Bovine pancreatic PLA2 (phospholipase A2) 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 °C, whereas complete denaturation was seen after 2 h at 70 °C and within 30 min at 80 °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 PLA2 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 (ΔGwater) 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, Δ(ΔGwater), of 6.2 kcal/mol was the conformational stability contributed by the ‘native-framework’ of seven disulphides. Using bovine PLA2 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.

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