Involvement of molten globule state on the folding process of secretary proteins

人血清白蛋白（HSA）、卵黄转铁蛋白（N-OVT） n端半分子和卵清蛋白（OVA）在二硫苏糖醇和8 M尿素的存在下被还原变性。用中性缓冲液稀释样品，用cd -光谱评价蛋白质构象。对于HSA和N-OVT，发现再折叠，二硫还原形式采取部分折叠的熔融球状构象。当加入氧化形式的谷胱甘肽（GSSG）时，链内蛋白二硫键再生。这些数据表明，HSA和N-OVT在氧化再折叠过程中以熔融球状态作为中间体。相比之下，OVA在其二硫化物还原状态下显示出类似天然的构象。然而，半胱氨酸巯基的反应活性在天然形式和再折叠的二硫还原形式之间有显著差异：前者没有检测到活性巯基，而后者检测到两个巯基。通过将GSSG添加到还原态，卵清蛋白转化为与天然半胱氨酸配对（Cys73-CYsl2O）的二硫键形式。从这些数据中，我们得出结论，具有多个二硫化物和结构域结构的蛋白质，如HSA和N-OVT，采用熔融球状构象作为氧化再折叠的中间产物，而具有单一二硫化物和单一结构域的蛋白质，如OVA，在二硫化物还原状态下采用类似天然的构象。

Human serum albumin(HSA), N-terminal half-molecule of ovotransferrin(N-OVT), and ovalbumin(OVA)were reduced and denatured in the presence of dithiothreitol and 8 M urea. The samples were diluted with a neutral buffer, and protein conformation was evaluated by CD-spectrum. With regards to HSA and N-OVT, the refolded, disulfide-reduced form was found to take a partially folded molten globule-like conformation. When oxidized form of gultathione(GSSG)was added to the state, the intrachain protein disulfide bonds were regenerated. These data indicated that HSA as well as N-OVT take a molten globulelike state during oxidative refolding as an intermediate. In contrast, OVA showed the native-like conformation in its disulfide-reduced state as evaluated by CD-spectrum. The reactivity of cysteine sulfhydryls, however, was significantly different between the native form and the refolded, disulfide-reduced form : no reactive sulfhydryl was detected in the former form, but two sulfhydryls were detected in the latter form. By the addition of GSSG to the reduced state, ovalbumin was transformed to the disulfide bonded form with native cyteine pairing(Cys73-CYsl2O). From these data, we concluded that a protein with many disulfide and domain structure, such as HSA and N-OVT, takes a molten globule-like conformation as a intermediate for oxidative refolding, while a protein with single disulfide and single domain, such as OVA, takes a naive-like conformation in the disulfide-reduced state.