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 (VR) 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, VR 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, VR 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. Graphic Abstract

Primary recovery strategies of low‐molecular‐weight toxins from Crotalus molossus nigrescens and Crotalus atrox using aqueous two‐phase and three‐phase partition systems

“…It is important to remark that some toxin families, such as Dis, MP, and SP, have a broad range of molecular masses, and in SDS-PAGE it is common to observe a toxin family represented by different bands. 20,28,[35][36][37][38] The abundance percentage of the SDS-PAGE bands was calculated using the following equation:…”

“…This partition phenomenon also was observed by Enriquez-Ochoa et al, where ATPS generated the partition of MPs to the PEG-rich phase. 20 Figure 6. SDS-PAGE visualization of partitioned Crotalus molossus nigrescens crude venom using PEG 6000-citrate systems.…”

“…This behavior could be generated due to its structural diversity, as the SP can have a wide range of pI (3-9) and molecular weight, and moderate hydrophobicity. 20,46 Three-phase partition system (TPP) As an alternative strategy for the enrichment of proteins and peptides, a three-phase partition system was explored in this study, using t-butanol and ammonium sulfate (20%, 40%, and 60% saturation). Figure 4 shows the partition behavior of proteins and peptides for both venoms through SDS-PAGE using Tris-glycine and Tris-tricine gels.…”

“…Only polyethylene glycol (PEG)-phosphate, PEG-sulfate, and alcohol-salt systems have been tested for the partition of proteases and phospholipases A 2 from Bothrops alternatus, B. jararacussu, and Crotalus molossus nigrescens (CMN) venoms. [17][18][19][20] On the other hand, TPP is a fast and easy separation method that allows concentration and partial purification of different molecules. 21 Three-phase separation occurs when the biological sample is mixed with ammonium sulfate, at a given saturation percentage, and t-butanol.…”

Investigating and Optimizing Insulin Partitioning with Conjugated Au Nanoparticles in Aqueous Two-Phase Systems Using Response Surface Methodology

“…There is a direct correlation between the partitioning of biomolecules, which is quantified by the partition coefficient ( K ), and their physicochemical properties. These properties include isoelectric points, sizes, surface hydrophobicity, polymer molecular weight, polymer/salt concentrations, pH, and temperature. ,, The separations’ selectivity in ATPS has been enhanced under various conditions, which are problematically associated with the need to make structural changes in ATPSs or to modify biomolecules. , It is therefore becoming more necessary to find techniques that do not require the reformulation of system structures or biomolecules. By creating a mechanism that facilitates the transfer of the biomolecule between the two phases, biomolecules partitioning enhances in ATPS. , Nanoparticles fulfill this demand and improve biomolecule partitioning.…”