DOI: 10.2478/JAS-2020-0015 J. APIC. SCI. VOL. 64 NO. 1 2020J. APIC. SCI. Vol. 64 No. 1 2020 Original Article BIOCHEMICAL ISOLATION AND CHARACTERIZATION OF HYALURONIDASE ENZYME FROM VENOM OF EGYPTIAN HONEY BEE APIS MELLIFERA LAMARCKII Mohammed M. Abdel-Monsef1* Hind A. Zidan2 Doaa A. Darwish1 Hassan M. Masoud1 Mohamed S. Helmy1 Mahmoud A. Ibrahim1 1Molecular Biology Department, National Research Centre, Dokki, Giza, Egypt 2Plant Protection Research Institute, Agricultural Research Center *corresponding author: [email protected] Received: 04 December 2019; accepted 03 March 2020 Abstract The hyaluronidase enzyme has been used in many such fields of medicine as ophthalmology, orthopaedia, internal medicine, gynecology, surgery, oncology and dermatology. In this study, the hyaluronidase enzyme was purified and characterized for the first time from Egyptian bee venom homogeneously using DEAE-cellulose and Sephacryl S-300 columns. Bee venom hyaluronidase specific activity was 411.7 units/ mg protein with 49.9% yield and 3.23-fold purification. The molecular weight of the purified bee venom hyaluronidase native form was 37 kDa. The purified enzyme was found homogeneous on native PAGE and SDS-PAGE, with two congruent subunits of 18.4 kDa and isoelectric point (pI) of 8.6- 8.8. The enzyme was found to be stable over a wide range of temperature (20-60°C) and pH (4.5-6.5), and its optimum activity at 37°C, pH 5.4 and 0.15 M NaCl. Km for bee venom hyaluronidase was 0.029 mg/ml hyaluronic acid and its activity was elevated in presence of MgCl2 and ZnCl2 and lowered in presence of FeCl2. Heparin inhibited the hyaluronidase enzyme noncompetitively with a Ki value of 2.9 units heparin and one binding site on the enzyme molecule. Keywords: bee venom, characterization, hyaluronidase, purification INTRODUCTION of inflammations, different nervous diseases, cancer, antiviral activities and human immune Bee keeping in Egypt has been an important disorders (Wehbe et al., 2019). Hyaluronidase, type of farming since ancient Egyptian. Bee found in all kinds of animal venoms, hydrolyzes venom products are important in the pharma- hyaluronic acid, one of the major components ceutical industry and drug formulations. The of extracellular matrix, and causes local tissue venom produced by the Egyptian honey bee damage (Girish et al., 2004; Kemparaju, & Girish, (Apis mellifera lamarckii) has a high biological 2006; Senff-Ribeiro et al., 2008; Bordon et al., activity and toxicity and is the best form for 2015). potential pharmacological sources (Kokot et Hyaluronidase is the major allergen in the al., 2009). Apitherapy is an alternate kind of venoms of bees, hornets, wasps and scorpions therapy which utilizes honey bee constituents that stimulate lethal systemic IgE-interme- especially bee venom in treating various human diated anaphylactic responses in humans illnesses. Bee venoms can be introduced into the (Kolarich et al., 2005). In microbes, hyaluroni- human body manually through injection or by dases are virulence agents included in patho- immediate bee sting. It includes many energetic genesis and disease advancement brought on molecules including peptides and enzymes by pathogens. The hyaluronidase degradation which have a beneficial ability in the treatment of the host tissue components facilitates the 153 Abdel-Monsef et AL. Hyaluronidase from venom of Egyptian honey bee infestation of pathogens. Increased permeabil- MATERIAL AND METHODS ity of tissues appears to play a principle role in wound infections, meningitis, pneumonia MATERIALS and bacteremia (Matsushita & Okabe, 2001; Venom Collection Makris et al., 2004). Additionally, hyaluronidase The colonies of honeybees Apis mellifera increases membrane permeability, so thera- lamarckii (Egyptian subspecies) were obtained peutically it is used to enhance injected fluid from the Asuit governorate and the venom absorption and excess-fluid resorption, increase was extracted from 500 forager workers. The local anesthesia effectiveness and decrease bees were caught at the colony entry and im- tissue destruction in subcutaneous and intra- mobilized through rapid freezing at -20°C. Indi- muscular injection (Farr et al., 1997). viduals were dissected; sting device and venom The hyaluronidase of sperms plays an important reservoir were removed and disrupted in tube role in fertilization success in humans and with 2.5 ml H2O, followed by five-minute cen- mammals (Klocker et al., 1995). Mammalian trifugation at 12000 xg at 4°C. The superna- sperm hyaluronidase is located on the sperm tants were saved and designated as the crude surface, where it facilitates the penetration venoms extracts. through the ovum and is also necessary for fertilization (Zheng et al., 2001). It can be used Chemicals to decrease myocardial infarction (Muckensch- Hyaluronic acid, cetyltrimethylammonium nabel et al., 1996). Hyaluronidase may have bromide, alcian blue, phenymethylsulfonyl- essential anticancer impacts and may repress fluoride (PMSF), 1,4 dithiothreitol (DTT), 1,10 tumor growth, treatment with it was stated to phenanthroline, sephacryl S-300, bovine serum block tumor cells invasion to lymph node in T cell albumin (BSA), blue dextran, gel filtration kits lymphoma (Zahalka et al., 1995). It was used as for molecular weight marker, (DEAE-cellulose) di- a chemotherapeutic-drug additive for anticancer ethylaminoethyl cellulose and standard markers activity augmentation (Baumgartner, 1998). The mixture pI 3.6 - 9.3 for isoelectric focusing (IEF) hyaluronidases of bee venom preparations have were products of Sigma Chemical Company. been used successfully in medicine combined Marker proteins for molecular weight SDS with anticancer drugs (Moga et al., 2018) and for were the product of Pharmacia Fine Chemicals scar resorption in treating wounds and burns of Company. Other chemicals were of analytical skins and mucosa surfaces (Krylov, 1995). grade. There have been studies on the hyaluronida- se from the venom of the honey bee (Gmachl METHODS & Kreil, 1993; Topchiyeva & Mammadova, Assay of hyaluronidase enzyme activity 2016), the scorpion Palamneus gravimanus The activity of hyaluronidase is measured tur- (Morey, Kiran, & Gadag, 2006), the funnel web bidometrically according to Pukrittayakamee spider Hippasa partita (Nagaraju, Devaraja, & et al. (1988). The assay mixture consisted of Kemparaju, 2007), the Egyptian horned viper 0.5 ml 0.2 M acetate buffer, pH 5.5 containing Cerastes cerastes (Wahby et al., 2012), the 0.15 M NaCl, 50 mg hyaluronic acid and venom social wasp Polybia paulista (Justo Jacomini et extract. It was incubated for fifteen minutes al., 2013) and the spider Vitalius dubius (Sutti at 37°C then stopped by adding 1 ml of 2.5% et al., 2014). Given the limited information cetyltrimethylammonium bromide in 2% NaOH regarding Egyptian bee venom components and and absorbance was read at 400 nm. Turbidity its important roles, the purpose of this study is reducing units (TRU) were defined as the the purification and characterization of hyalu- amount of hyaluronidase necessary for hydro- ronidase from Egyptian bee venom for uses in lyzing 50% of hyaluronic acid. One unit was various therapeutic, cosmetic and pharmaceuti- the amount of hyaluronidase that caused 50% cal applications. reduction in turbidity. 154 J. APIC. SCI. Vol. 64 No. 1 2020 Hyaluronidase activity staining on poly- Brilliant Blue R-250. acrylamide gels Samples were electrophoresed on 7% native- Protein determination PAGE that co-polymerized with 0.17 mg/ml of Protein determination utilizing Coomassie hyaluronic acid. Following electrophoresis, gel Brilliant Blue G-250 relied on the existence of was incubated in 20 mM Tris-HCL buffer, pH Coomassie dye in two different color forms, red 7.5, containing 150 mM NaCl for three hours at and blue. On dye-protein binding, the red color 37°C, followed by incubation in 100 mM sodium was converted to blue. 0.5 ml of the protein acetate buffer pH 4.0 overnight at 37°C. Gels reagent (Coomassie dye) was added to 0.5 ml were then stained for two hours at room tem- dH2O and the protein sample. The absorbance perature with 0.5% alcian blue in 3% acetic acid was recorded at 595 nm against a blank control. and destained in 70% acetic acid. The activity A calibration curve was constructed using in gel was detected by a lack of color in the bovine serum albumin (BSA) as a standard digested area against a blue background dis- protein (Bradford, 1976). tinctive for undigested hyaluronic acid (Gunten- höner, Pogrel, & Stem, 1992). RESULTS Purification of hyaluronidase enzyme from Purification of hyaluronidase from bee bee venom venom DEAE-cellulose column chromatography The starting specific activity of hyaluronidase Crude venom extract underwent chromatogra- in the crude venom extract was 127.43 units / phy on DEAE-cellulose column (12 cm x 2.4 cm i.d.) mg protein. One peak containing the hyaluroni- equilibrated previously with 0.02 M Potassium dase activity was resolved and eluted from the phosphate buffer pH 7.4. The adsorbed proteins DEAE-cellulose column by equilibration buffer were eluted with NaCl stepwise gradient (0 - 1 (Fig. 1a). The bee venom hyaluronidase specific M) in the preceding buffer at 60 ml / hr flow activity of the pooled fraction of DEAE-cellulose rate. Fractions of 3 ml volumes were collected peak increased 1.84 fold over crude venom with and those containing hyaluronidase activity a recovery of 84.6%. Then, the chromatography were combined and concentrated. of this DEAE-cellulose concentrate on Sephacryl S-300 column gave one hyaluronidase activity Sephacryl S-300 column chromatography peak (Fig. 1b) with the elevation of specific The DEAE-cellulose concentrated proteins of activity to 411.7 units / mg protein showing the peak including hyaluronidase activity were 3.23 fold and 49.9% yield (Tab. 1). The molecular loaded to a Sephacryl S-300 column (142 cm weight of the bee venom hyaluronidase enzyme X 1.75 cm i.d.) that equilibrated with 0.02 M was determined from its elution volume using Potassium phosphate buffer pH 7.4, two ml the gel filtration column as 37 kDa.