Isolation, Purification and Characterization of Hyaluronidase and Phospholipase a 2 Enzymes of Echis Ocellatus and Naja Nigricollis Snake Venoms

doi:10.21203/rs.3.rs-1725908/v1

Issues related to snakebite has for long been of high economic and medical importance. Management and treatment of snake envenomation has always been an issue, especially in remote areas, where anti-snake venom and facilities for its storage are not available, coupled with the high specificity and instability of anti-venom. Venom enzymes are usually responsible for almost all the activity of snake venom after bite. This research was aimed at evaluation and characterization of Hyaluronidase and Phospholipase A₂ (PLA2) Enzymes of Echis ocellatus and Naja nigricollis snake venoms. Isolation and purification of these enzymes were done using a two-step process which included gel filtration on Sephadex G-75, active fractions were applied to ion–exchange chromatography on DEAE (Diethylaminoethyl) cellulose. Individual active fractions of each venom, were then subjected to SDS–PAGE for molecular weight extrapolation. Enzyme characterization was done on the two isolated enzymes for the two snake venom used. N. nigricollis enzymes were revealed to have an optimum temperature of 35 ^oC and 55^oC, while that of E. ocellatus had 37 ^oC and 40^oC, with a pH of 3.5 and 8.0 for both the venom enzymes. Velocity kinetics carried out shows that N. nigricollis PLA₂ has the highest V_max value of 30.567mg/min, while E. ocellatus PLA₂ however has the highest K_m value of 4.5378mg/ml. Purification and characterization done in this research has revealed/confirmed that these venoms contains Hyaluronidase and PLA₂ enzymes, giving a better perception on this enzymes which will aids in the management and treatment of snake envenomation from these snakes.

Envenomation by snake usually causes around 100,000 deaths annually, which in turns leads to some chronic, physical detrimental changes. Snakebite, however still remains a serious medical, economic and social problem. The risk of envenomation by snake is usually higher in rural areas than in urban areas, due to the fact that most of this snakes resides in rural environments where many farms, mountains and so many natural environments are found. Snake venom is a crude mixture of compounds of numerous kinds of enzymes (such as; protease, phospholipase A, hyaluronidase, L-amino oxidase and phosphodiesterase) and other toxic proteins. It is usually produced and stored in snake’s venomous gland, which is a modification of the salivary gland. The glands are however situated on each side of the head below and behind the eye of venomous animals [1].

Snakes are elongated, legless, carnivorous reptiles of the suborder Serpentes. Like all squamates, snakes are ectothermic, amniote vertebrates covered in overlapping scales [2]. Many species of snakes have skulls with several more joints than their lizard ancestors, enabling them to swallow prey much larger than their heads with their highly mobile jaws [3]. Snakebite detrimental hemostatic effects were shown to be directly involved in proteolytic events caused by the protease enzymes present in snake venom, which subsequently results in blood coagulation, fibrinolysis and platelet aggregation among others. Snake venom proteolytic enzymes interfere with many biological processes, immune system and cause organs inflammation [4].

More than 90% of snake venom is protein, of more than a hundred different kinds including enzymes (constituting 80–90% of viperid and 25–70% of elapid venoms), non-enzymatic polypeptide toxins and non-toxic proteins such as nerve growth factor [5].

E. ocellatus is one of the venomous viper snake species predominantly found in West Africa, commonly known as the West African carpet viper. It is locally known as ‘Kububuwa’ in hausa in northern Nigeria [6]. Its envenomation causes the most fatalities, more than all the other African snake species combined. The venom contains a prothrombin activating procoagulant, haemorrhagin and cytolytic fractions which cause haemorrhage, incoagulable blood, shock and local reactions/ necrosis. Another snake of high economic and medical importance is the N. nigricollis which is also known as the black-necked spitting cobra. This species of spitting cobra is normally found mostly in sub-Saharan Africa. They are moderately sized snakes that can grow to a length of 1.2 to 2.2 m. Their coloration and markings can vary considerably. They prey primarily on small rodents. They possess medically significant venom, like other spitting cobras, they can eject venom from their fangs when threatened (one drop over 7metres and more in perfect accuracy). Their neurotoxic venom irritates the skin, causing blisters and inflammation and can cause permanent blindness if the venom makes contact with the eyes and is not washed off immediately [7]. This research work was there for carried out to isolation, purification and characterization of hyaluronidase and phospholipase a₂ enzymes of E. ocellatus and N. nigricollis snake venoms.

Snake Venom Sample Collection

Lyophilized venom of E. ocellatus and N. nigricollis (400mg each) was purchased from the snake lab of Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Kaduna Nigeria and was aseptically transported and stored at -4⁰C until used.

Enzyme Isolation

Purification of PLA₂ Enzyme

200mg of each of the snake venom sample was dissolved in 10ml equilibration buffer (0.05M Tris-HCl, pH 6.8); and was loaded on Sephadex G-75 column (2.6 X 50Cm). The sample dissolving buffer was used in equilibration of the sephadex column and elution of the loaded samples. Fraction of 3ml was then collected at a flow rate of 30ml/hr. using fraction collectors. Fraction with PLA₂ activity was recovered, pooled and directly applied to a DEAE cellulose column (1.6 X 25Cm) pre-equilibrated with the same buffer and eluted with a linear NaCl gradient from 0 to 1.2M in the same buffer [1].

PLA₂ Activity Assay

This was carried out as described by [8]. Here, 0.5 ml of egg yolk suspension (4 mg ml^− 1) was introduced into a clean test tube containing 50µL of 1mM CaCl₂. To this, 100µL of 20 mgml^− 1 venom solution was added and incubated at 37°C for 1 h. Thereafter, the activity was stopped, by heating at 100°C for 2 min. A drop of phenolphthalein was added and then titrated against 2mM NaOH solution to an end point. The same procedure was carried out in the absence of the enzyme, to obtain the titre value for the blank, for an adequate comparison to deduce the effect of the enzyme on the yolk (deduction of any FFA released). The activity of phospholipase A₂ was defined as the amount of enzyme required to hydrolyse 1mg of FFA from the lecithin present in the egg yolk under the standard conditions [9].

Hyaluronidase Enzyme Purification

Hyaluronidase from the two venom were isolated and purified using a combination of two purification steps; gel filtration chromatography on sephadex G-75 and ion-exchange chromatography on DEAE cellulose.

Gel-Filtration on Sephadex-G75

The gel was prepared by dissolving 20g of sephadex G-75 in 100ml sodium acetate buffer, pH 6 for 24 hours at room temperature and mixed with a glass rod to make the swollen particles form slurry. The slurry was then poured into a (10mm by 30cm) column packed with cotton wool at the bottom. The column was first equilibrated with 0.1M sodium acetate buffer, pH 6.0, before the sample was applied. Recovered supernatant from the previous step was loaded onto the glass column. Fractions of 3ml each was collected at a flow rate of 1 mL per 0.153 sec minute total protein and enzyme activity determined [10].

Ion Exchange Chromatography on DEAE Cellulose

DEAE-cellulose was prepared by dissolving 10g of the anion-exchanger in 100 ml acetate buffer, pH 6.0. The slurry was poured into a 300mm column. The pooled sample (3ml) obtained from the gel-filtration step was loaded onto the DEAE cellulose column equilibrated with sodium acetate buffer pH6.0. The fractions was then washed and eluted under a linear concentration gradient of sodium chloride solution (1.0M). 3ml fractions were collected at a flow rate of 1ml per 1.33 minute and analyzed for total protein and enzyme activity. The fractions showing high specific activity were analyzed on SDS PAGE [10].

Determination of Hyaluronidase Activity

A hyaluronidase standard curve was prepared by making dilutions of the hyaluronic acid standard (10mg) dissolved in 25ml of 0.1M sodium acetate buffer, pH 6 with 0.15M sodium chloride and different concentration were pipetted into test tubes. The tubes were placed in a boiling water bath for 5 minutes, cool to room temperature and 9.0ml of albumin reagent was added and allowed to stand for 10 minutes. The absorbance was read at 540 nm against a reagent blank. Absorbance of hyaluronic acid versus mg of hyaluronic acid was plotted to form a standard curve. A 0.5ml of a 0.4mg/ml hyaluronic acid solution was pipetted into series of test tubes. The tubes were incubated at 37°C for 4–5 minutes to achieve temperature equilibrium. One blank tube was incubated with 1ml of 0.1M sodium phosphate buffer pH 5.3 and 0.15M sodium chloride at timed interval (30mins), 0.5ml of appropriately diluted enzyme or standard was added to the respective tubes. The tubes were then incubated for 10 minutes and cooled in an ice bath to room temperature. A 9.0ml of albumin reagent was then added to each tube and incubated at room temperature for 10 minutes. Absorbance was read at 540nm [1].

SDS PAGE Electrophoresis

According [11], 12.5% polyacrylamide slab gel with a Tris-HCl buffer at pH 8.9. SDS PAGE was carried out using a 0.1% SDS-14% polyacrylamide slab gel. The gel was stained with 0.1% coomassie Brilliant blue R-250 in 50% methanol 7% acetic acid and the background of the gel then de-stained with 7% acetic acid.

Characterization of the Partially-purified Enzymes

Effect of Temperature

The effect of temperature 20◦C to 60◦C on the activity of the enzymes. In which the activity of each enzyme is taken at every limiting step respectively, to ascertain the optimum temperature at which the venoms enzymes are more active and viable [1].

Effect of pH

The effect of pH on the activity of partially purified enzymes was measured at various pH (2, 3.5, 5 and 8). In which the activity of each enzyme is taken at every limiting step respectively, to ascertain the optimum pH at which the venoms enzymes are more active and viable [1].

Initial velocity studies

The enzymes velocity studies were analyzed using standard procedures as described by Nwune, (2016). The activities of hyaluronidase and phospholipase A₂ (V₀) were determined in the presence of various concentrations of substrates (0.4, 0.6, and 0.8mg/ml). The data obtained was then used to plot the Lineweaver-Burk plot to determine the kinetic parameters K_m and V_max [1].

Statistical Analysis

Some of the data obtained were presented as mean ± standard deviation of three determinations. The analysis of variance was used to compare the paired means; the P < 0.05 was considered statistically significant.

Result for Hyaluronidase and PLA₂ enzymes isolation from E. ocellatus and N. nigricollis snakes venoms are shown in Figs. 1a/1b, Figs. 2a/2b, Figs. 3a/3b and Figs. 4a/4b respectively. Using a two way step purification (gel filtration chromatography on sephadex G-75 and ion-exchange chromatography on DEAE cellulose). Purified Hyaluronidase from E. ocellatus was shown to have a final total protein, enzyme and specific activities of 0.050956mg/ml, 1.257338TRU/mg and 22.115236TRU respectively. While that of N. nigricollis was 0.067343mg/ml, 1.490221TRU/mg and 22.12882TRU. While that of N. nigricollis was revealed to have 0.077761mg/ml, 2.49615(µmol/min), 32.10028(mol/min/mg), 3.05 and 86 respectively Enzymes purification step analysis is shown in Tables 1 to 4. Isolated enzymes from the two venoms were run on SDS-PAGE to estimate and extrapolate their molecular weight as shown on Fig. 5a and 5b. Results from the characterization of the partially-purified enzymes is shown on Figs. 6–7, and Table 5.

Table 1: shows the purification steps for enzyme Hyaluronidase isolated from E. ocellatus venom using a two-step purification protocol (Gel filtration and ion exchange chromatography).

Table 1

Purification Table for Hyaluronidase from *E. ocellatus* Venom
Enzyme	Step	Total Protein (mg/ml)	Total Enzyme Activity (TRU/mg)	Specific Activity (TRU)	Purification Fold	Yield (%)
Hyaluronidase	Crude	0.208383	2.131154	10.227101	1	100
	Gel filtration on sephadex G-75	0.124358	1.969923	15.840742	1.5	92
	Ion exchange on DEAE − 52 Celullose	0.050956	1.257338	22.115236	1.4	52

Table 2: shows the purification steps for enzyme PLA₂ isolated from E. ocellatus venom using a two-step purification protocol (Gel filtration and ion exchange chromatography).

Table 2

Purification table for PLA₂ from *E. ocellatus* Venom
Enzyme	Step	Total Protein (mg/ml)	Total Enzyme Activity (µmol/min)		Purification Fold	Yield (%)
PLA₂	Crude	0.354742	4.267348	12.029441	1	100
	Gel filtration on sephadex G-75	0.184431	3.95723	21.456425	1.70	93
	Ion exchange on DEAE − 52 Celullose	0.086305	3.746543	46.15385	2.15	86

Table 3

Purification Table for Hyaluronidase from *N. nigricollis* Venom
Enzyme	Step	Total Protein (mg/ml)	Total Enzyme Activity (TRU/mg)	Specific Activity (TRU)	Purification Fold	Yield (%)
Hyaluronidase	Crude	0.258383	3.879022	15.0126	1	100
	Gel filtration on sephadex G-75	0.185397	2.954326	15.93513	1.1	76
	Ion exchange on DEAE − 52 Celullose	0.067343	1.490221	22.12882	1.5	50

Table 4

Purification Table for PLA₂ from *N. nigricollis* Venom
Enzyme	Step	Total Protein (mg/ml)	Total Enzyme Activity (µmol/min)	Specific Activity (mol/min/mg)	Purification Fold	Yield (%)
PLA₂	Crude	0.383654	4.027221	10.49701	1	100
	Gel filtration on sephadex G-75	0.164453	3.184615	19.36489	1.844801	79
	Ion exchange on DEAE − 52 Celullose	0.077761	2.49615	32.10028	3.05804	62

Table 5

Result for Enzyme Velocity Studies
Enzymes	Kinetic Parameters	*
E. ocellatus Hyaluronidase	K_m (mg/ml)	0.7492
E. ocellatus Hyaluronidase	V_max (tru/min)	2.2563
N. nigricollis Hyaluronidase	K_m (mg/ml)	1.3860
N. nigricollis Hyaluronidase	V_max (tru/min)	4.2125
E. ocellatus PLA₂	K_m (mg/ml)	4.5378
E. ocellatus PLA₂	V_max (mg/min)	20.534
N. nigricollis PLA₂	K_m (mg/ml)	1.1895
N. nigricollis PLA₂	V_max (mg/min)	30.567

Snake envenomation has been one of man’s greatest concerns for quite a long time, causing countless number of fatalities, infections and numerous health defects. Even though snakes are of different species and types, one sure fact about them is their venomous nature. Snake venom from different snake species all have one single peculiar feature, which is the proteinous aggregative nature. This series of enzymes are responsible for all detrimental effects encountered from snakebite. PLA₂ enzyme for example have been shown to possess anticoagulant properties, hence inhibiting blood coagulation. PLA₂ also exhibits quite a high edematogenic and myotoxic activities, demonstrating its capacity to contribute to tissue damage after snake bite [12]. Another important enzyme found in snake venoms of utmost importance is the Hyaluronidase, which aids in tissue degradation and necrosis, hence providing way/entry point for the other toxins enzymes into the body. Hyaluronidase and Phospholipase A₂ enzymes were isolated, purified and characterized from the venoms of E. ocellatus and N. nigricollis. Using a two way step purification (gel filtration chromatography on sephadex G-75 and ion-exchange chromatography on DEAE cellulose). Purified Hyaluronidase from E. ocellatus was shown to have a final total protein, enzyme, specific activities, purification fold and yield of 0.050956mg/ml, 1.257338TRU/mg, 22.115236TRU, 1.4 and 52 respectively. While that of N. nigricollis was revealed to have 0.067343mg/ml, 1.490221 TRU/mg, 22.12882 TRU, 1.5 and 50. Purified PLA₂ from E. ocellatus was shown to have a final total protein, enzyme, specific activities, purification fold and yield of 0.086305mg/ml, 3.746543(µmol/min), 46.15385(mol/min/mg), 2.15 and 86 respectively. While that of N. nigricollis was revealed to have 0.077761mg/ml, 2.49615(µmol/min), 32.10028(mol/min/mg), 3.05 and 86 respectively. This shows that the E. ocellatus venom enzymes has higher activity and viability than those from N. nigricollis. Which implies that E. ocellatus bite will be more dangerous than that of N. nigricollis.

Isolated enzymes from the two venoms were run on SDS-PAGE analysis to estimate and extrapolate their molecular weight as shown on Fig. 5a and 5b. In which Hyaluronidase and PLA₂ from E. ocellatus were extrapolated to have a molecular weight of 30 and 14KDa respectively on SDS-PAGE. While those from N. nigricollis were shown to have 26 and 12.5KDa. These correspond with the studies done by [13], in which he revealed that PLA₂ isolated has 15KDa molecular mass and that of [14], which reported that at the PLA₂ isolated from snake has molecular mass 15.6KDa. [9] and [15] also report a similar extrapolation of snake venom hyaluronidase with the ones revealed in this research.

Understanding the characteristics of Hyaluronidase and PLA₂ from snake venoms is important for venom researchers, as it would help the production and management of effective therapeutic antivenom, considering the role of the enzyme in envenomation. That’s one of the reason why enzyme characterization was done in this research on the two isolated enzymes for the two snake venom used. Temperature optimization assay was done, in which Hyaluronidase and PLA₂ from E. ocellatus were found out to be more active and viable at optimum temperatures of 37^oC and 40^oC for Hyaluronidase and PLA₂ respectively. While those of N. nigricollis was shown to have an optimum temperatures of 35^oC and 55^oC respectively. Also the optimal pH range for hyaluronidase and PLA₂ from E. ocellatus was shown to be more active and viable in a pH of around 3.5 and 8.0 respectively. While that of N. nigricollis was shown to have an optimum pH viability of 3.5 and 8.0 too for same enzymes respectively. This come in agreement with the work done by [16], which show Hyaluronidase and PLA₂ to have an optimal temperature of 35^oC and 7.0^oC and a pH of 4 and 8. Thus the increase in body temperature of snake bitten victims above the physiological temperature could also make the condition favorable for PLA₂ to exert its hydrolytic function effectively.

Kinetic of enzyme help in revealing the various enzyme kinetics velocity parameters in the presence and absence of substrate or inhibitor. Velocity kinetics carried out shows that N. nigricollis PLA₂ had the highest V_max value of 30.567mg/min, while E. ocellatus Hyaluronidase has the lowest V_max of 2.2563tru/min. E. ocellatus PLA₂ however has the highest K_m value of 4.5378mg/ml as shown in Table 5. This was compared with work done by [17], which had a similar research outcome with this. This research will aids in giving a clearer vision on how inhibitors can acts on enzymes as the key to the management and treatment of snake envenomation lies with a better perception of how this enzymes are and their kinetic conditions and viabilities.

Both E. ocellatus and N. nigricollis snake venoms contains Hyaluronidase and PLA₂ enzymes, which contributes to the various detrimental snake bite effects. Results from purification and characterization done in this research has revealed a better perception on this enzymes which will aids in the management and treatment of snake envenomation from these snakes.

FUNDING

This research work was funded by the TETFUND IBR, Nigerian Defence Academy, 2021

(TETF/DR&D/CE/NDA/KADUNA/IBR/2020/VOL.1).

DATA AVAILABILITY

All data generated or analyzed during this study are included in this published article [and its supplementary information files.

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No competing interests reported.

Isolation, Purification and Characterization of Hyaluronidase and Phospholipase a 2 Enzymes of Echis Ocellatus and Naja Nigricollis Snake Venoms

Status:

Version 1

Abstract

Figures

Introduction

Materials And Method

Snake Venom Sample Collection

Enzyme Isolation

Purification of PLA₂ Enzyme

PLA₂ Activity Assay

Hyaluronidase Enzyme Purification

Gel-Filtration on Sephadex-G75

Ion Exchange Chromatography on DEAE Cellulose

Determination of Hyaluronidase Activity

SDS PAGE Electrophoresis

Characterization of the Partially-purified Enzymes

Effect of Temperature

Effect of pH

Initial velocity studies

Statistical Analysis

Result

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Isolation, Purification and Characterization of Hyaluronidase and Phospholipase a 2 Enzymes of Echis Ocellatus and Naja Nigricollis Snake Venoms

Status:

Version 1

Abstract

Figures

Introduction

Materials And Method

Snake Venom Sample Collection

Enzyme Isolation

Purification of PLA2 Enzyme

PLA2 Activity Assay

Hyaluronidase Enzyme Purification

Gel-Filtration on Sephadex-G75

Ion Exchange Chromatography on DEAE Cellulose

Determination of Hyaluronidase Activity

SDS PAGE Electrophoresis

Characterization of the Partially-purified Enzymes

Effect of Temperature

Effect of pH

Initial velocity studies

Statistical Analysis

Result

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Purification of PLA₂ Enzyme

PLA₂ Activity Assay