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Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii

Mohammed M. Abdel-Monsef
Mohammed M. Abdel-Monsef
, 
Hind A. Zidan
Hind A. Zidan
, 
Doaa A. Darwish
Doaa A. Darwish
, 
Hassan M. Masoud
Hassan M. Masoud
, 
Mohamed S. Helmy
Mohamed S. Helmy
 and 
Mahmoud A. Ibrahim
Mahmoud A. Ibrahim
   | Jul 02, 2020
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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.

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): 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.

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 (pI) marker proteins and (2) the purified hyaluronidase.
(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 (pI) marker proteins and (2) the purified hyaluronidase.

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.
(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.

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 (Ki) value for the inhibition of the purified hyaluronic acid by Heparin. The plotted slope values were determined from the lines of the reciprocal plots of the different inhibitor concentrations.
(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 (Ki) value for the inhibition of the purified hyaluronic acid by Heparin. The plotted slope values were determined from the lines of the reciprocal plots of the different inhibitor concentrations.

A typical purification scheme of honey bee venom hyaluronidase

Purification stepsTotal protein (mg)Total Activity (unit)Specific ActivityYield (%)Fold Purification
Crude honey bee venom27.23466.2127.431001.0
DEAE-Cellulose
Hyalurodinase (0 M NaCI)12.52932.8234.6284.61.84
Sephacryl S-300
Hyalurodinase4.21729.1411.749.93.23

Effect of divalent cations on honey bee venom hyaluronidase

ReagentFinal Concentration (mM)Residual activity (%)
Control-----100.0
CaCl22.096.4
5.089.9
CoCl22.049.7
5.028.1
CuCl22.098.0
5.093.1
FeCl22.018.8
5.011.2
MgCl22.0109.0
5.0115.0
MnCl22.044.5
5.020.7
NiCl22.052.4
5.027.5
ZnCl22.0112.0
5.0118.0

Effect of various inhibitors on honey bee venom hyaluronidase

ReagentFinal Concentration (mM)Inhibition (%)
Control-----0.0
1,10 Phenanthroline2.072.8
5.085.1
β-Mercaptoethanol2.037.6
5.048.4
Citric acid2.050.9
5.069.3
Dithiothreitol (DTT)2.015.6
5.023.3
EDTA2.052.2
5.071.8
Heparin5 U47.85
10 U85.73
Hydrogen peroxide (H2O2)2.030.2
5.043.6
Iodoacetamide2.021.3
5.029.1
Potassium cyanide (KCN)2.012.4
5.015.8
Sodium azide (NaN3)2.016.5
5.022.1
Phenyl methyl sulfonyl fluoride (PMSF)2.026.7
5.031.8
Reduced glutathione2.08.20
5.012.4
Sodium dodecyl sulphate2.06.20
5.09.60
Urea2.078.3
5.089.9
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