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 . 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 , in which he revealed that PLA2 isolated has 15KDa molecular mass and that of , which reported that at the PLA2 isolated from snake has molecular mass 15.6KDa.  and  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 37oC and 40oC for Hyaluronidase and PLA2 respectively. While those of N. nigricollis was shown to have an optimum temperatures of 35oC and 55oC 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 , which show Hyaluronidase and PLA2 to have an optimal temperature of 35oC and 7.0oC 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 Vmax value of 30.567mg/min, while E. ocellatus Hyaluronidase has the lowest Vmax of 2.2563tru/min. E. ocellatus PLA2 however has the highest Km value of 4.5378mg/ml as shown in Table 5. This was compared with work done by , 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.