Structural Changes and Aggregation Process of Cu/Containing Amine Oxidase in the Presence of 2,2,2-Trifluoroethanol

Abstract

Conformational and structural changes of lentil seedlings amine oxidase (LSAO) were studied in the presence of trifluoroethanol (TFE) by spectroscopic and analytical techniques. At TFE concentrations up to 5%, the induction of a structural transition from beta-sheet to alpha-helix and up to 10% TFE a structural transition from alpha-helix to beta-sheet as well as inactivation of the enzyme are observed. At TFE concentrations between 10-35%, LSAO proves to be prone to aggregation and beyond 35% TFE leads to a non-native protein structure with a high alpha-helix content. The obtained results revealed that the aggregation of LSAO is strongly linked to the nature of secondary structures.

External Factors Affecting Protein Aggregation

“…TFE above 35% promoted a nonnative structure of lentil seedling amine oxidase with a high alpha -helix content and between 10% and 35% facilitated aggregation of the protein. 272 TFE at 5 -70% (v/v) promoted aggregation of CT at pH 7.0 at 25 ° C, and maximal aggregation was observed around 32% TFE. 179 TFE at 25% (v/v) induced partial unfolding (within a few seconds) of human muscle AcP at 25 ° C with subsequent formation of globular aggregates (60 -200 nm in diameter), and 5X dilution (to 5% TFE) led to rapid refolding of partially unfolded monomeric proteins and disaggregation of 60 -200 nm globular aggregates.…”

Stabilization challenges and aggregation in protein-based therapeutics in the pharmaceutical industry

“…Under normal physiological conditions, aggregation typically does not occur, but it can be induced by certain metal ions. 111,112 Metal ions like Zn 2+ , Cu 2+ and Pb 2+ can inhibit or accelerate aggregation. Overall, understanding the precise impact of cations on protein aggregation remains a challenge, and further research is needed to establish a consensus.…”

Impact of Residual Impurities and Contaminants on Protein Stability

“…Indeed, 2,2,2‐trifluoroethanol (TFE) was found to enhance α‐helical conformations with a simultaneous increase in aggregation of bovine serum albumin (BSA) because of the promotion of intermolecular cluster formation . TFE was also shown to increase the aggregation of several other proteins, including α‐chymotrypsin, protein L, and human muscle acylphosphatase, and lentil seedling amine oxidase . In many cases, however, proteins can tolerate a significant amount of organic solvents such as human and bovine IgGs at an acetonitrile concentration up to 50%–60% and lysophospholipase in a 0%–50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile .…”

“…118 TFE was also shown to increase the aggregation of several other proteins, including "-chymotrypsin, 119 protein L, 120 and human muscle acylphosphatase, 121 and lentil seedling amine oxidase. 122 In many cases, however, proteins can tolerate a significant amount of organic solvents such as human and bovine IgGs at an acetonitrile concentration up to 50%-60% 123 and lysophospholipase in a 0%-50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. 124 In some cases, organic solvent, such as DMSO can help to reduce protein aggregation, as observed for arginine kinase aggregation during refolding 125 or convert fibrils to monomers such as 50% AcOH/water for fibrillated human calcitonin.…”