Photometric determination of enzymatic activity

Enzymatic assays of intestinal alkaline phosphatase (iAP), xanthine oxidase (XOD) and glutathione-S-transferase (GST) were analyzed by means of photometric methods.

3.3.1. General methodology

Enzymatic activities were determined using a SLT Spectra plate reader (SLT Instruments, Crailsheim, Germany). Assay concentrations and additives, i.e. inhibitors, PF extracts or organic solvents can be found in Table 3. In all cases, assay components except for the enzyme were pipetted into a reaction tube or directly into a 96-well plate. Reaction was then started by addition of the respective enzyme. Unless stated otherwise all assays were conducted in 10 mM NH₄Ac pH 7.4. Suitable control measurements were performed in order to verify the absorption signal changes are due to enzymatic activity. These included the detection of all assay component combinations without the presence of the respective enzyme as well as the determination of assay formulation background absorption.

26 Table 3 Overview of photometric determination of iAP, XOD and GST assays (ACN = acetonitrile, IPA = isopropyl alcohol, ACE = acetone).

Formulation of stock solutions Assay concentrations Additives Enzyme Substrate(s) Enzyme Substrate(s) Inhibitor / Extract /

Solvent

Data were analyzed using Microsoft Office Excel 2007 (Microsoft Inc). Data interpretation was conducted by comparing the slopes of trend lines, which were applied within the linear range of either substrate degradation or product generation curve at the onset of the enzymatic assay detection. Possible enzymatic activity changes in the presence of e.g.

organic solvents or PF extracts were assessed by comparing assays containing those additives with the respective control assays without additives. Average values of control

27 assays were set as 100%. Further details can be found in the Materials & Methods chapter of Appendix I.

3.3.2. Intestinal alkaline phosphatase

Activity of iAP was determined with its physiological nucleotide substrates ATP, ADP and AMP in 10 mM NH₄Ac pH 6.0, 7.4 or 9.0. The enzymatically released inorganic phosphate (Pi) was detected by means of a color reaction at seven time points within 90 min. Detailed information about chemicals, assay procedure, instrumentation, data evaluation and statistics can be found in Appendix I.

3.3.3. Xanthine oxidase

Xanthine oxidase activity was determined either in 200 mM phosphate buffer pH 7.4 or 10 mM NH4Ac pH 7.4 using photometric detection. In order to estimate the amount of nitrite generated by the reaction of NH2OH and the secondary enzymatic product superoxide a calibration curve was prepared beforehand.

Calibration

Calibration was performed by preparing NaNO₂ stock solutions (Sigma-Aldrich) in 10mM NH₄Ac pH 7.4 or 200 mM phosphate buffer pH 7.4 ( ≥ each) (Table 4). 80 µL of the respective NaNO₂ concentration was added to 80 µL 0.02% sulfanilamide (Merck Chemicals, Darmstadt, Germany) solution followed by the addition of 80 µL 1% NED solution (Merck Chemicals) to a 96-well plate. After thorough mixing, the plate was incubated in the dark for 15 minutes at room temperature. The absorption was determined at 492 nm.

Table 4 Overview of chemicals and concentrations utilized for calibration measurements

Chemicals Solved in Final concentrations

NaNO2 10 mM NH4Ac pH 7.4

or 200 mM phosphate buffer pH 7.4

100, 50, 25, 12.5, 6.25, 3.13 1.56, 0 µM

Sulfanilamide 0.2 mg/mL (0.02%)

8% HCl 0.067 mg/mL

N-(1-naphthyl)ethylendiamine (NED) 10 mg/mL (1%)

H₂O (bidest.) 3.33 mg/mL

28 Assay detection

XOD activity was determined in aqueous solution 10 mM NH₄Ac pH 7.4 or 200 mM phosphate pH 7.4, respectively, by detecting enzymatically generated nitrite after 30 minutes with a XOD concentration of 0.0125 U/mL and 0.04 U/mL XOD (solely in 10 mM NH₄Ac pH 7.4 ≥ . Generated nitrite quantitywas calculated by means of the respective calibration curves either prepared in 200 mM phosphate buffer pH 7.4 or 10 mM NH₄Ac pH 7.4.

Progress of xanthine degradation was furthermore assessed by means of capturing the generation of nitrite in 10 mM NH₄Ac pH 7.4 at time points 0, 20, 40 and 60 minutes. After starting the enzymatic reaction, the assays were incubated at 37°C. 80 µL of the respective assay corresponding to the time point of interest were withdrawn from the tube and added to 80 µL 0.02% sulfanilamide to stop the reaction. The addition of 80 µL 1% NED solution then started the color reaction. The plate was incubated in the dark at room temperature for 15 minutes and the absorption was measured at 492 nm. Controls containing xanthine substrate and/or NH2OH but not the enzyme and vice versa were performed to control the results.

3.3.4. Glutathione S-Transferase

GST activity was investigated using CDNB and GSH as substrates. The impact of different organic solvents and solvent concentrations as well as increasing concentrations of PF extracts on the generation of the enzymatically generated GSH-CDNB conjugate was tested.

Due to its hydrophobicity CDNB stock was solved in 100% EtOH. Consequently all GST assays contain a small proportion of 2.5% EtOH, which was kept constant in all experiments.

Determination of enzyme kinetics

GST kinetics was determined by measuring various concentrations of CDNB with a constant concentration of GSH and vice versa (Table 3). Data were plotted as Lineweaver-Burk diagram with 1/v corresponding to the slopes of linear trend lines applied within the initial linear range of substrate generation.

29 GST assay in the presence of organic solvents

10, 20 and 30% of either MeOH, acetonitrile (ACN), isopropyl alcohol (IPA), acetone (ACE) or EtOH were added to the assay and the activity was determined in comparison to standard assays in 10 mM NH₄Ac pH 7.4.

GST assay in the presence of PF extracts

PF extracts used were prepared by means of extraction method 1 (Table 1). For the conduction of GST assays two extracts per treatment (water, 90% EtOH or 90% MeOH, 0.5%

FAc) were redissolved in 200 µL 80% MeOH each and pooled. Thus extract of 40 mg freeze dried and milled PF leaves was redissolved in 400 µL 80% MeOH (≙ g / µL . Since the final GST assay volume was 250 µL, the addition of 0.2, 0.5, 0.8 or 1.0 % (v/v) redissolved PF e t a t g /400 µL corresponded to extract prepared from 0.05 mg, 0.1025 mg, 0.2 mg or 0.25 mg freeze-dried & milled leaves respectively. Control experiments included the assessment of GST activity in the presence of the respective proportions of 80% MeOH as well as the progress of absorption of GSH and CDNB individually and combined in the absence of the enzyme.