A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.     

Structural stability of the PsbQ protein of higher plant photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.     

Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.     

FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: "hidden" intermediates at low denaturant concentrations

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.     

Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.     

Role of amino-terminal residues in the folding of the constant fragment of the immunoglobulin light chain

Three constant fragments with different amino terminals, CL(105-214), CL(109-214), and CL(113-214), were obtained by limited proteolysis with trypsin or papain of a type lambda immunoglobulin light chain. The conformations of the three CL fragments were indistinguishable on the basis of circular dichroism and tryptophyl fluorescence spectra. The stability to heat and guanidine hydrochloride of CL(105-214) was almost the same as that of CL(109-214), but the stability of CL(113-214) was slightly lower than that of CL(105-214) or CL(109-214). The midpoint of the thermal unfolding transition at pH 7.5 was at 60.0 degrees C for CL(105-214), 60.4 degrees C for CL(109-214), and 57.5 degrees C for CL(113-214). The midpoint of the unfolding transition by guanidine hydrochloride at pH 7.5 and 25 degrees C was 1.2 M for CL(105-214) and CL(109-214) and at 1.0 M for CL(113-214). The kinetics of unfolding and refolding by guanidine hydrochloride of these CL fragments were analyzed on the basis of the three-species mechanism, U1 in equilibrium with U2 in equilibrium with N, where U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. It was found that only the microscopic unfolding rate constant for CL(113-214) is 2-3 times greater than that for CL(105-214) or CL(109-214) and that the other microscopic rate constants for the three CL fragments are all the same. These findings indicated that the amino-terminal residues, Gly-109-Lys-112, or a part of them, stabilize the CL(113-214) fragment by decreasing only the unfolding rate, that the transition state of the folding of the CL fragment is independent of the presence or absence of this peptide, and that, at the last step of folding, the peptide is incorporated into the globular domain, thus stabilizing it.     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.     

Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride.

The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.     

Nuclear magnetic resonance studies of the unfolding of pancreatic ribonuclease. II. Unfolding by urea and guanidine hydrochloride.

The unfolding of ribonuclease by urea and guanidine hydrochloride has been studied by ¹H nuclear magnetic resonance spectroscopy, under conditions where the unfolding is fully reversible and concentration-independent. Both urea and guanidine produce marked changes in the chemical shift of the histidine C₍₂₎H resonances, together with small changes in other regions of the spectrum, at concentrations (0.1 to 1.0 m) far below those which are required for gross unfolding of the protein. The changes in area of the histidine C₍₂₎H resonances through the major unfolding transition produced by these denaturants give evidence for the existence of at least two intermediates in the unfolding process. The “order of unfolding” of the histidine residues is closely similar for both urea and guanidine hydrochloride unfolding, and also similar to that found for thermal unfolding at low pH (see Benz &; Roberts, 1975) accompanying paper.     

Conformational stability of 17 beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

The functional activities of proteins are closely related to their molecular structure and understanding their structure-function relationships remains one of the intriguing problems of molecular biology. We investigated structural changes in 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) induced by pH, temperature, salt, urea, guanidine hydrochloride, and coenzyme NADPH binding. At 25 degrees C and within the relatively narrow pH range of 7.0-9.0, 17beta-HSDcl exists in its native conformation as a dimer. This native conformation is thermally stable up to 40 degrees C in this pH range. At 25 degrees C and pH 2.0 in the presence of 150-300 mM NaCl, 17beta-HSDcl forms soluble aggregates enriched in alpha-helical and beta-sheet structures. At higher temperatures and NaCl concentrations, these soluble aggregates start to precipitate. The denaturants urea and guanidine hydrochloride unfold 17beta-HSDcl at concentrations of 1.2 and 0.4 M, respectively. Binding of the coenzyme NADPH to 17beta-HSDcl causes local structural changes that do not significantly affect the thermal stability of this protein.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters

The stability parameters delta Gst, delta Hst and delta Sst of native basic pancreatic trypsin inhibitor (BPTI) have been characterized by microcalorimetric unfolding studies in various buffer solutions, at different pH values and in the presence of guanidine hydrochloride. The unfolding enthalpy of BPTI, in contrast ot other globular proteins, exhibits a very small dependence on temperature, which results in a characteristic different temperature dependence of the Gibbs energy of stabilization. BPTI has a very high specific Gibbs energy of stabilization, which renders the slow exchange rates of amide protons understandable. Comparison of the unfolding entropy of BPTI at 110 degrees C with corresponding values of other proteins, revealed that the delta S values of BPTI are lower by 2.9 J/(K X residue). This lower value of the unfolding entropy is in good agreement with predictions of a theoretical study by Poland & Scheraga (1965) where the influence of crosslinks on the configurational entropy has been studied. Additionally, we were able to calculate an interaction enthalpy per site of -5.6 kJ/mol based on the measurements of unfolding of BPTI in 6 M-guanidine hydrochloride.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

Differentiation between conformational and autoproteolytic stability of the neutral protease from Bacillus stearothermophilus containing an engineered disulfide bond.

The introduction of a disulfide bond into the neutral protease from Bacillus stearothermophilus by the double mutation G8C/N60C had resulted in an extremely thermostable enzyme with a half-life of 35.9 min at 92.5 degrees C [Mansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., van den Burg, B., Venema, G., Ulbrich-Hofmann, R. & Eijsink, V.G. (1997) J. Biol. Chem. 272, 11152-11156]. The study in guanidine hydrochloride of this enzyme and the respective wild-type enzyme allowed us to distinguish between the stability toward global unfolding and autoproteolysis. At low protease concentrations (20 microg.mL-1) and short periods of incubation with guanidine hydrochloride (5 min), transition curves without the interference by autoproteolysis could be derived from fluorescence emission measurements. The effect of the disulfide bond on the global unfolding of the protein proved to be smaller than expected. In contrast, the measurement of autoproteolysis at higher protein concentrations (100 microg.mL-1) by quantitative evaluation of the bands of intact protein on SDS/PAGE revealed a strong stabilization toward autoproteolytic degradation by the disulfide bond. The rate of autoproteolysis in guanidine hydrochloride was found to be much lower than that of thermal denaturation, which can be attributed to the inhibition of the proteases by this denaturant. The results suggest that the disulfide bond stabilizes the protease against autoproteolysis more than against global unfolding. Autoproteolysis starts as soon as the cleavage sites in flexible external structural regions become accessible. It is suggested that the stabilizing effect of the disulfide bond is caused by the fixation of the crucial loop region 56-69 or by hindrance of the primary cleavage in this region by the amino acid exchanges.