Singh R Rajesh, Chang Jui-Yoa
Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, U.S.A.
Biochem J. 2004 Feb 1;377(Pt 3):685-92. doi: 10.1042/BJ20030968.
Bovine pancreatic PLA(2) (phospholipase A(2)) is a 14 kDa protein whose structure is highly cross-linked by seven disulphide bonds. We investigated the structural stability of this enzyme by the method of 'disulphide-scrambling' with denaturants such as urea, GdmCl (guanidine hydrochloride), GdmSCN (guanidine thiocyanate) and at high temperatures in the presence of 2-mercaptoethanol (0.2 mM) as thiol initiator. Reverse-phase HPLC was used to follow denaturation. To denature 50% of the native protein, 1.25 M GdmSCN, approx. 3 M GdmCl and higher than 8 M urea were required. Only 20% of the protein was denatured after 2 h at 60 degrees C, whereas complete denaturation was seen after 2 h at 70 degrees C and within 30 min at 80 degrees C. A distinct enhancement of stability was observed when denaturation was conducted in the presence of 10 mM calcium chloride, which has not been reported previously. CD studies of GdmCl denaturation of bovine PLA(2) showed that 2.5 M GdmCl was required to denature 50% of the protein in the presence of 0.2 mM 2-mercaptoethanol (in agreement with the HPLC analysis), whereas 6.4 M GdmCl was necessary to denature 50% of the protein in the absence of a thiol initiator. Conformational stability (Delta G (water)) was estimated to be 8.7 kcal/mol (1 cal=4.184 J) by 'disulphide-intact' denaturation (where 'native' disulphide framework was unaffected) and 2.5 kcal/mol by 'disulphide-scrambling' denaturation (involved breaking of native disulphides and formation of 'non-native' ones). The difference, Delta(Delta G (water)), of 6.2 kcal/mol was the conformational stability contributed by the 'native-framework' of seven disulphides. Using bovine PLA(2) as an example, we have demonstrated a novel comparative technique, where the conformational stability study of a disulphide-containing protein, with a common denaturant, in both the presence and absence of catalytic amounts of a thiol initiator can be used as a convenient method to estimate selectively and quantitatively the actual contribution of the 'native disulphide bond network' towards the global conformational stability of the protein.
牛胰磷脂酶A₂（PLA₂）是一种14 kDa的蛋白质，其结构通过七个二硫键高度交联。我们通过“二硫键重排”方法，在变性剂如尿素、盐酸胍（GdmCl）、硫氰酸胍（GdmSCN）存在的情况下，以及在高温下并以2 - 巯基乙醇（0.2 mM）作为硫醇引发剂来研究这种酶的结构稳定性。采用反相高效液相色谱法跟踪变性过程。使50%的天然蛋白质变性，需要约1.25 M GdmSCN、约3 M GdmCl和高于8 M的尿素。在60℃下2小时后只有20%的蛋白质变性，而在70℃下2小时以及80℃下30分钟内可观察到完全变性。当在10 mM氯化钙存在下进行变性时，观察到稳定性有明显增强，这在以前未见报道。对牛PLA₂进行GdmCl变性的圆二色性研究表明，在0.2 mM 2 - 巯基乙醇存在下使50%的蛋白质变性需要2.5 M GdmCl（与高效液相色谱分析结果一致），而在没有硫醇引发剂时使50%的蛋白质变性则需要6.4 M GdmCl。通过“完整二硫键”变性（其中“天然”二硫键框架未受影响）估计构象稳定性（ΔG（水））为8.7 kcal/mol（1 cal = 4.184 J），通过“二硫键重排”变性（涉及天然二硫键断裂并形成“非天然”二硫键）估计为2.5 kcal/mol。两者差值Δ（ΔG（水））为6.2 kcal/mol，这是七个二硫键的“天然框架”所贡献的构象稳定性。以牛PLA₂为例，我们展示了一种新颖的比较技术，即对于含二硫键的蛋白质，在有和没有催化量硫醇引发剂的情况下，使用一种常见变性剂进行构象稳定性研究，可以作为一种方便的方法，选择性地、定量地估计“天然二硫键网络”对蛋白质整体构象稳定性的实际贡献。
