Temperature Dependence of Benzyl Alcohol- and 8-Anilinonaphthalene-1-sulfonate-Induced Aggregation of Recombinant Human Interleukin-1 Receptor Antagonist

Abstract

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.

Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

“…In a Bacillus subtilis strain, the lethal concentration of BzA increased the membrane fluidity (43). For protein structures, BzA induced the aggregation of proteins, such as human interleukin (44) and interleukin-1 receptor antagonist (45), by partially unfolding these proteins (46,47). Unfolding actions were also reported for short-chain alcohols (48).…”

External Factors Affecting Protein Aggregation

“…The adverse effect of benzyl alcohol was also observed during reconstitution of lyophilized rhIL1ra, where reconstitution of the dried solid with 0.9% (w/v) benzyl alcohol caused a greater degree of protein aggregation than reconstitution with water. 237 Later, Roy et al 114 found the effect of benzyl alcohol is temperature dependent. While benzyl alcohol at 0.9% accelerated aggregation of rhIL -1ra at 37 ° C (almost no monomers after 7 days), it showed no effect at 25 ° C, corresponding to temperature -dependent perturbation in the tertiary structure of rhIL -1ra in the presence of benzyl alcohol.…”

“…Increasing the temperature slightly above room temperature may cause some proteins to aggregate on much shorter timescales. At 37 ° C, many proteins aggregate easily, including GCSF 1.0 -1.5 mg/mL at pH 7, 18,108 recombinant human keratinocyte growth factor (rhKGF) at 0.5 mg/mL at pH 7, 109 bovine insulin at pH 2.5, 110 tubulin at pH 6.4, 111 recombinant human platelet -activating factor acetylhydrolase (rhPAF -AH, pI = 6.7) after reconstitution (also at room temperature), 112 rhIL -1ra, 113,114 IgG molecules at different pH values, 61,115,116 and mutant transthyretin ( ∆ N50) at pH 7.5. 66 Other examples include aggregation of human serum albumin (HSA) at 40 ° C, 117 aggregation of fi refl y luciferase in 25 mM tricine at pH 7.5 at 42 ° C, 80 and aggregation of recombinant factor VIIa (rFVIIa) at different pH values at 45 ° C. 118 In addition to the infl uence on protein stability, higher temperature increases protein diffusion, frequencies of molecular collisions, and hydrophobic interactions, promoting protein aggregation.…”

“…66 Other examples include aggregation of human serum albumin (HSA) at 40 ° C, 117 aggregation of fi refl y luciferase in 25 mM tricine at pH 7.5 at 42 ° C, 80 and aggregation of recombinant factor VIIa (rFVIIa) at different pH values at 45 ° C. 118 In addition to the infl uence on protein stability, higher temperature increases protein diffusion, frequencies of molecular collisions, and hydrophobic interactions, promoting protein aggregation. 13,25 Roy et al 114 demonstrated that increasing the temperature from 25 to 37 ° C changes the nature of the interaction between 8 -anilinonaphthalene -1 -sulfonate and rhIL -1ra from electrostatic to both electrostatic and hydrophobic, and increasing temperature also increased the degree of hydrophobic interaction between benzyl alcohol and rhIL -1ra. Vetri and Militello 30 found that the aggregation rate of β -lactoglobulin gradually increased with incubation temperature from 10 to 80 ° C mainly through hydrophobic interactions.…”

“…236 Presumably due to the hydrophobic nature of the interaction, the degree of interactions increased with temperature. 114 Benzyl alcohol is perhaps the most studied preservative in protein formulation literature. Its effect was evaluated in several studies on the aggregation of rhIL -1ra.…”