Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere

An enzymatic method for measuring the O(2) concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2,3-dioxygenase from Pseudomonas putida. With this system, the amount of O(2) in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction. The product was stable and its anion exhibited strong absorption around 380 nm (molar absorption coefficient of 4.3 x 10(4) M(-1) cm(-1), pK value of 5.4). A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol. When the O(2) concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O(2) could be successfully measured by the HPLC method. We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon. By applying these devices, the submicromolar O(2) concentration of an anoxic solution and the back diffusion of O(2) from the atmosphere could be directly determined for the first time. The K(m) values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 microM (at pH 6.5), respectively, at 25 degrees C.