Kynurenlnase Ubbink et al 1991

Kynureninase is involved in the oxidative metabolism of tryptophan. It catalyzes the conversion of L-kynurenine to anthranilic acid. The enzyme also converts L-3-hydroxykyneurenine to 3-hydroxyanthranilic acid. The latter compound has a high fluorescence, which is the basis for detection in this assay.

The reaction mixture was separated on a Whatman Partisphere Qg column (4.6 mm X 150 mm, 5 /xm). The mobile phase at a flow rate of 1 mL/min was 0.1 Af potassium phosphate buffer (pH 5.8) containing 1% acetonitrile. The fluorescence detector was set for excitation and emission wavelengths of 322 and 414 nm, respectively.

Homogenates of lymphocytes were preincubated at either 15 or 30°C for 10 minutes before the reaction was started, by addition of 3-hydroxykynure-nine to a final concentration of 1 mAf. When the assay was performed at 30°C, the presence of 4 /¿Af pyridoxal-5'-phosphate was needed to obtain a linear reaction. The reaction was terminated after 5 minutes by the addition of 150 fiL of 10% trichloroacetic acid. HPLC analysis was carried out on 30 fiL of the supernate obtained after centrifugation.

Lymphocytes were isolated from human blood collected with EDTA as anticoagulant. Packed lymphocytes (10 fiL) were resuspended in 225 fiL of ice-cold sodium 5,5-diethylbarbiturate-HCL buffer (pH 8.4). After addition

Figure 9-53 Determination of lymphocyte kynureninase activity levels using HPLC. Enzyme activity is measured by quantification of formation of the product, 3-hydroxyanthranilic acid (3-HAA). (A) 3-HAA standard (12.0 nmol/L). (fl), Lymphocyte homogenate blank. (C) Lymphocyte 3-HAA production after 5 min of incubation in presence of 3-hydroxy-kynurenine. Peaks: 1,3-HAA; unmarked peaks are unidentified components . (From Ubbink et al., 1991.)

Figure 9-53 Determination of lymphocyte kynureninase activity levels using HPLC. Enzyme activity is measured by quantification of formation of the product, 3-hydroxyanthranilic acid (3-HAA). (A) 3-HAA standard (12.0 nmol/L). (fl), Lymphocyte homogenate blank. (C) Lymphocyte 3-HAA production after 5 min of incubation in presence of 3-hydroxy-kynurenine. Peaks: 1,3-HAA; unmarked peaks are unidentified components . (From Ubbink et al., 1991.)

of 25 nL of 10% Triton X-100, the suspension was sonicated to produce the homogenate.

Figure 9.53 shows chromatograms.

Was this article helpful?

0 0

Post a comment