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Assay Method Information |
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| | Steady-State Kinetic Assay |
| Description: | Initial rates of amine oxidation were measured by monitoring oxygen consumption using an oxygen electrode (model 5300A, Yellow Springs Instruments, Yellow Springs, OH) at 25 °C. The buffers were 0.1 M sodium Hepes at pH 7-8, 0.1 M sodium CHES at pH 8.5-10, and 0.1 M sodium CAPS at pH 10.5−11; all buffers also contained 0.1 M NaCl. For solvent isotope effects, buffers and substrates were made up inD2O. The concentrations of all the enzymes were determined using the ε445 value of 11.3 mM−1 cm−1 for the wild-type enzyme,8 because there were no significant changes in the visible absorbance spectra of the variants. The use of the flavinabsorbance to determine the enzyme concentration meant that only the holoenzyme was included in the enzyme concentration. The concentration of oxygen was varied by bubbling the desired concentration of oxygen (62 μM to 1.25 mM) into the oxygen electrode cell for ∼10 min. Assays typically contained 0.1 μM enzyme, with th |
| Affinity data for this assay | |
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