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

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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.