Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of α-chymotrypsin

Rezaei‐Ghaleh, Nasrollah; Ebrahim‐Habibi, Azadeh; Moosavi-Movahedi, Ali Akbar; Nemat‐Gorgani, Mohsen

doi:10.1016/j.abb.2006.10.031

Cited by 49 publications

(64 citation statements)

References 45 publications

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“…Rezaei -Ghaleh et al 179 found that the log initial rate of TFE -induced aggregation of CT at 25 ° C decreased rather linearly with the square root of ionic strength (10 -40 range), using several salts including NaCl, KCl, NH 4 SCN, and so on. Certain lipids do not infl uence the aggregation of insulin at 0.05% (w/w) at pH 1.9 at 60 ° C, but in the absence of 0.1 M NaCl at pH 1.9, they enhanced the rate of aggregation.…”

Section: Ionic Strength and Other Stability -Infl Uencing Factorsmentioning

confidence: 99%

“…271 While glycerol at 10% reduced the amount of aggregates of Fc fusion protein during elution at pH 3.6, 10% propylene glycol signifi cantly increased the rate of aggregation. 160 Trifl uoroethanol as an alpha -helix enhancer induces aggregation of several proteins, such as CT, 179 protein L, 82 and human muscle AcP. 258 Its effects on protein aggregation vary signifi cantly depending on its concentration.…”

Section: Organic Solventsmentioning

confidence: 99%

“…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. 107 TFE at 10% did not promote aggregation of BSA at 70 ° C even with a signifi cant reduction in T m from 65 to 53 ° C but at 20% inhibited the aggregation process dramatically.…”

Section: Organic Solventsmentioning

confidence: 99%

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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The behavior of biotherapeutic proteins is signifi cantly different from small molecule drugs both in liquid and solid states, especially in terms of physical stability. One such property is the high tendency of protein molecules to aggregate under a variety of conditions. The aggregation tendency is arguably the most common and troubling manifestation of protein instability during the development of protein biotherapeutics. 1 Protein aggregates usually exhibit either reduced or, in many cases, no biological activity. 2 -5 A clear correlation was established between loss of recombinant factor VIII (rFVIII ) and its degree of aggregation as shown in Fig. 4.1 . 6 To achieve effective prevention or inhibition of protein aggregation, it is of paramount importance to understand all the possible factors that affect or control the protein aggregation process.Many factors have been identifi ed and reported in the literature affecting the process and rate of protein aggregation. These factors can be roughly divided into two major categories: internal (protein structure related) and external (protein environment related). Chapter 3 in this book has focused extensively on factors that belong to the fi rst category. This chapter focuses on the external factors that affect the process and rate of protein aggregation. To facilitate discussion, these external factors are further divided into the following sections: (1) temperature; (2) solution conditions and composition (pH, buffer type and concentration, ionic strength, excipients and level, protein concentration, metal ions, denaturing and reducing agents, impurities, organic solvents, containers/closures, sources of proteins, sample treatment, and analytical methodologies); (3) processing steps (fermentation/expression,

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Section: Ionic Strength and Other Stability -Infl Uencing Factorsmentioning

confidence: 99%

Section: Organic Solventsmentioning

confidence: 99%

Section: Organic Solventsmentioning

confidence: 99%

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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“…Such conditions promote aggregation because they result in the partial unfolding of structured proteins and they allow the sampling of locally unfolded native-like states, both of which are possibly amyloidogenic states [7]. It has been demonstrated experimentally that the surface charges play an important role in the aggregation of proteins [8,9].…”

Section: Introductionmentioning

confidence: 99%

Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG

Kotormán

Simon

Borics

et al. 2015

PPL

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Abstract:The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 o C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol.

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“…In addition, at 45 % v/v TFE a 2-fold enhancement in the intensity of this dichroic signal is observed. The position of this new minimum is typical of largely extended β-sheet conformation, a structure prevalent in amyloid fibrillar aggregates [13,31]. Conversely, at 65 % v/v TFE the spectrum shows a change in its shape toward one with two negative bands: one at ~222 nm and an incipient band at ~208 nm, a feature typical of α-helical structure.…”

Section: Effect Of Tfe On Protein Conformationmentioning

confidence: 90%