Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

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.