0.1 M phosphate buffer solutions (PBS) with various pH values were prepared by dissolving an appropriate amount of [Na.sub.2]HP[O.sub.4] in water and adjusting the pH values with 0.1 M HCl or NaOH solutions.
Samples were diluted with 10 mM phosphate pH 7.0 for native conditions and with 10 mM phosphate and 5 M GdnHCl buffer solution pH 7.0 for denaturing conditions, while for denaturing-reducing conditions samples were treated with 10 mM DTT for 30 min at 25[degrees]C and buffer exchanged using NAP-5 columns against 10 mM phosphate buffer solution and GdnHCl 0.5 M pH 3.0.
When the signal of the oxygen electrode stabilized in the 50 ml phosphate buffer solution, the certain amounts of calibration solution were added to achieve known [BOD.sub.7] values to the measurement solution.
Addition of 10 [micro]L of urine to 50 [micro]L of TMB (tetramethylbenzidine) solution, followed by addition of 500 [micro]L of 0.1 M phosphate buffer solution, caused a dramatic color change of the specimen to dark brown.
In additional test series, this control sample was supplemented with 100 [micro]L volumes of a phosphate buffer solution containing 8 and 10 nmol of Trp (final concentrations, 62.2 and 72.2 [micro]mol/L) or 0.8 and 1.0 nmol of Kyn (final concentrations, 4.81 and 5.81 [micro]mol/L).