Enzymatic activity in the presence of regulatory compounds

The regulation of enzymes involved in the development and progress of diseases is a common therapeutical means for a successful alleviation, e.g the suppression of XOD activity with allopurinol for the treatment of gout [87]. Inhibition kinetics are usually investigated via

54 well-established and robust photometric approaches, which also offer the possibility for an automatized HTS. Those methods however lack important information in terms of observing all assay components. Therefore efforts have been made to use MS detection for the sensitive and quantitative HTS of enzymatic regulators [20, 21, 240, 241]. By utilizing MS detection a more comprehensive picture of enzymatic regulation can be obtained. This is of special interest with regard to multi-component assays, which include the generation of several intermediates (Figure 11).

Figure 11 Direct comparison of individual iAP assay traces ATP, ADP, AMP and Ado in the presence (dotted traces) and in the absence (solid traces) of 200 µM GSH (left) as well as determination of IC₅₀ value with increasing GSH concentrations (right). Assay composition was as follows: iAP assay with 0.2 U/mL iAP and 40 µM ATP in 10 mM NH4Ac pH 7.4.

The stepwise ATP substrate dephosphorylation by iAP results in the generation of ADP and AMP intermediates as well as the final enzymatic product adenosine. Changes in the progress of individual nucleotide traces could be continuously followed, whereupon a distinctly decelerated degradation of ATP and ADP as well as generation of ADP, AMP and

55 concentrations to iAP assays revealed an IC50 of about 200 µM with the here applied conditions (Figure 11, right).

Besides the investigation of effects of single molecules on enzymatic activity, the addition of complex mixtures like e.g. plant extracts to enzymatic assays provides insight into possible regulatory effects. Although drawing conclusions about individual regulators is not possible in this case, the addition of a plant extract of interest to an enzymatic assay is an easy means to assess a mixtures regulatory potential. In this regard PF extracts, prepared with different organic solvents, were added to photometrically measured GST assays in 4 different concentrations (Figure 12, A). In contrast to EtOH extract, which lacked inhibitory effects, water and MeOH extracts were found to distinctly suppress GST activity. Due to the polarity of extraction solvents used, GST is inhibited rather by polar than moderately polar PF compounds as they would be present in the here applied non-inhibitory EtOH extract.

Rohman et al. investigated the activity of different GSTs in the presence of onion bulb extracts and detected the most pronounced effects in the presence of a polar water extract compared to only minor inhibition caused by a non-polar hexane extract [242]. In contrast, no correlation between compound polarity and regulatory activity was found by Iio et al., who detected a distinct GST inhibition after addition of individual and mainly poorly water-soluble flavonols and flavones to the assay [243].

However, as fast and easy photometric determination of enzymatic activity may be, it can also lead to ambiguous results. The addition of complex mixtures or even individual compounds might be capable of interfering with photometric assay detection, either by overlaying absorption of mixture compounds at the wavelength of assay detection or by affecting the formation of a colored complex. Moreover, considering the photometric investigation of XOD assay, a distinction between an actual enzymatic inhibition and the scavenging of released superoxide by extract molecules is not feasible with the applied assay procedure. MS detection, which directly observes xanthine substrate and uric acid product may therefore help to elucidate enzymatic action without relying on the release of superoxide (Figure 10, A(2)). Xanthine oxidase assay was therefore measured in the presence of increasing concentrations of VS PF water extracts. Data evaluation revealed an extract concentration-dependent enzymatic inhibition (Figure 12, B), which is reflected by decreasing substrate degradation and product generation.

56 Figure 12 A: Photometrically determined GST activity with 0.1 U/mL GST, 0.2 mM GSH, 1 mM CDNB in 10 mM NH₄Ac pH 7.4 without extract (left dark grey colum) or 0.2, 0.5, 0.8 or 1.0% (v/v) of extract redissolution solvent 80% MeOH as controls = MeOH [%] or 0.2, 0.5, 0.8 or 1.0% (v/v) water, 90% EtOH or 90% MeOH, 0.5% FAc PF extract (= ate e t a t [%] , EtOH e t a t [%] o MeOH e t a t [%] respectively). PF extracts were prepared using method 1 described in Table 1. B: Mass spectrometrically determined xanthine degradation and uric acid generation. Depicted columns represent the slopes of trend lines applied to the initial linear decrease (xanthine) and increase (uric acid). Assay composition were as follows, 0.004 U/mL XOD and 25 µM xanthine in 90% 10 mM NH₄Ac pH 7.4, 10% IPA (v/v) in the presence of increasing quantities of VS PF water extract redissolved in water in a total assay volume of 500 µL. PF extract quantities given here refer to the weight of the powder, which is obtained after extraction and extraction solvent evaporation.

Finding promising enzymatic regulation after applying a complex mixture consequently results in the isolation of single compounds, which are then further tested for their individual regulatory potential. The procedure mainly employed for this purpose is the so-called bioassay guided fractionation (Appendix III, Figure 5). This technique has been used e.g. by Huo et al. for the isolation of PF molecules, whereupon they were added to XOD assay to assess their impact on the enzymatic activity, which resulted in the finding of several inhibitory compounds including caffeic acid, rosmarinic acid and apigenin [244]. The employed procedure usually involves repeated fractionation, followed by the fractions exposure to the enzyme. Therewith the quantity of extracts compounds can be gradually narrowed down to eventually isolate enzyme-regulatory substances [191].

0 20 40 60 80 100 120 140

0.2 0.5 0.8 1.0 0.2 0.5 0.8 1.0 0.2 0.5 0.8 1.0 0.2 0.5 0.8 1.0 Assay MeOH [%] EtOH extract [%] MeOH extract [%] Water extract [%]

Relative product formation [absorption*time-1]

A BB

-150 -100 -50 0 50 100 150

0 1.25 2.5 6.25 12.5 25 50 Relative xanthine degradation / uric acid generation [intensity*time-1]

VS Perilla water extract in assay [µg]

Xanthine Uric acid Xanthine Uric acid

57