title: Mechanism for benzyl alcohol-induced aggregation of recombinant human interleukin-1 receptor antagonist in aqueous solution.
abstract: Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in aqueous solution. The loss of native monomer during incubation at 37 degrees C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE-HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H-D exchange was accelerated and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL-1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation-competent species. Sucrose partially inhibited benzyl alcohol-induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation-prone forms.
authors.affiliation: Department of Pharmaceutical Science, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shouvik
authors.initials: S
authors.lastname: Roy
authors.forename: Latoya S
authors.initials: LS
authors.lastname: Jones
authors.forename: Sampathkumar
authors.initials: S
authors.lastname: Krishnan
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Kerwin
authors.forename: Byeong S
authors.initials: BS
authors.lastname: Chang
authors.forename: Mark C
authors.initials: MC
authors.lastname: Manning
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
mesh_terms: D019905:Benzyl Alcohol / Q000494:pharmacology*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000032:analysis / Q000378:metabolism*; D012996:Solutions; D014867:Water / Q000378:metabolism
chemical_list: C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D012996:Solutions; D014867:Water; D019905:Benzyl Alcohol