Colorimetric assay – Morgan-Elson assay

For the investigation of the potential enzyme inhibitors described in this thesis, the hyaluronidase activity was determined by a modified colorimetric assay, which is based on the method ofGacesa et al. (Gacesa et al. 1981) and Reis-sig et al. (ReisReis-sig et al. 1955) and reported in detail byMuckenschnabel et al.

(Muckenschnabel et al. 1998). The colorimetric assay (Reissig assay, Morgan-Elson assay) is based on the reaction of theN-acetyl-D-glucosamine (GlucNAc) at the reducing ends of hyaluronan and its fragments with p-dimethylamino-benzaldehyde resulting in a red coloured product.

As shown in Fig. 1, the postulated main product of the degradation of hyaluro-nic acid by the bovine testicular hyaluronidase is a tetrasaccharide with N-acetyl-D-glucosamine at the reducing end. The chromogens I and II are formed under alkaline conditions (100 °C, pH 9) of the Morgan-Elson reaction.

The chromogen III, which is formed by elimination of water under acidic condi-tions (conc. HCl / glacial acetic acid) react in the final step with p-dimethyl-aminobenzaldehyde (Ehrlich´s reagent) to give the instable red - coloured product, which can be photometrically measured at 586 nm. The described structure of the red product was postulated by Muckenschnabel et al. in 1998 (Muckenschnabel et al. 1998) on the basis of HPLC-MS investigations.

Methods and assays for the determination of hyaluronidase activity at the reducing end of sugars

Fig. 1: Mechanism of the Morgan-Elson reaction - reaction of the N-acetyl-D-glucosamine with p-dimethylaminobenzaldehyde resulting in the red coloured product postulated byMuckenschnabel et al. (Muckenschnabel et al. 1998).

Methods and assays for the determination of hyaluronidase activity

25

Effect of organic solvents on the enzyme activity

Our examined compounds were not soluble in the citrate-phosphate buffer, but soluble in organic solvents such as dimethylformamide, dimethylsulphoxide, methanol and ethanol. To find the appropriate solvent and the tolerated con-centration bovine testicular hyaluronidase and hyaluronate lyase were investi-gated in the presence of different concentrations of various organic solvents.

For this purpose the activities of the two enzymes were determined as a func-tion of the solvent concentrafunc-tion in the incubafunc-tion mixture. The effects of the solvents on the bovine testicular hyaluronidase are shown in Fig. 2. Whereas for all four solvents concentrations were accepted up to 4% higher concentra-tions resulted in weak or strong inhibition of the enzyme depending on the type of solvent.

0 20 40 60 80 100

2 3 4 5 10 15 22,2

[%] (v/v) organic solvent

Activity[%]

DMF DMSO MeOH EtOH

Fig. 2: Effect of DMF, DMSO, MeOH and EtOH on the activity of the bovine testicular hyaluronidase at optimum pH (3.6)

The effects of the four organic solvents on the hyaluronate lyase are shown in Fig. 3. Whereas the inhibitory effect of dimethylsulphoxide, methanol and ethanol at concentrations up to 4 % were negligible, concentrations higher than 5 % affected the enzyme activity negatively. The inhibitory effect of DMF was already significant at a concentration of 3 %.

Methods and assays for the determination of hyaluronidase activity

26

0 20 40 60 80 100

2 3 4 5 10 15 22,2

[%] (v/v) organic solvent

Activity[%]

DMF DMSO MeOH EtOH

Fig. 3: Influence of the solvents DMF, DMSO, MeOH and EtOH on the activity of the bacterial hyaluronidase at optimum pH (5.0)

Because of the good solubility of the test compounds in DMSO and the low in-hibitory effect of DMSO (< 4%) on the two enzymes, dimethylsulphoxide was used as solvent for pharmacological investigations.

Measurement of hyaluronidase activity

The procedure of the Morgan-Elson assay described in the literature was scaled down to minimise the required amounts of test compounds and en-zymes. The enzyme concentrations and the incubation periods were optimised both for investigations at pH optimum and physiological pH. The incubation mixture, the enzyme concentrations, the incubation periods etc. of the modified assay are described in the following.

The test compounds (0.1 µM - 20 mM) dissolved in DMSO (7 µl), were incu-bated at 37 °C in an incubation mixture containing 60 µl of citrate-phosphate buffer (solution A: 0.1 M Na2HPO4 / 0.1 M NaCl, solution B: 0.1 M citric acid / 0.1 M NaCl; solution A and B were mixed in appropriate portions to adjust the required pH), 40 µl BSA solution (0.2 mg BSA per ml of water), 20 µl substrate solution (2 mg or 5 mg hyaluronic acid from rooster comb or Streptococcus zooepidemicus per ml of water), 33 µl H2O and 20 µl enzyme solution (equiac-tive concentration: 8 IU BTH (pH 3.6), 40 IU BTH (pH 7.4), 0.4 IU hyalB4755(pH

Methods and assays for the determination of hyaluronidase activity

27

5.0 and 7.4)). The pH of the incubation mixture was adjusted to the pH opti-mum of the enzymes (for BTH pH value of 3.6 and for hylB4755pH value of 5.0) and to physiological pH (7.4). The incubation time varied with the pH of the in-cubation mixtures: an inin-cubation period of 1.5 h at optimum pH and 3 h at physiological pH for both enzymes.

The enzyme reaction was stopped by addition of 45 µl of alkaline borate solu-tion and subsequent heating for 4.5 min in a boiling water bath. The alkaline borate solution was prepared immediately before use from the borate solution (17.3 g H3BO4 and 7.8 g KOH in 100 ml water) and the potassium carbonate solution (8.0 g K2CO3 in 10 ml water). After cooling on ice for 1 min 600 µl of N,N-dimethylaminobenzaldehyde (20.0 g N,N-dimethylaminobenzaldehyde dis-solved in 25 ml concentrated hydrochloric acid and 75 ml glacial acetic acid; the solution was diluted with 4 volumes of glacial acetic acid immediately before use) was added and the mixture was incubated at 37 °C for 20 min. The re-sulting solution was transferred to 96 well plates and the absorbance of the col-oured product was photometrically measured at 590 nm.

Enzyme activity was calculated from the formation of the red coloured product measured at 590 nm. The effect of the inhibitors on the enzyme activity was calculated according to the equation:

A % = (B - C) / (D - E) A: calculated enzyme activity

B: absorbance of the incubation mixture containing inhibitor

C: absorbance of the incubation mixture containing inhibitor in absence of the enzyme (enzyme solution replaced with buffer)

D: absorbance of the incubation mixture in absence of the inhibitor (inhibitor solution replaced with DMSO)

E: absorbance of the incubation mixture in absence of both enzyme and in-hibitor (enzyme solution replaced with buffer, inin-hibitor solution replaced with DMSO)

Methods and assays for the determination of hyaluronidase activity

28

The IC50 ± SEM values were calculated using the standard curves analysis of SigmaPlot^TM (version 8.0) and are the means of three independent experiments performed in duplicate.

The Morgan-Elson assay is a useful method for the determination of hyaluroni-dase activity in the presence of inhibitors. With respect to the required incuba-tion times, the assay is practicable and reproducible for the quantitaincuba-tion of en-zyme activities. However, not all compounds can be examined with this assay.

For instance, this assay is not suitable for the investigation of indole derivatives since the indole ring reacts with the Ehrlich´s reagent (p-dimethylaminobenz-aldehyde) to a coloured product which is also detectable at 590 nm and falsifies the quantification of the red coloured product of the Morgan-Elson reaction (cf.

chapter 9)