Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii
Article Category: Original Article
Published Online: Jul 02, 2020
Page range: 153 - 164
Received: Dec 04, 2019
Accepted: Mar 03, 2020
DOI: https://doi.org/10.2478/jas-2020-0015
Keywords
© 2020 Mohammed M. Abdel-Monsef et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Bee keeping in Egypt has been an important type of farming since ancient Egyptian. Bee venom products are important in the pharmaceutical industry and drug formulations. The venom produced by the Egyptian honey bee (
Hyaluronidase is the major allergen in the venoms of bees, hornets, wasps and scorpions that stimulate lethal systemic IgE-intermediated anaphylactic responses in humans (Kolarich et al., 2005). In microbes, hyaluronidases are virulence agents included in pathogenesis and disease advancement brought on by pathogens. The hyaluronidase degradation of the host tissue components facilitates the infestation of pathogens. Increased permeability of tissues appears to play a principle role in wound infections, meningitis, pneumonia and bacteremia (Matsushita & Okabe, 2001; Makris et al., 2004). Additionally, hyaluronidase increases membrane permeability, so therapeutically it is used to enhance injected fluid absorption and excess-fluid resorption, increase local anesthesia effectiveness and decrease tissue destruction in subcutaneous and intramuscular injection (Farr et al., 1997).
The hyaluronidase of sperms plays an important role in fertilization success in humans and mammals (Klocker et al., 1995). Mammalian sperm hyaluronidase is located on the sperm surface, where it facilitates the penetration through the ovum and is also necessary for fertilization (Zheng et al., 2001). It can be used to decrease myocardial infarction (Muckenschnabel et al., 1996). Hyaluronidase may have essential anticancer impacts and may repress tumor growth, treatment with it was stated to block tumor cells invasion to lymph node in T cell lymphoma (Zahalka et al., 1995). It was used as a chemotherapeutic-drug additive for anticancer activity augmentation (Baumgartner, 1998). The hyaluronidases of bee venom preparations have been used successfully in medicine combined with anticancer drugs (Moga et al., 2018) and for scar resorption in treating wounds and burns of skins and mucosa surfaces (Krylov, 1995).
There have been studies on the hyaluronidase from the venom of the honey bee (Gmachl & Kreil, 1993; Topchiyeva & Mammadova, 2016), the scorpion
The colonies of honeybees
Hyaluronic acid, cetyltrimethylammonium bromide, alcian blue, phenymethylsulfonyl-fluoride (PMSF), 1,4 dithiothreitol (DTT), 1,10 phenanthroline, sephacryl S-300, bovine serum albumin (BSA), blue dextran, gel filtration kits for molecular weight marker, (DEAE-cellulose) diethylaminoethyl cellulose and standard markers mixture
The activity of hyaluronidase is measured turbidometrically according to Pukrittayakamee et al. (1988). The assay mixture consisted of 0.5 ml 0.2 M acetate buffer, pH 5.5 containing 0.15 M NaCl, 50 mg hyaluronic acid and venom extract. It was incubated for fifteen minutes at 37°C then stopped by adding 1 ml of 2.5% cetyltrimethylammonium bromide in 2% NaOH and absorbance was read at 400 nm. Turbidity reducing units (TRU) were defined as the amount of hyaluronidase necessary for hydrolyzing 50% of hyaluronic acid. One unit was the amount of hyaluronidase that caused 50% reduction in turbidity.
Samples were electrophoresed on 7% native-PAGE that co-polymerized with 0.17 mg/ml of hyaluronic acid. Following electrophoresis, gel was incubated in 20 mM Tris-HCL buffer, pH 7.5, containing 150 mM NaCl for three hours at 37°C, followed by incubation in 100 mM sodium acetate buffer pH 4.0 overnight at 37°C. Gels were then stained for two hours at room temperature with 0.5% alcian blue in 3% acetic acid and destained in 70% acetic acid. The activity in gel was detected by a lack of color in the digested area against a blue background distinctive for undigested hyaluronic acid (Guntenhöner, Pogrel, & Stem, 1992).
Crude venom extract underwent chromatography on DEAE-cellulose column (12 cm × 2.4 cm i.d.) equilibrated previously with 0.02 M Potassium phosphate buffer pH 7.4. The adsorbed proteins were eluted with NaCl stepwise gradient (0 – 1 M) in the preceding buffer at 60 ml / hr flow rate. Fractions of 3 ml volumes were collected and those containing hyaluronidase activity were combined and concentrated.
The DEAE-cellulose concentrated proteins of the peak including hyaluronidase activity were loaded to a Sephacryl S-300 column (142 cm X 1.75 cm i.d.) that equilibrated with 0.02 M Potassium phosphate buffer pH 7.4, two ml fraction volumes were collected with 30 ml / hr flow rate.
Electrophoresis was performed using 7% PAGE as described by Smith (1969). SDS-PAGE was carried out with 12% PAGE as described by Laemmli (1970). The hyaluronidase molecular weight was determined by SDS-PAGE according to Weber & Osborn (1969). Isoelectric focusing was performed as described by O’Farrell (1975). Proteins were stained with 0.25% Coomassie Brilliant Blue R-250.
Protein determination utilizing Coomassie Brilliant Blue G-250 relied on the existence of Coomassie dye in two different color forms, red and blue. On dye-protein binding, the red color was converted to blue. 0.5 ml of the protein reagent (Coomassie dye) was added to 0.5 ml dH2O and the protein sample. The absorbance was recorded at 595 nm against a blank control. A calibration curve was constructed using bovine serum albumin (BSA) as a standard protein (Bradford, 1976).
The starting specific activity of hyaluronidase in the crude venom extract was 127.43 units / mg protein. One peak containing the hyaluronidase activity was resolved and eluted from the DEAE-cellulose column by equilibration buffer (Fig. 1a). The bee venom hyaluronidase specific activity of the pooled fraction of DEAE-cellulose peak increased 1.84 fold over crude venom with a recovery of 84.6%. Then, the chromatography of this DEAE-cellulose concentrate on Sephacryl S-300 column gave one hyaluronidase activity peak (Fig. 1b) with the elevation of specific activity to 411.7 units / mg protein showing 3.23 fold and 49.9% yield (Tab. 1). The molecular weight of the bee venom hyaluronidase enzyme was determined from its elution volume using the gel filtration column as 37 kDa.
Fig. 1
(a): A typical elution profile for the chromatography of the crude honey bee venom on DEAE-cellulose column (12 cm × 2.4 cm i.d.) previously equilibrated with 0.02 M potassium phosphate buffer, pH 7.4. (b): A typical elution profile for the chromatography of the concentrated pooled DEAE-cellulose fractions containing hyaluronidase enzyme activity on Sephacryl S-300 column (142 cm × 1.75 cm i.d.) previously equilibrated with 0.02 M potassium phosphate buffer pH 7.4. The blue line indicates the protein b (Absorbance at 280 nm) present on the left vertical axis, while the red line indicates the hyaluronidase activity (Unit / ml) present on the right vertical axis.
A typical purification scheme of honey bee venom hyaluronidase
| Purification steps | Total protein (mg) | Total Activity (unit) | Specific Activity | Yield (%) | Fold Purification |
|---|---|---|---|---|---|
| Crude honey bee venom | 27.2 | 3466.2 | 127.43 | 100 | 1.0 |
| DEAE-Cellulose | |||||
| Hyalurodinase (0 M NaCI) | 12.5 | 2932.8 | 234.62 | 84.6 | 1.84 |
| Sephacryl S-300 | |||||
| Hyalurodinase | 4.2 | 1729.1 | 411.7 | 49.9 | 3.23 |
*One unit of hyaluronidase enzyme activity is identified as the amount of enzyme required to cause 50% turbidity reduction as one unit of international standard preparation. Turbidity reducing units (TRU) are expressed as the amount of enzyme required to hydrolyze 50% of the hyaluronic acid.
*Specific activity is expressed as units / mg protein.
Purification steps samples, crude venom, DEAE-cellulose and Sephacryl S-300 portions were electrophoresed on 7% native PAGE. A single protein band matched with the hyaluronidase activity band emphasized the hyaluronidase purity (Fig. 2a and 2b). Electrophoretic analysis of purified hyaluronidase on SDS-PAGE was compared to the protein markers and its subunit molecular weight was determined to be 18.4 kDa (Fig. 2c). Isoelectrofocusing of bee venom hyaluronidase showed a single molecular species with an isoelectric point (
Fig. 2
(a) Electrophoretic analysis of hyaluronidase protein pattern of the different purification steps on 7% native polyacrylamide gel: (1) crude venom, (2) DEAE-cellulose fraction and (3) Sephacryl S-300 purified fraction of honey bee venom hyaluronidase. (b) Electrophoretic analysis of hyaluronidase isoenzyme pattern of the different purification steps on 7% native polyacrylamide gel: (1) crude venom, (2) DEAE-cellulose fraction and (3) Sephacryl S-300 purified fraction of honey bee venom hyaluronidase. (c): Subunit molecular weight determination by electrophoretic analysis of purified hyaluronidase on 12% SDS-polyacrylamide gel: (1) molecular weight marker proteins and (2) purified hyaluronidase. (d): Isoelectrofocusing; (1) Isoelectric point (
The pH effect on the activity of purified hyaluronidase was examined using 0.02 M sodium acetate buffer, pH (3.2 – 5.6) and Tris-HCl buffer, pH (5.8 – 8.6). It displayed the optimum activity at pH 5.4 (Fig. 3a). The temperature's effect on hyaluronidase enzymatic activity determined after incubating the enzyme at different temperature values (20–100°C). The optimum hyaluronidase activity was attained at a temperature of 37–40°C with a relatively sharp decrease at 60–70°C and complete inactivation at 85°C (Fig. 3b). The NaCl effect on hyaluronidase enzymatic activity was determined after pre-incubating the enzyme with different concentrations (0 – 0.5 M) of NaCl. The optimum hyaluronidase enzymatic activity was attained at 0.15 M NaCl and was enhanced in the presence of 0.1 – 0.2 M NaCl and inhibited in 0.25 – 0.5 M NaCl (Fig. 3c). The Lineweaver-Burk plot of the reciprocal of the reaction velocity (1/V) and substrate concentration (1/[S]) was constructed (Fig. 3d) detecting the
Fig. 3
(a) Effect of pH on the purified honey bee venom hyaluronidase using 0.02 M sodium acetate buffer, pH (3.2 – 5.6) and Tris-HCl buffer, pH (5.8 – 8.6). (b) Effect of temperature on the purified honey bee venom hyaluronidase. (c) Effect of NaCl concentration on the purified honey bee venom hyaluronidase. (d) Lineweaver-Burk plot relating the reciprocal of the reaction velocity of the purified hyaluronidase to hyaluronic acid concentration in mg/ml.
The effect of divalent cations on the activity of hyaluronidase was examined after a pre-incubation with 2 and 5 mM solutions of every cation at 37°C. A control without cation was considered to be 100% activity. ZnCl2 and MgCl2 elevated hyaluronidase activity of while CoCl2, FeCl2 and NiCl2 inhibited it (Tab. 2). The different inhibitors’ effect on the hyaluronidase activity was examined after pre-incubating with 2 and 5 mM solutions of every inhibitor or five and ten units of heparin at 37°C. A control without inhibitor was considered to be 100% activity. Heparin, 1,10 Phenanthroline, β-Mercaptoethanol, Citric acid, EDTA and Urea were inhibitors for bee venom hyaluronidase activity (Tab. 3).
Effect of divalent cations on honey bee venom hyaluronidase
| Reagent | Final Concentration (mM) | Residual activity (%) |
|---|---|---|
| Control | ----- | 100.0 |
| CaCl2 | 2.0 | 96.4 |
| 5.0 | 89.9 | |
| CoCl2 | 2.0 | 49.7 |
| 5.0 | 28.1 | |
| CuCl2 | 2.0 | 98.0 |
| 5.0 | 93.1 | |
| FeCl2 | 2.0 | 18.8 |
| 5.0 | 11.2 | |
| MgCl2 | 2.0 | 109.0 |
| 5.0 | 115.0 | |
| MnCl2 | 2.0 | 44.5 |
| 5.0 | 20.7 | |
| NiCl2 | 2.0 | 52.4 |
| 5.0 | 27.5 | |
| ZnCl2 | 2.0 | 112.0 |
| 5.0 | 118.0 |
Effect of various inhibitors on honey bee venom hyaluronidase
| Reagent | Final Concentration (mM) | Inhibition (%) |
|---|---|---|
| Control | ----- | 0.0 |
| 1,10 Phenanthroline | 2.0 | 72.8 |
| 5.0 | 85.1 | |
| β-Mercaptoethanol | 2.0 | 37.6 |
| 5.0 | 48.4 | |
| Citric acid | 2.0 | 50.9 |
| 5.0 | 69.3 | |
| Dithiothreitol (DTT) | 2.0 | 15.6 |
| 5.0 | 23.3 | |
| EDTA | 2.0 | 52.2 |
| 5.0 | 71.8 | |
| Heparin | 5 U | 47.85 |
| 10 U | 85.73 | |
| Hydrogen peroxide (H2O2) | 2.0 | 30.2 |
| 5.0 | 43.6 | |
| Iodoacetamide | 2.0 | 21.3 |
| 5.0 | 29.1 | |
| Potassium cyanide (KCN) | 2.0 | 12.4 |
| 5.0 | 15.8 | |
| Sodium azide (NaN3) | 2.0 | 16.5 |
| 5.0 | 22.1 | |
| Phenyl methyl sulfonyl fluoride (PMSF) | 2.0 | 26.7 |
| 5.0 | 31.8 | |
| Reduced glutathione | 2.0 | 8.20 |
| 5.0 | 12.4 | |
| Sodium dodecyl sulphate | 2.0 | 6.20 |
| 5.0 | 9.60 | |
| Urea | 2.0 | 78.3 |
| 5.0 | 89.9 |
How different concentrations heparin affect hyaluronidase activity is examined (Fig. 4a). There was a linear relationship between the heparin concentration and the inhibition of hyaluronidase activity. In the Hill plot, log Vi / Vmax – Vi values were plotted against log [I] values of the heparin and a straight line was obtained with a slope of 0.91 (Fig. 4b). The type of inhibition of hyaluronidase by heparin was examined by using Lineweaver-Burk plot (Fig. 4c). A constant amount of hyaluronidase activity was measured in the presence and absence of three various heparin concentrations with varying substrate concentrations. The plot indicated that the inhibition of hyaluronidase by heparin was noncompetitive. The determination of
Fig. 4
(a) Inhibition of the purified honey bee venom hyaluronidase by varying concentrations of Heparin. (b) Hill plot for inhibition of the purified hyaluronidase by varying concentrations of Heparin where Vmax is the enzyme activity in absence of inhibitor, Vi is the enzyme activity in presence of inhibitor and [I] is inhibitor concentration in units of Heparin. (c) Lineweaver-Burk plots showing the type of inhibition of the purified honey bee venom hyaluronidase by Heparin. The activity of the purified hyaluronidase was measured with varying concentrations of the substrate hyaluronic acid in absence and presence of three various concentrations of Heparin. (d) Determination of the inhibition constant (
Hyaluronidase has many such medical applications as in embryo implantation, surgery, wound healing and drug delivery. This study presents a simple method for hyaluronidase purification from Egyptian honey bee
Various specific activities and purification folds were reported; hyaluronidase from venom of the honey bee
Various molecular weights for hyaluronidases were reported; 41 kDa for venom of the honeybee
In contrast FeCl2 was found to be a potent inhibitor of hyaluronidase activity, while CoCl2 and NiCl2 were moderate inhibitors (Table 2) which is similar to the Egyptian horned viper
In conclusion, this study presents for the first time a reproducible and simple procedure for the purification of well characterized hyaluronidase from Egyptian honey bee venom. The characterizations of the purified enzyme established the optimum conditions for its activity and will help in its uses in different applications with maximum efficiency.