3.2.1 Materials
• Photometer: An Aminco DW-2 dual beam spectral photometer equipped with a magnetic stirrer and connected to a computer.
• HPLC-systems:
I) Jasco components: PU-980 pump, UV-975 fast scanning detector, LG-980-02 low pressure mixer. Solvent degaser unit SDU 2003 from Lab-source and helium pressure. This system was connected to a computer with the Borwin-HPLC-software.
II) LKB components: 2150 pump, 2152 LC controller. Spectra Physics components: spectra focus forward scanning detector, SP4290 integrator.
Equipped with helium pressure.
• Chemicals were purchased from Merck, Sigma, Lancaster or Aldrich and were from the highest purity available. The dimethyl-uric acid derivatives and dithio-purine and -pyrimidine were purchased from Sigma, Germany.
Ebselen was a kind gift from Prof. Wendel (Universit¨at Konstanz). TEM-PONE and TEMPO were purchased from Alexis.
3.2.2 Inhibitors of Phenol Nitration
5 mM phenol and 800 µM PN were incubated in 0.5 M K-phosphate buffer pH 7 at 25 ◦C. The formation of 2- and 4-nitro-phenol was followed photometrically at 405 nm (after addition of 20µl saturated NaOH to 980µl reaction solution) or by separation of 2- (tR=28 min) and 4-nitrophenol (tR=16 min) by HPLC (system II, 1 ml/min, 30 % acetonitril and 75 % 0.1 M citrate buffer pH 2, Bischoff C18-Nucleosil-100-5 250x4.6, detection at 280 nm). For the determination of the IC50-values, the scavengers were added before addition of PN by vortex-mixing and IC50-values were the concentrations of scavengers, where the 405 nm absorbance or nitro-phenol peakarea reached the half amount compared to the reaction without scavengers.
In another experiment 5 mM phenol were incubated either with 655 µM PN in 0.2 M K-phosphate buffer pH 6 (1 min) or with 400 µM PN at pH 9 (10 min) and 37 ◦C. At pH 6 the formation of 2- (tR=15.6 min) and 4-nitro-phenol (tR=12.8 min) was monitored by HPLC (system I, 1 ml/min, 10 % acetonitril and 90 % 0.05 M citrate buffer pH 2, Macherey Nagel C4-Nucleosil-300-5 250x4.6,
3 MATERIALS AND METHODS 36 detection at 280 nm). At pH 9 the formation of 4-nitroso-phenol (tR=11 min) was either followed photometrically at 395 nm or by HPLC (system I, 1 ml/min, 10 % acetonitril and 90 % 0.05 M citrate buffer pH 2, Macherey Nagel C18 -Nucleosil-100-5 250x4.6, detection at 300 nm). Once more the IC50-values were estimated as described above.
3.2.3 Inhibitors of Microperoxidase-catalyzed Phenol Nitration The same method as in the phenol system at pH 6 was used, but with 5 µM microperoxidase (MP-11) (a porphyrin with 11 amino acids originating from the degradation of cytochrome c). MP-11 like other heme containing enzymes and iron-porphyrins leads to a strong increase in nitration of phenol. No IC50-values were estimated, but the effect of 100 µM scavenger on nitration was monitored using HPLC.
3.2.4 Inhibitors for the Inactivation of ADH by PN
The activity of 26 nM ADH with 300 µM NAD+ for the conversion of 172 mM ethanol to acetaldehyde in 0.1 M K-phosphate buffer pH 7.6 was followed spec-troscopically at 37 ◦C. The formation of NADH at 340 nm was measured time dependently and the basic specific activity was calculated from the slope of the linear area (first 25 sec) and was found to be 0.044±0.007 U/mg. In a next step the amount of PN was estimated that led to a complete inhibition of the en-zyme.In this case the ethanol and NAD+ were added after complete reaction of ADH with PN (5 min) and the activity was measured. 20 µM PN quantitativly inhibited the enzyme, therefore this value was used for the following experiments.
In a following step we determined the IC50-values of several PN-scavengers in this systems, by addition of different concentrations to the enzyme, prior to addition of PN by vortexing carefully. Subsequently the activity was determined. The IC50-value given for the scavenger corresponded to the concentration, that inhib-ited 50 % of the ADH-activity.
3.2.5 Kinetics of PN-decomposition
Decay of PN was followed by UV/Vis spectroscopy at 302 nm and the reference wavelength at 400 nm. The samples were measured in a special cuvette which was equipped with a magnetic stirrer. Because of inadequate mixing during the first 2-3 sec the decomposition curves were only compared qualitatively and no kinetic constants could be obtained from these measurements. 400-800 µM PN were injected into a stirred pH 7, 8 or 9 buffer solution which contained the
scavenger in different concentrations. The velocity of decay was then compared with the one in controls.
3.2.6 Investigation of the Reaction of Uric Acid with PN
In a first attempt we chose dimethyl analogues of UA, because they were easier to handle on HPLC and 13C-NMR-data should be easier to interprete, due to the methyl signals. 2 mM 1,3-dimethyl-uric acid were incubated with 8 mM PN in 1 M K-phosphate buffer pH 7. The reaction solution (1 ml) was injected on system II equipped with a semipreparative column (4 ml/min, A: 0.2 M KCl/HCl buffer B: 2-propanol, gradient: 0-10 min 100 % A, 10-23 min linear to 35 % B, 23-27 min linear to 0 % B, Merck C18-LiChrosorb-7 250x10, detection at 210 nm). The products were isolated from 20 runs, each of them concentrated and purified once more with the same HPLC-system, but water as the mobile phase. This time the isolated peaks were evaporated to dryness and dissolved in D6-DMSO. From this solutions 1H- and 13C-NMR (also one DEPT) spectra were made on a Bruker NMR DRX 600, the frequence for 1H was 600MHz, for
13C 150 MHz. Also EI-MS and FAB-MS spectra were made on a Varian MAT 312 respectively Varian MAT 312/amd 5000 (EI: 3 kV / 70 eV; FAB: primary ions (133Cs) 10 kV, secondary ions 6 kV).
In a second attempt 0.5-1 mM uric acid were incubated with 250-2000µM PN in 0.2 M K-phosphate buffer pH 6-9 (1-10 min). 20 µl of the reaction solutions were injected on system I (0.7 ml/min, 0.2 M K-phosphate buffer pH 4.5, two Macherey Nagel C18-Nucleosil-100-5 250x4.6 columns connected in series, detec-tion at 220 nm). The HPLC system was modified from the descripdetec-tion in [205].
From each product a UV spectrum was taken and the already described oxidation products of uric acid (allantoin, allantoic acid, alloxanthin, oxonic acid, cyanuric acid and alloxan) were used as external and internal standards [206, 207]. Some reaction solutions were additionally incubated with uricase, to see whether some peaks dissapeared, which could mean that the basic structure was recognized by uricase. Allantoin and allantoic acid were estimated by hydrolysis (0.12 M NaOH) of allantoin to allantoate, further hydrolysis to glyoxylic acid and its cou-pling to 2,4-dinitro-phenyl-hydrazine and identification of the coucou-pling product on HPLC as described [208]. Urea was quantified by the method of urea degra-dation to ammonia by urease and the colorimetric measurement of the ammonia-pH-shift with phthalein purple (o-cresol-phthalein complexone) [209] or by the o-phthaldialdehyde method [210]. Reagent A consisted of 30 mM Tris, 5 mM
3 MATERIALS AND METHODS 38 complexone, 10 mM edta, 30.8 mM sodium azide and the pH was adjusted to 7.4. Reagent B consisted of 150 mM sodium chloride and 20 U/ml urease (Sigma, U1500) in water. For assays 250 µl sample were mixed with 125 µl reagent A and/or 125 µl reagent B respectively in assays without urease, the amount of reagent B was replaced by equal amounts of 150 mM NaCl-solution.
3.2.7 Reaction of Ebselen withPeroxynitrite
3.2.7.1 Materials Two HPLC systems were used: I) Jasco PU-980 pump, UV-975 variable-wavelength detector and LG-980-02 low-pressure gradient mixer.
II) LKB 2150 pump, Spectra Physics spectra focus detector and SP4290 integra-tor. In both systems solvents were gassed with helium. Kinetics were recorded on an Aminco DW-2 UV/Vis spectrophotometer in the dual-wavelength mode, equipped with a magnetic stirrer.
3.2.7.2 Separation of ebselen, GSH-ebselen and ebselenoxide System I was used with a C4-Nucleosil-300-5 column 250x4.6 (Macherey-Nagel, D¨uren, Germany), the mobile phase consisted of 80 % 0.1 M K-phosphate buffer pH 4.5 and 20 % acetonitrile. The flow was 1.2 ml/min and the products were detected at 270 nm. Spectra were taken from each peak. Solutions of 500 µM ebselen (from a 10 mM stock solution of ebselen in acetonitrile) in 0.1 M K-phosphate buffer at pH 7 and 9 were treated with different concentrations of GSH (0.05-5 mM) and were then reacted with 500 µM PN. 50 µl of each sample were injected on the HPLC.
3.2.7.3 Determination of free ebselen concentrations in bovine aortic microsomes and tissue-containing solutions System II was used with a C4 -Nucleosil-300-5 column 250x4.6 (Macherey-Nagel, D¨uren, Germany), the mobile phase consisted of 80 % 0.1 M K-phosphate buffer pH 4.5 and 20 % acetonitril for the tissue-samples (1) and 75 % water and 25 % acetonitril with 0.1 % added trifluoroacetic acid in the microsomal experiments (2). The flow was 1.2 ml/min and the products were detected at 270 nm. (1) 4.3 µM ebselen were incubated with 0.134 g dry weight (first sample) and 0.077 g (second sample) of bovine coronary tissue in 10 ml PBS-buffer and aliquotes of 50 µl were taken after 1, 15, 30 and 35 min and injected on HPLC. Ebselen and GSH-ebselen were identified by external standards. (2) 500 µl bovine aortic microsomes (1 mg/ml protein) were incubated with 500 µl 1 mM DTNB for 10 min in 0.2 M K-phosphate buffer pH 7.5. The solution turned yellow, indicating the reaction of DTNB with free thiol-groups. After centrifugation the supernatant was removed and 1 ml
fresh K-phosphate buffer was added. The sample was vortexed and then 20 or 50 µM ebselen were added and incubated for 5 min. 50 µl of this solution were injected on HPLC. The same procedure was performed for the non-DTNB-treated microsomes. Concentrations of ebselen were quantified by external standards.
3.2.7.4 Protective effects of ebselen, GSH and ebselen-GSH on phenol- and BSA-nitration Solutions of 5 mM phenol in 0.1 M K-phosphate buffer at pH 7 or 9 were supplemented with ebselen, GSH or both and were re-acted with 1 mM PN. The yield of nitro-phenols (pH 7) or 4-nitroso-phenol (pH 9) was determined from the absorbance at 400 nm (after addition of NaOH(aq)).
PN- and ebselen decomposition kinetics were recorded at 320 against 370 nm.
At this wavelength PN as well as ebselen absorb, but not ebselenoxide and the ebselen-GSH-adduct has a very low absorbance. PN was always added last to the stirred solution in the cuvette by a syringe through a septicum. The de-composition of 100 µM PN and 50 µM ebselen with 0, 20 or 40 µM BSA in 0.2M K-phosphate buffer at pH 10 was recorded and also the decomposition of 250 or 500 µM PN and 200 µM ebselen with 0, 200 and 400 µM GSH in 0.2 M K-phosphate buffer pH 8, 9 and 10.