Figure 3.

Effect of heat, proteolytic activity of venom and a reducing agent (DTT) on MetHb production. (A) N. nigricollis venom was heat treated at 65 °C or 95 °C for 10 min prior to cooling the venom to room temperature and mixing with ovine Hb. The level of absorbance at 500 nm and 630 nm was measured after 16 h. (B) SDS-PAGE (Coomassie stained) gel showing the protein profile of Hb before (as a control) and after incubation with different concentrations of N. nigricollis venom for 16 h. Lanes, MW—molecular weight marker, Lane 1 and 4: Venom (12 µg) + haemoglobin (3 µg). Lane 2 and 5: Venom (6 µg) + haemoglobin (3 µg). Lane 3 and 6: Venom (3 µg) + haemoglobin (3 µg). Lanes 1-3; after 16 h incubation and lanes 4–6; as controls at 0 h. Lane 7: N. nigricollis venom (3 µg) alone and Lane 8: Hb (3 µg) alone. The image shown is representative of three separate experiments. (C) N. nigricollis venom was treated with various concentrations (0.5 mM, 1 mM or 5 mM) of reducing agent, DTT for 10 min prior to mixing with ovine Hb and incubating for another 16 h. The level of absorbance was measured as shown above. Data represent mean ± SEM (n = 3). The p values shown were as calculated by two-way ANOVA followed by post-hoc Tukey’s test using GraphPad Prism 7 (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). The heated (A) or DTT (C)-treated samples were all compared with the respective positive controls (untreated venom), and the negative control used was PBS.