Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.
J Pharm Sci. 2012 Apr;101(4):1391-9. doi: 10.1002/jps.23039. Epub 2011 Dec 28.
The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional (15) N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein's structure, the fine structure of the desorbed protein was nearly identical to the protein's structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption.
本研究旨在深入探究一种模型抗原——噬菌体 T4 溶菌酶的半胱氨酸缺陷突变体——在与含铝佐剂相互作用后的精细结构命运。荧光光谱法和差示扫描量热法被用于比较蛋白质在溶液中和吸附到含铝佐剂上时的热稳定性。使用一种外在荧光探针，8-苯胺-1-萘磺酸（ANS），研究了可及的疏水面区的差异。与其他模型抗原一样，吸附到佐剂上后，蛋白质的表观热稳定性降低。ANS 光谱表明，吸附到佐剂上导致蛋白质的疏水区暴露增加。静电相互作用驱动了吸附，而用高离子强度缓冲液破坏这些相互作用有助于从佐剂中收集释放的蛋白质的二维（15）N 异核单量子相干核磁共振数据。尽管吸附蛋白的稳定性变化表明蛋白结构发生了变化，但解吸蛋白的精细结构与无佐剂配方中的蛋白结构几乎相同。因此，在解吸时，未观察到吸附蛋白的结构变化导致的热稳定性降低。
