Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed. The authors used the scrambled disulfide bond technique to generate conformationally-altered isomers of the catalytic domain of mouse PCSK9. The focus was on the immune response of four X-isomers and their effects on plasma cholesterol and triglyceride levels in both C57BL/6J and Apoe-/- mice. The authors showed that the four immunogens produced significant immunogenicity against native PCSK9 to day 120 after immunization of C57BL/6J and Apoe-/- mice. This resulted in significantly decreased plasma cholesterol levels in C57BL/6J mice, and to a lesser degree in Apoe-/- mice. The X-PCSK9-B1 treated mice had increased LDL receptor mRNA and protein levels at day 120 after treatment. Thus, this study provides a new, potentially promising approach that uses long-term immunotherapy for a treatment of hypercholesterolemia.

Physical and functional antagonism between tumor suppressor protein p53 and fortilin, an anti-apoptotic protein.

Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.

Diverse pathways of oxidative folding of disulfide proteins: underlying causes and folding models.

The pathway of oxidative folding of disulfide proteins exhibits a high degree of diversity, which is manifested mainly by distinct structural heterogeneity and diverse rearrangement pathways of folding intermediates. During the past two decades, the scope of this diversity has widened through studies of more than 30 disulfide-rich proteins by various laboratories. A more comprehensive landscape of the mechanism of protein oxidative folding has emerged. This review will cover three themes. (1) Elaboration of the scope of diversity of disulfide folding pathways, including the two opposite extreme models, represented by bovine pancreatic trypsin inhibitor (BPTI) and hirudin. (2) Demonstration of experimental evidence accounting for the underlying mechanism of the folding diversity. (3) Discussion of the convergence between the extreme models of oxidative folding and models of conventional conformational folding (framework model, hydrophobic collapse model).

Targeted disruption of the gene encoding the murine small subunit of carboxypeptidase N (CPN1) causes susceptibility to C5a anaphylatoxin-mediated shock.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1(-/-) mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1(-/-)/C5aR(-/-) but not in CPN1(-/-)/C3aR(-/-) mice. Moreover, CPN1(-/-) mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1(-/-) mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation.

Folding of small disulfide-rich proteins: clarifying the puzzle.

The process by which small proteins fold to their native conformations has been intensively studied over the past few decades. The particular chemistry of disulfide-bond formation has facilitated the characterization of the oxidative folding of numerous small, disulfide-rich proteins with results that illustrate a high level of diversity in folding mechanisms, differing in the heterogeneity and native disulfide-bond content of their intermediates. Information from folding studies of these proteins, together with the recent structural determinations of predominant intermediates, has provided new molecular insights into oxidative folding and clarifies the major rules that govern it.

Synthesis and immune response of non-native isomers of vascular endothelial growth factor.

Native proteins often lack immunogenicity and thus limit vaccine and mAb development. We described here a unique method to enhance the immunogenicity of native proteins. This is achieved by creating non-native isomers of disulfide proteins (X-isomers) using the method of disulfide scrambling. X-isomers have the potential to be developed as vaccines and effective immunogens, as they are capable of breaking the immune tolerance and eliciting antibodies that cross-react with the native protein. In this report, we describe production of X-isomers of vascular endothelial growth factor (X-VEGF). The aim is to develop X-VEGF for cancer immunotherapy targeting reduction of VEGF. The production of mouse X-VEGF is achieved by expressing the short version of VEGF (1-110) commonly shared by all VEGF isoforms, with two Cys --> Ala mutations at Cys(51) and Cys(60) to generate R-VEGF(110) (R stands for fully reduced). R-VEGF(110) was then allowed to undergo oxidative folding in the absence of denaturant to form N-VEGF(110) (N stands for native) or in the presence of denaturant to generate five fractions of X-VEGF(110) isomers. While N-VEGF(110) exhibits only marginal immunogenicity in mice, all five fractions of X-VEGF(110) isomers were shown to elicit high titers of antibodies that cross-react with N-VEGF(110). In sera of immunized mice, the amounts of anti-N-VEGF antibodies elicited by X-VEGF(110) isomers range from 54 to 186 mug/mL, which are compatible with or greater than the concentration required for effective therapy using anti-VEGF MAbs. The underlying mechanism of enhanced immunogenicity of X-VEGF(110) is investigated and elaborated. These data suggest that X-VEGF(110) isomers are potential compounds in developing active immunotherapy for treatment of VEGFR bearing tumors and the wet form of age-related macular degeneration.

Rapid and irreversible reduction of protein disulfide bonds.

This report describes the development of a method that enables a rapid (less than 20s), quantitative, and irreversible reduction and inactivation of disulfide-containing proteins at room temperature (20 to 25 degrees C). The formula comprises the ingredients of optimized concentrations of denaturant, reductant, and hydroxide ion. The novelty of this formula is the application of a potent hydroxide ion in the concoction. The component of hydroxide ion serves two major functions. (1) It accelerates the cleavage of disulfide bonds mediated by the reducing agent and denaturant, leading to an instant and quantitative reduction of disulfide proteins. (2) It triggers a rapid covalent destruction of sulfhydryl groups and disulfide bonds via the mechanism of base-catalyzed beta-elimination, thus leading to the irreversible and permanent abolition of disulfide bonds. The usefulness of this formula has been demonstrated here with the effective and rapid reduction of numerous highly stable disulfide-containing proteins, including cardiotoxin and prion aggregates.

Structural heterogeneity of 6 M GdmCl-denatured proteins: implications for the mechanism of protein folding.

An in vitro experiment with protein folding is typically initiated with 6 M GdmCl-denatured proteins, which are generally considered fully unfolded. However, studies conducted by various laboratories have shown that many 6 M GdmCl-denatured proteins are structurally heterogeneous and still retain nativelike residual structures. The extent of conformational heterogeneity of the 6 M GdmCl-denatured protein has significant implications for the folding landscape as well as the interpretation of the observed early stage folding mechanism. Using the method of disulfide scrambling, we are able to gain rough insight into the diverse structural properties of 6 M GdmCl-denatured proteins. It demonstrates that most 6 M GdmCl-denatured proteins are approximately fully denatured, but partially unfolded. Most of them comprise diverse conformational isomers. We review here the cumulative evidence obtained from various laboratories and also provide experimental data obtained in our laboratory.

Conformational isomers of denatured and unfolded proteins: methods of production and applications.

Conformational isomers of denatured-unfolded proteins are rich in numbers and varied in shapes. They represent an opulent resource of biological molecules that have remained unexploited. The major obstacle in utilizing this untapped potential is that it is inherently difficult to isolate and characterize pure conformational isomers, not only because of the excessive large number, but also because of their instability and rapid inter-conversion. Our lab has developed a method for trapping selected conformational isomers of denatured proteins that are amenable to isolation, characterization and further applications. The method has potential usefulness, ranging from the comprehensive structural characterization of denatured proteins, to the elucidation of pathways of protein unfolding-folding, to the production of unlimited structurally defined non-native protein isomers for biomedical applications.

Diversity of folding pathways and folding models of disulfide proteins.

Comprehensive understanding of the mechanism of protein folding requires the elucidation of both a folding pathway and a folding model. This entails characterization of the properties and structures of folding intermediates populated along the folding pathway, as well as the formation and interplay of secondary structures and tertiary structures along the course of folding. Using the conventional unfolding-refolding technique, there are limitations of acquiring these data in detail because of the inherent difficulty of trapping and analysis of folding intermediates. The technique of oxidative folding, in contrast, permits trapping, isolation, and further structural characterization of folding intermediates at any stage of the folding process. In this brief review, we present the potential of the technique of oxidative folding for concurrent analysis of both folding pathways and folding models.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo.

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

Conformational stability of secretory leucocyte protease inhibitor: a protein with no hydrophobic core and very little secondary structure.

Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymotrypsin. The EMBO J. 7, 345-352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (DeltaG(H20)) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.

Oxidative folding of hirudin in human serum.

Human serum contains factors that promote oxidative folding of disulphide proteins. We demonstrate this here using hirudin as a model. Hirudin is a leech-derived thrombin-specific inhibitor containing 65 amino acids and three disulphide bonds. Oxidative folding of hirudin in human serum is shown to involve an initial phase of rapid disulphide formation (oxidation) to form the scrambled isomers as intermediates. This is followed by the stage of slow disulphide shuffling of scrambled isomers to attain the native hirudin. The kinetics of regenerating the native hirudin depend on the concentrations of both hirudin and human serum. Quantitative regeneration of native hirudin in undiluted human serum can be completed within 48 h, without any redox supplement. These results cannot be adequately explained by the existing oxidized thiol agents in human serum or the macromolecular crowding effect, and therefore indicate that human serum may contain yet to be identified potent oxidase(s) for assisting protein folding.

Entropic folding pathway of human epidermal growth factor explored by disulfide scrambling and amplified collective motion simulations.

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extra-cellular domains. Human EGF is a small, single-chain protein comprising three distinct loops (A, B, and C), which are connected by three disulfide bridges (Cys6-Cys20, Cys14-Cys31, and Cys33-Cys42). These disulfide bridges are essential for structural stability and biological activity. EGF was extensively studied by disulfide scrambling, an experimental technique for the conformational entrapment of intermediate states, which allows us to study the folding pathway of proteins containing disulfide bonds. The experimental results showed that there is a major 2-disulfide intermediate (denoted EGF-II) and that the native disulfide bonding pattern is less prevalent in one of the mutants. In this article, we investigated for the first time the solution conformations of wild-type EGF, EGF-II, and the mutant S9C through extensive molecular dynamics (MD) simulations in water using both the standard MD technique and a recently developed amplified-collective-motion (ACM) sampling method. Compared to standard MD simulations, we achieved a much more enhanced sampling by the ACM simulations, and the structures were sufficiently relaxed to estimate configurational entropies. The simulation results suggest a predominantly entropic folding pathway governed by the disorder of three functional loop regions. Although EGF-II exhibits two native disulfide bonds (Cys14-Cys31 and Cys33- Cys42), its large configurational entropy inhibits a direct transition to the native structure in the folding process. When Ser9 is mutated into Cys, a non-native disulfide bridge Cys9- Cys20 is slightly more favorable than the native Cys6-Cys20 because a less constrained N-terminus affords larger entropy. Isomers that are functionally less active also exhibit a more localized dynamics of the functional loop regions, which may suggest a possible mechanism for the modulation of EGF activity.

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III.

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.

Pathway of oxidative folding of a 3-disulfide alpha-lactalbumin may resemble either BPTI model or hirudin model.

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. alphaLA-IIIA is a 3-disulfide variant of alpha-lactalbumin (alphaLA) with a 3-D conformation essentially identical to that of intact alphaLA. alphaLA-IIIA contains 3 native disulfide bonds of alphaLA, two of them are located at the calcium binding beta-subdomain (Cys61-Cys77 and Cys73-Cys91) and the third bridge is located within the alpha-helical domain of the molecule (Cys28-Cys111). We investigate here the pathway of oxidative folding of fully reduced alphaLA-IIIA with and without stabilization of its beta-subdomain by calcium binding. In the absence of calcium, the folding pathway of alphaLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of beta-sheet domain by calcium binding, the folding pathway of alphaLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of alphaLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.

Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling.

Human alpha-thrombin is a very important plasma serine protease, which is involved in physiologically vital processes like hemostasis, thrombosis, and activation of platelets. Knowledge regarding the structural stability of alpha-thrombin is essential for understanding its biological regulation. Here, we investigated the structural and conformational stability of alpha-thrombin using the techniques of disulfide reduction and disulfide scrambling. alpha-Thrombin is composed of a light A-chain (36 residues) and a heavy B-chain (259 residues) linked covalently by an inter-chain disulfide bond (Cys(1)-Cys(122)). The B-chain is stabilized by three intra-chain disulfide bonds (Cys(42)-Cys(58), Cys(168)-Cys(182), and Cys(191)-Cys(220)) (Chymotrypsinogen nomenclature). Upon reduction with dithiothreitol (DTT), alpha-thrombin unfolded in a 'sequential' manner with sequential reduction of Cys(168)-Cys(182) within the B-chain followed by the inter-chain disulfide, generating two distinct partially reduced intermediates, I-1 and I-2, respectively. Conformational stability of alpha-thrombin was investigated by the technique of disulfide scrambling. alpha-Thrombin denatures by scrambling its native disulfide bonds in the presence of denaturant [urea, guanidine hydrochloride (GdmCl) or guanidine thiocyanate (GdmSCN)] and a thiol initiator. During the process, cleavage of the inter-chain disulfide bond and release of the A-chain from B-chain was the foremost event. The three disulfides in the B-chain subsequently scrambled to form three major isomers (designated as X-Ba, X-Bb, and X-Bc). Complete denaturation of alpha-thrombin was observed at low concentrations of denaturants (0.5 M GdmSCN, 1.5 M GdmCl, or 3 M urea) indicating low conformational stability of the protease.

Unfolding and breakdown of insulin in the presence of endogenous thiols.

Native insulin denatures and unfolds in the presence of thiol catalyst via disulfide scrambling (isomerization). It undergoes two transient non-native conformational isomers, followed by an irreversible breakdown of the protein to form oxidized A- and B-chain. Denaturation and breakdown of native insulin may occur under physiological conditions. At 37 degrees C, pH 7.4, and in the presence of cysteine (0.2 mM), native insulin decomposes with a pseudo first order kinetic of 0.075 h(-1). At 50 degrees C, the rate increases by 5-fold. GdnCl and urea induced denaturation of insulin follows the same mechanism. These results demonstrate that stability and unfolding pathway of insulin in the presence of endogenous thiol differ fundamentally from its reversible denaturation observed in the absence of thiol, in which native disulfide bonds of insulin were kept intact during the process of denaturation.

Investigating conformational stability of bovine pancreatic phospholipase A2: a novel concept in evaluating the contribution of the 'native-framework' of disulphides to the global conformational stability of proteins.

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.