Chang B S, Beauvais R M, Arakawa T, Narhi L O, Dong A, Aparisio D I, Carpenter J F
Department of Pharmaceutics, Amgen Inc., Thousand Oaks, California 91320-1789, USA.
Biophys J. 1996 Dec;71(6):3399-406. doi: 10.1016/S0006-3495(96)79534-6.
The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.
对重组人白细胞介素-1受体拮抗剂（rhIL-1ra）在30℃水溶液中储存期间形成的降解产物进行了表征。储存样品的阳离子交换色谱显示在天然蛋白之前（P1）和之后（L2）洗脱的两个主要新峰，它们是可相互转化的。尺寸排阻色谱和电泳证明P1和L2组分都是通过非共价相互作用形成的不可逆二聚体。白细胞介素-1竞争试验表明，以每个单体计，P1和L2二聚体保留了天然单体约三分之二的活性。红外和远紫外圆二色光谱表明，P1二聚体形成时二级结构仅有微小变化。然而，近紫外圆二色光谱的变化表明P1二聚体中存在天然蛋白中不存在的二硫键。羧甲基化前后的质谱分析和胰蛋白酶图谱分析表明，P1二聚体在Cys-66和Cys-69之间含有分子内二硫键。虽然天然蛋白在单独缓冲液中转化为P1二聚体是不可逆的，但通过用盐酸胍使P1二聚体变性并通过透析使其复性，可以重新获得天然单体，这表明分子内二硫键不干扰重折叠。对30℃储存期间P1形成的时间进程分析表明，该过程遵循一级动力学而非二级动力学，这表明限速步骤不是二聚化。有人提出单体的构象变化是P1二聚体降解产物形成的限速步骤。蔗糖使天然单体对该过程具有稳定性。这一结果可以用该添加剂的一般稳定机制来解释，这是由于其优先从蛋白质表面排除。
