Hong Dong-Pyo, Gozu Masayo, Hasegawa Kazuhiro, Naiki Hironobu, Goto Yuji
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2002 Jun 14;277(24):21554-60. doi: 10.1074/jbc.M200188200. Epub 2002 Apr 9.
Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.
β2-微球蛋白（β2-m）是透析相关淀粉样纤维的主要成分，其链内二硫键埋藏于天然结构内部。我们通过分析二硫键对还原试剂二硫苏糖醇的反应性，研究了β2-m淀粉样纤维的构象。尽管天然结构中的二硫键对还原具有高度抗性，但在pH 2.5条件下制备的淀粉样纤维中的二硫键，在pH 8.5时会被50 mM的二硫苏糖醇逐步还原。由于已知在酸性条件下制备的β2-m淀粉样纤维在中性pH下会解聚，我们研究了解聚与二硫键还原之间的关系。结果表明，淀粉样纤维中的二硫键对还原具有抗性，且还原发生在解聚过程中。另一方面，未成熟细丝（在pH 2.5的高盐条件下制备的细而柔韧的细丝）中的二硫键在pH 8.5时比淀粉样纤维中的二硫键更容易被还原，这表明二硫键暴露于溶剂中。综上所述，酸变性后一旦暴露于溶剂中的二硫键，在淀粉样纤维形成过程中可能会逐渐埋藏于其内部。
