Hirano Atsushi, Maruyama Takuya, Shiraki Kentaro, Arakawa Tsutomu, Kameda Tomoshi
Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan.
Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan.
J Chromatogr A. 2014 Dec 19;1373:141-8. doi: 10.1016/j.chroma.2014.11.032. Epub 2014 Nov 18.
A multimodal chromatography column containing 4-mercaptoethylpyridine (4-MEP) as a resin ligand is capable of binding antibodies. Arginine has been proven effective for eluting bound antibodies from the 4-MEP resin column. In this study, the effect of arginine on the binding of antibodies to 4-MEP resin was examined through equilibrium adsorption experiments using bovine γ-globulin (BGG). Arginine was found to efficiently reduce antibody adsorption onto 4-MEP resin more than solvents containing guanidine, urea and NaCl. Organic solvents, including ethylene glycol and glycerol, were also found to be effective for antibody desorption. These results suggest that the attraction between BGG and 4-MEP is based on hydrophobic interactions that can be weakened by arginine or the organic solvents. The use of arginine yielded similar results with globular proteins. Molecular dynamics simulations used to illuminate the mechanism of binding of an arginine molecule to a 4-MEP resin ligand molecule showed that the alkyl chain of the arginine side chain primarily interacts with the pyridine ring of the 4-MEP resin ligand through hydrophobic interactions. Interaction of the backbone or guanidinium group of arginine with the pyridine ring of the 4-MEP resin ligand played an insignificant role in their bindings. The hydrophobic interaction of the arginine side chain with the pyridine ring reduces the attraction between the antibodies and 4-MEP, leading to the efficient dissociation of the antibodies from the 4-MEP resin in the presence of arginine-containing solutions. Thus, utilization of arginine as an eluent can improve antibody purification by multimodal column chromatography.
一种以4-巯基乙基吡啶（4-MEP）作为树脂配体的多模式色谱柱能够结合抗体。已证明精氨酸可有效从4-MEP树脂柱上洗脱结合的抗体。在本研究中，通过使用牛γ-球蛋白（BGG）的平衡吸附实验，研究了精氨酸对抗体与4-MEP树脂结合的影响。发现精氨酸比含有胍、尿素和氯化钠的溶剂更能有效地减少抗体在4-MEP树脂上的吸附。还发现包括乙二醇和甘油在内的有机溶剂对抗体解吸有效。这些结果表明，BGG与4-MEP之间的吸引力基于疏水相互作用，而精氨酸或有机溶剂可削弱这种相互作用。使用精氨酸对球状蛋白也产生了类似的结果。用于阐明精氨酸分子与4-MEP树脂配体分子结合机制的分子动力学模拟表明，精氨酸侧链的烷基链主要通过疏水相互作用与4-MEP树脂配体的吡啶环相互作用。精氨酸的主链或胍基与4-MEP树脂配体的吡啶环之间的相互作用在它们的结合中起的作用微不足道。精氨酸侧链与吡啶环的疏水相互作用降低了抗体与4-MEP之间的吸引力，导致在含精氨酸的溶液存在下抗体从4-MEP树脂上有效解离。因此，使用精氨酸作为洗脱剂可通过多模式柱色谱法改善抗体纯化。
