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

Abstract BACKGROUND Rattlesnake venoms are a complex source of different toxin families with biotechnological and biomedical applications. However, the complexity of the sample and the low abundance of some toxins can restrict their study and applications. Toxin isolation usually requires multiple chromatographic steps, implying elevated operative costs and processing times. This raises the need for recovery and enrichment of those proteins and peptides. For that reason, we tested two liquid–liquid separation methods – aqueous two‐phase systems (ATPS) and three‐phase partition (TPP) – since these two alternatives have shown great potential for the primary recovery of different toxin families from rattlesnake venoms. RESULTS Polyethylene glycol (PEG)–citrate and ionic liquid (IL)–potassium phosphate ATPS and TPP based on t ‐butanol and ammonium sulfate were used to partition different toxin families from Crotalus molossus nigrescens and C. atrox venoms. PEG–citrate allowed a selective partition of proteins between both phases. The best enrichment case was observed with a myotoxin band (6.6 kDa) in the PEG‐rich phase from 2% to 41.3% of abundance. In IL–potassium phosphate systems, proteins showed a preference towards the IL‐rich phase with no selective partition. In TPP, proteins were partitioned mainly to the bottom phase and interphase, but serine proteases (29.1 kDa) were enriched from 3.7% to 56.6% in the top phase. CONCLUSION The present study demonstrated that PEG–citrate ATPS and TPP separation methods can generate a selective partition of some toxin families from rattlesnake venoms. These enrichment methods will facilitate the isolation of toxins, from animal venoms, that are relevant for biotechnological and biomedical applications. © 2024 Society of Chemical Industry (SCI).