FGC opens the way for its application to functional characterization studies in the post-genom era.5.Crystallization and preliminary X-ray diffraction studies of Mpc-2-HPNO complex have been carried out.