Development of aqueous two-phase systems-based approaches for the selective recovery of metalloproteases and phospholipases A2 toxins from Crotalus molossus nigrescens venom

Abstract

Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA₂) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA₂ from Crotalus molossus nigrescens venom. A system with PEG 400 g mol^-1, volume ratio (V_R) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA₂ recovery. In systems with PEG 400 g mol^-1, V_R 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol^-1, V_R 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol-salt systems were also evaluated, however, failed to differentially partition PLA₂ and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses.

Keywords: Crotalus molossus nigrescens; Aqueous two-phase systems; Metalloproteases; Phospholipases A2; Recovery; Venom.

Fig. 1

Effect of PEG–potassium phosphate systems’ variables on the recovery of MPs and PLA₂. A Principal component analysis (PCA) of the recovery of MPs and PLA₂ in systems with a PEG molecular weight of 400, 1000 and 3350 g mol⁻¹, TLL 15, 25, 35 and 45% w/w and V_R 0.33, 1 and 3. Clear circles (○) denote the selected systems with the best partition characteristics. B SDS-PAGE of 15 μg of C. m. nigrescens venom (CMNv) and molecular weight marker (MWM). C SDS-PAGE of top and bottom phases (T and B, respectively) of selected ATPS from PCA analysis

Fig. 2

NaCl concentration effect on the recovery of MPs and PLA₂ in PEG–potassium phosphate systems. A Principal component analysis (PCA) analysis of the recovery metalloproteases (MPs) and phospholipases A2 (PLA₂) in selected systems at different NaCl concentrations (1, 3 and 5% w/w). Clear circles (○) denote the selected ATPS with the best partition characteristics. B SDS-PAGE of top and bottom phases (T and B, respectively) of selected ATPS from PCA analysis, 15 μg of C. m. nigrescens venom (CMNv) and molecular weight marker (MWM)

Fig. 3

Effect of pH on the recovery of MPs and PLA₂ in PEG–salt systems. A Principal component analysis (PCA) analysis of the recovery of MPs and PLA₂ in selected systems at different pH values (7.5–10). Clear circles (○) denote the selected ATPS with the best partition characteristics. B Effect of pH on MPs and PLA₂ activities. C SDS-PAGE of top and bottom phases (T and B, respectively) of selected ATPS from PCA analysis, 15 μg of C. m. nigrescens venom (CMNv) and molecular weight marker (MWM)

Fig. 4

Recovery of MPs and PLA₂ in ethanol–salt systems. A Principal component analysis (PCA) analysis of the recovery and purification of MPs and PLA₂ in ethanol–salt systems at different volume ratio (V_R) (0.33, 1 and 3). B SDS-PAGE of top and bottom phases (T and B, respectively) of ethanol–salt systems, 15 μg of C. m. nigrescens venom (CMNv) and molecular weight marker (MWM)