Recombinant protein A/G (PAG) has a sequence coding for eight IgG binding sites and has enhanced interspecies affinity. High-frequency sampling of a PAG titration with IgG produces concentration profiles that are sensitive to the kinetic availability of the binding sites. The full kinetic model developed here for IgG binding sequentially to PAG shows only two distinct kinetic processes, describing an initial rapid association of two antibodies to PAG with a rate constant k-fast = (1.86 ± 0.08) × 10⁶ M^–1 s^–1 and a slower antibody binding process to all remaining sites, k-slow = (1.24 ± 0.05) × 10⁴ M^–1 s^–1. At equilibrium (after 1 h), the maximum IgG occupancy of PAG is 2.8 ± 0.5, conflicting with the genetic evidence of eight binding sites and suggesting significant steric hindrance of the neighboring IgG binding sites. The phosphate-buffered saline (PBS) solution defines a standard system setting, and this may be compared with other settings. The mean association rate of PAG–IgG_n in the standard setting is 282 ± 20% higher than when PAG is tethered to a surface. A systems biology approach requires that a model parameter set that defines a system in a standard setting should be transferable to another system. The transfer of parameters between settings may be performed using activity coefficients characterizing an effective concentration of species in a system, a_i = γ_ic_i. The activity correction, γ, for the eight-site occupancy is γ = 0.35 ± 0.06, and mapping from the standard setting to the solution setting suggests γ_PAG–IgG = 0.4 ± 0.03. The role of activity coefficients and transferability of kinetic parameters between system settings is discussed.

中文翻译：

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A / G蛋白与IgG之间多价配体结合相互作用的动力学分析：标准系统设置

重组蛋白A / G（PAG）具有编码八个IgG结合位点的序列，并且具有增强的种间亲和力。用IgG进行PAG滴定的高频采样会产生对结合位点的动力学利用率敏感的浓度曲线。此处开发的针对IgG依序与PAG结合的完整动力学模型仅显示了两个不同的动力学过程，描述了两种抗体与PAG的初始快速缔合，其速率常数k -fast =（1.86±0.08）×10 ⁶ M ^–1 s ^{– 1}和较慢的抗体与所有剩余位点的结合过程，k -slow =（1.24±0.05）×10 ⁴ M ^–1 s ^–1。在平衡时（1小时后），PAG的最大IgG占用率为2.8±0.5，与八个结合位点的遗传证据相矛盾，并暗示了相邻IgG结合位点的显着空间位阻。磷酸盐缓冲盐水（PBS）溶液定义了标准系统设置，可以将其与其他设置进行比较。在标准设置中，PAG–IgG _n的平均缔合率比将PAG拴在表面上的平均缔合率高282±20％。系统生物学方法要求以标准设置定义系统的模型参数集应可转移到另一个系统。可使用系统中的表征物种的有效浓度活度系数来执行的参数设置之间的转移，一个_我=γ_我Ç_我。八个位置的活度校正值γ= 0.35±0.06，从标准设置到溶液设置的映射表明， _γPAG–IgG = 0.4±0.03。讨论了活度系数的作用和动力学参数在系统设置之间的传递性。