Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis

Abstract

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL −1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL −1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors.

Water-Based Extraction of Bioactive Principles from Blackcurrant Leaves and Chrysanthellum americanum: A Comparative Study

“…Among 10 major peaks detected in CA (see Figure 4 and Table 5 ), one of them (peak 1, Chlorogenic acid) was unambiguously identified by comparing the UV spectra maximum values, the retention times, and ESI-MS(-), MS/MS data with the reference standards used in our previous study [ 4 ]. The peaks 8 a , 8 b , 9, 10, 11, 12, 13, 14, and 15 were tentatively identified, respectively to: Eriodicyol-7-O-glucoside (8 a ), 6,8-C,C-Diglucosylapigenin (8 b ), Isookanin-7-O-glucoside (Flavanomarein) (9), Maritimetin-6-O-glucoside (Maritimein) (10), Luteolin-7-O-glucuronide (11), Di-caffeoylquinic acid (12), Apigenin-7-glucuronide (14), Di-caffeoylquinic acid isomers (13, 15); based on the data from the literature [ 17 , 32 , 33 , 34 , 35 , 36 ] and their fragmentation patterns from ESI-MS(-), MS/MS data as presented in Table 5 . Compound peaks 8 a , 9, and 10 have been described by Shimokoriyama and Honore-Thorez [ 18 , 47 ] and those peaks showed UV maximum absorption bands at 282, 284, and 414 nm, respectively, very similar to Eriodicyol-7-O-glucoside, Flavanomarein, and Martitimein, respectively.…”

“…The effects of the different plant extracts were evaluated towards hyaluronidase activity using an optimized protocol based on a CE/PDA assay previously described by some of us [ 33 ]. Briefly, 25 µL of incubation buffer was preincubated with 10 µL of HA (at 4 mg mL −1 ) and 10 µL of plant extract (at 5 mg mL −1 ) at 37 °C for 10 min.…”

“…The final concentration of the enzyme in the mixture was 0.2 mg mL −1 . Reactions were incubated for 180 min at 37 °C, then they were stopped by increasing the temperature to 90 °C for 10 min using a water bath [ 33 , 34 ]. Results were compared to those obtained with a referenced inhibitor of hyaluronidase (EGCG) at 1 mg mL −1 in the mixture [ 34 , 35 ].…”

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments

“…In our previous work [13], the kinetics of BTH was followed at two HA concentrations (0.3 and 1 mg/mL). In addition, for each HA concentration, the reaction mixture contained different quantity of the PEG crowder (0, 10, 17 % w/w).…”

“…Recently, we have investigated the degradation of HA catalyzed by Bovine testicular Hyal (BTH) in the presence of PEG 6000 crowders using a CE-based enzymatic assay [13]. BTH specifically cleaves the β 1,4-glucosaminidic bond between C1 of the glucosamine moiety and C4 of the glucuronic acid [14] and the reaction products (oligomers) thus contain 2m monomers (m =1,2,3 …).…”