©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Properties of Scrambled Hirudins (*)

(Received for publication, June 26, 1995; and in revised form, August 8, 1995)

Jui-Yoa Chang (§)

From the Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel CH-4002, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Scrambled hirudins serve as relevant folding intermediates during in vitro renaturation of the reduced/denatured hirudin. Eleven species of scrambled hirudins were isolated and structurally identified. Their properties, including their relative stability and the kinetics of their consolidation (disulfide reshuffling) to attain the native structure, have been investigated and are reported here.


INTRODUCTION

Scrambled structures of cystine-containing proteins are fully oxidized (disulfide formed quantitatively) species that contain at least two non-native disulfide pairings. They were first observed during the renaturation of ribonuclease A (RNase A) performed by Anfinsen and colleagues (Anfinsen et al., 1961; Haber and Anfinsen, 1962; Anfinsen, 1973). When reduced/denatured RNase A was allowed to refold in the presence of a denaturant (8 M urea), 99% of the protein was recovered as inactive scrambled species. Despite the finding (Anfinsen et al., 1961) that they were able to self reorganize to acquire the native conformation, scrambled proteins have been generally regarded as dead-end products of abortive folding and largely ignored by protein chemists.

However, from our recent refolding experiments performed with hirudin (49 amino acids, 3 disulfides), potato carboxypeptidase inhibitor (PCI) (^1)(39 amino acids, 3 disulfides), and human epidermal growth factor (53 amino acids, 3 disulfides), scrambled species have been found to exist along the folding pathway in all three cases and under a wide range of folding conditions (Chatrenet and Chang, 1993; Chang et al., 1994, 1995a). Their foldings undergo an initial stage of nonspecific disulfide pairing that leads to the formation of scrambled species as essential intermediates. This is followed by disulfide reshuffling of scrambled species in the presence of thiol catalyst to reach the native structure. The accumulation of scrambled species thus can be enhanced through manipulation of folding conditions by either accelerating the speed of disulfide formation or halting the process of disulfide reshuffling. The mechanism of halting disulfide reshuffling has been demonstrated in the example of folding carried out in the buffer alone. Under these conditions, free cysteines of 1- and 2-disulfide intermediates act as thiol catalyst during the early stage of folding. As the process advances, free cysteines begin to deplete because more cysteines become involved in disulfide pairing and less are available as thiol catalyst for disulfide reshuffling, therefore scrambled species become trapped and accumulated in an exponential manner. By this method, about 50-60% of the protein was consistently recovered as stable scrambled species (Chang, 1994). Alternatively, a high yield of scrambled species could be obtained by promoting the efficiency of disulfide formation. For instance, when folding of PCI was performed in the presence of 0.5 mM of cystine, more than 99% of the total protein was trapped as scrambled species before trace amounts of the native PCI even appeared (Chang et al., 1994).

The reproducible appearance of scrambled species in such significant concentrations indicates that they could not be simply dismissed as by-products or abortive structures of ``off-pathway'' folding. As a matter of fact, they have also been observed during the productive folding of ribonuclease A (Creighton, 1979) and pro-BPTI (Weissman and Kim, 1992) that involved no denaturants. In our opinion, scrambled species represent logical intermediates in a folding process that is governed by thermodynamic principles (Anfinsen, 1973). Their conversion to the native structure, which is apparently driven and guided by noncovalent specific interactions, accounts for the major event of protein folding. There are 14 possible scrambled species for a protein containing three disulfides. In the case of hirudin, 11 of them have been isolated and structurally identified (Chang et al., 1995b). In this report, we investigate their relative stability and the kinetics of their conversion to attain the native structure.


EXPERIMENTAL PROCEDURES

Preparation of Scrambled Hirudins

Scrambled hirudins were prepared by allowing reduced/denatured hirudin core domain, positions 1-49 of hirudin (0.5 mg/ml) to refold in the sodium bicarbonate buffer (50 mM, pH 8.3) for 24 h at 22 °C (Chang, 1994). About 60% of the sample was reproducibly trapped as scrambled species under these conditions. Ten fractions of scrambled hirudins were subsequently isolated by HPLC using the conditions described in the legend of Fig. 1.


Figure 1: End products of hirudin folding carried out in the alkaline buffer alone. About 60-65% of the protein was trapped as scrambled species, unable to convert spontaneously to the native structure due to the absence of free thiols as catalyst. Ten fractions of scrambled hirudins were identified, and their disulfide structures have been characterized. Fraction c contains two scrambled species with approximately equal concentration. The sample was analyzed by HPLC using the following conditions. Solvent A was water containing 0.1% trifluoroacetic acid. Solvent B was acetonitrile/water (9:1, v/v) containing 0.1% trifluoroacetic acid. The gradient was 14-32% solvent B linear in 50 min. The column was Vydac C-18 for peptides and proteins (4.6 mm, 10 µm). Column temperature was 22 °C.



Unfolding and Refolding of Hirudins in the Presence of Denaturant

Unfolding

The native hirudin was dissolved in Tris-HCl buffer (0.1 M, pH 8.5) containing GdmCl (3-8 M) or urea (5-8 M) and in the presence of beta-mercaptoethanol (0.25 mM). The sample was allowed to incubate at 22 °C for 16 h and then quenched by mixing with 2 volumes of 4% aqueous trifluoroacetic acid.

Refolding

The native hirudin was first reduced and denatured in the Tris-HCl buffer (0.5 M, pH 8.5) containing 5 M GdmCl and 50 mM dithiothreitol (Chatrenet and Chang, 1993), desalted by a PD-10 column, and then reconstituted in the Tris-HCl buffer (0.1 M, pH 8.5) in the presence of 6 M GdmCl and beta-mercaptoethanol (0.25 mM). The folding intermediates were trapped in a time-course manner by mixing with double volumes of 4% aqueous trifluoroacetic acid.

Consolidation of Scrambled Hirudins to Form the Native Structure

Isolated fractions of scrambled hirudin were dissolved in the Tris-HCl buffer (0.1 M, pH 8.5). To initiate the disulfide reshuffling, Cys (1 mM), reduced glutathione (1 mM), or beta-mercaptoethanol (0.25 mM) was introduced. The final concentration of hirudin was 80 µM. At different time intervals, aliquots of the sample were mixed with 2 volumes of 4% trifluoroacetic acid and subsequently analyzed by HPLC to monitor the progress of consolidation.

Stability of Scrambled Hirudins

Stability of the native and scrambled hirudins was compared and judged by their resistance to reduction of disulfide bonds or enzymatic digestion of peptide bonds. Hirudins (80 µM) were dissolved in the Tris-HCl buffer (0.1 M, pH 8.5) containing various concentrations of dithiothreitol, in the absence or presence of 6 M GdmCl. The process of reduction was trapped in a time-course manner by mixing aliquots of the sample with 2 volumes of 4% trifluoroacetic acid, followed by HPLC analysis.

For enzymatic digestion, hirudins (100 µM) were incubated at 22 °C with Glu-C protease (20:1 or 10:1, substrate/enzyme, w/w) in the ammonium bicarbonate buffer (50 mM, pH, 8.0). Aliquots of the digest were removed in a time-course manner, and amino termini generated by the digestion were quantitatively evaluated by the dimethylaminoazobenzene isothiocyanate method (Chang, 1988).


RESULTS

Heterogeneity of Scrambled Hirudins

Scrambled hirudins are fully oxidized but biologically inactive species. Their disulfide content was determined by mass spectrometry and amino acid analysis. Both methods reveal that scrambled hirudins contain three intact disulfides (with standard deviation of 4%). Contamination of free cysteine (as carboxymethyl derivative) has not been detected. There are 15 possible 3-disulfide isomers of hirudin. One is native and the other 14 are scrambled species. 10 fractions of scrambled hirudins (designated a-h) were identified and isolated by HPLC (Fig. 1). Further analysis by capillary electrophoresis indicates that fraction c is composed of two sub-fractions with roughly equal concentrations. Together, 11 species of scrambled isomers have been found to serve as the folding intermediates of hirudin. Their disulfide structures have been characterized and reported (Chang et al., 1995b). The three isomers that should have contained Cys^14-Cys have not been found. Their absence is most likely due to the unfavorable steric constraint.

Scrambled Hirudins Exist in Equilibrium

Scrambled species of hirudin exist in a state of equilibrium along the folding pathway with constant molar ratios of 0.31 (a), 0.33 (a*), 1.0 (b), 0.15 (b*), 0.93 (c), 0.31 (d), 0.16 (e), 0.26 (f), 0.25 (g), and 0.18 (h) (Fig. 1). This pattern is independent of the stage of folding and remains indistinguishable regardless of whether folding was catalyzed by air oxidation or redox agents, such as oxidized glutathione or cystine. In practice, all 3-disulfide hirudins were present in a state of equilibrium. There is, however, a wide free energy gap between the native species and scrambled isomers. In the folding buffer (pH 8.0-8.5), the equilibrium predominantly favors the native conformation. Consequently, conversion of scrambled hirudins to the native structure proceeds spontaneously and irreversibly during the folding. Under these conditions, the equilibrium constant (molar ratio) of scrambled hirudins (quantitated collectively)/ native hirudin is smaller than 0.001, as judged by the detection limit of HPLC.

This state of equilibrium is perturbed in the presence of a denaturant. Denaturants display two important effects. First, they alter the equilibrium constant among scrambled species. For example, the concentrations of fractions a, e, f, g, and h (as compared with those of fractions a*, b, b*, c, and d) are diminished by 5-fold in the presence of 6 M GdmCl (Fig. 2). The extent of their decrease is directly related to the concentration of denaturant. Second, the presence of denaturant significantly narrows the free energy gap between the native and scrambled structures. In the presence of 4, 6, and 8 M GdmCl, the equilibrium constant of scrambled hirudins/native hirudin increases from <0.001 to 0.30, 1.45, and 5.25, respectively (Fig. 2). For instance, when the native hirudin was incubated in the Tris-HCl buffer containing 6 M GdmCl and beta-mercaptoethanol (0.25 mM), 60 ± 2% of the sample reshuffled its native disulfides and settled at the scrambled structures. Conversely, when the reduced/denatured hirudin was allowed to refold in the same solution, only 40 ± 2% of the protein eventually converted to the native structure (Fig. 3). The same equilibrium could be obtained by permitting scrambled hirudins (mixture) to consolidate in the same GdmCl solution. Along this process, scrambled hirudins reshuffle among themselves to establish a new state of equilibrium that is accompanied by a drastic decrease of the denaturant-sensitive species (a, e, f, g, and h) (Fig. 4).


Figure 2: The equilibrium constant of scrambled hirudins/native hirudin is dependent upon the concentration of denaturant. Native hirudin (1 mg/ml) was incubated in the Tris-HCl buffer containing 0.25 mM beta-mercaptoethanol and various concentrations of GdmCl. Control sample was dissolved in the same buffer containing 0.25 mM beta-mercaptoethanol without GdmCl. The reaction was allowed for 16 h. Samples were trapped by acid and analyzed by HPLC. End products contain a mixture of equilibrated scrambled/native (N) species. The equilibrium constant (K) obtained is highly reproducible with an average deviation smaller than 5%. The inset provides the equilibrium constants. The open arrow indicates the elution position of the fully reduced species. Another set of experiment demonstrated that the disulfide structure of native hirudin was not disrupted at all by GdmCl in the absence of beta-mercaptoethanol.




Figure 3: In the presence of 6 M GdmCl, scrambled hirudins and the native hirudin exist in equilibrium (K 1.45), regardless of whether the equilibration is driven from the end of native hirudin (unfolding) or the end of fully reduced/denatured species (R, refolding). Both folding and unfolding were performed at 22 °C in the Tris-HCl buffer (0.1 M, pH 8.5) containing 6 M GdmCl and 0.25 mM beta-mercaptoethanol.




Figure 4: Consolidation of scrambled hirudins in the absence (A) and the presence (B) of 6 M GdmCl. The reactions were performed in the Tris-HCl buffer containing 0.25 mM of beta-mercaptoethanol as catalyst. Only 38% of scrambled hirudins was able to convert to the native structure in the presence of 6 M GdmCl.



Similar studies were also conducted in the presence of varied concentrations of urea. The results demonstrate that urea is about 4-fold less potent in denaturing the native hirudin. In the presence of 8 M urea, the equilibrium constant of scrambled/native hirudins is 0.11 ± 0.01.

Kinetics of the Conversion of Isolated Scrambled Species to Form the Native Structure

Consolidation of scrambled hirudins occurs spontaneously but requires the presence of free thiols to jump-start and catalyze the reshuffling of non-native disulfides. The efficiency of this process has been shown to depend on the redox potential of the thiol catalyst (Chang, 1994).

When an isolated species of scrambled hirudin was allowed to reshuffle disulfides and consolidate, it advances simultaneously toward two directions. It equilibrates with the native structure as well as with other scrambled species (Fig. 5). Because the equilibration predominantly favors the native structure, in the end all scrambled species convert spontaneously to the native hirudin. These reactions were carefully analyzed by using either beta-mercaptoethanol, cysteine, or reduced glutathione as thiol catalyst. The kinetics and mechanisms involved in those reactions are enormously complicated, but a number of crucial observations can be concluded from these studies ( Fig. 5and Fig. 6). 1) The initial rate to equilibrate with other scrambled species is about 4-5 times faster than that of converting to the native species. This was observed with the time-course analysis of all 11 different scrambled species (Fig. 6). 2) An isolated scrambled species equilibrates faster with those that already share a common disulfide bridge. An obvious case is species b*, which converted to a* about 6-fold faster than to the remaining scrambled species (Fig. 6). a* and b* happen to be the only two species that share the largest disulfide loop Cys^6-Cys (Fig. 1). Another example is the rapid interconversion of species h and g, both of which contain Cys^6-Cys. 3) The denaturant-sensitive species (a, e, f, g, and h) are relatively less productive in converting to the native structure. This may in part reflect their better stability, which is also consistent with the finding that denaturant-sensitive species are more resistant to reduction (see Fig. 8). With Cys (1 mM) as catalyst, the most and least productive species display comparable rate constants of 0.072 (fraction b) and 0.038 min (fraction f), respectively. Using reduced glutathione (1 mM), the rate of consolidation is reduced by half to 0.035 (b) and 0.022 min (f). In the presence of beta-mercaptoethanol (0.25 mM), the rate constants range from 0.016 (b) to 0.01 min (f).


Figure 5: Consolidation of an isolated scrambled hirudin to acquire the native structure. Fraction d was reconstituted in the Tris-HCl buffer containing 1 mM of reduced glutathione as catalyst. Intermediates during the course of disulfide reshuffling were trapped by acid and analyzed by HPLC using the same conditions described in the legend for Fig. 1.




Figure 6: The intermediates (trapped at 5 min) during the course of consolidation of isolated scrambled hirudins. Cys (1 mM) was used as thiol catalyst in these experiments. The intermediates were trapped by acid. Values given on the left-hand side of the panel indicate the recoveries of native hirudin.




Figure 8: Reduction of scrambled hirudins. The reduction of scrambled hirudins was performed in the presence of 0.2 mM of dithiothreitol. R and N stand for fully reduced and the native hirudins, respectively. The elution positions of the scrambled species (III), 2-disulfide (II), and 1-disulfide (I) species are indicated at the 0-time and 60 min samples. The gradient systems of HPLC are as described in the legend for Fig. 1, except that a 5-µm instead of a 10-µm Vydac C-18 column was used here. With the 5 µm column, fractions e and d are baseline separated, but a* and b* co-elute with b and c, respectively.



The striking outcome is that all fractions of scrambled hirudins behave in a very similar manner. This includes the efficiency to generate the native structure and the rate to reach equilibrium with other scrambled species ( Fig. 5and Fig. 6). No scrambled species has been shown to be particularly productive in generating the native structure. These findings unambiguously demonstrate that scrambled hirudins consist of an assemblage of equilibrated isomers possessing a similar state of free energy.

The Native Disulfides Are About 500-fold More Stable than the Scrambled Non-native Disulfides

Stability of the disulfide bonds of the native and scrambled hirudins were measured by their ability to resist reduction. Judging from the relative concentrations of dithiothreitol required to achieve a similar efficiency of reduction, the native disulfides are roughly 500-fold more stable than the scrambled non-native disulfides (Fig. 7). In the presence of a denaturant (6 M GdmCl), the reduction of scrambled disulfides was hardly affected (data not shown), but the efficiency of reducing of the native disulfides was enhanced by nearly 100-fold. These data clearly demonstrate that the difference of stability between the native and scrambled hirudins is mainly attributed to the noncovalent specific interactions that are abrogated by the denaturant.


Figure 7: Recoveries of fully reduced hirudin obtained from the reduction of the native and scrambled hirudins. Conditions of reduction are described in the text, and concentrations of dithiothreitol employed are indicated along with each curve. A, scrambled hirudins. B, the native hirudin. C, the native hirudin in the presence of 6 M GdmCl. In the case of C, the fully reduced hirudin eventually swung back to the 1- and 2-disulfide species due to the oxidation of dithiothreitol.



The results of HPLC analysis reveal the small differences of stability among scrambled species. During the reduction, the decrease of species c and d is about 2-fold faster than that of denaturant-sensitive species e, f, g, and h (a, a*, b, and b* could not be measured here because they overlap with 2-disulfide species) (Fig. 8). Also, species f appears to be slightly more resistant against reduction than e, g, and h. All these are consistent with the sluggish behavior of denaturant-sensitive species during the process of consolidation (Fig. 6). Furthermore, 2- and 1-disulfide species were detected during the reduction of scrambled hirudins (Fig. 8). By contrast, reduction of the native disulfides adopts an ``all-or-none'' (collapse) pathway in which only 1-2% of 1- and 2-disulfide intermediates appear, a mechanism similar to that found during the reductive unfolding of RNase (Creighton, 1979) and observed at dithiothreitol concentrations ranging from 10 to 100 mM.

The Peptide Bonds of Scrambled Hirudins Are Freely Accessible to Enzymatic Digestion

The stability of scrambled hirudins was further evaluated by their resistance toward proteolytic digestion. Three peptide bonds, Glu^8-Ser^9, Glu-Gly^18, and Glu-Gly, which distribute evenly along the hirudin sequence, were selectively digested by different concentrations of Glu-C protease in a time-course manner. The rate of cleavage at each peptide bond was then determined by the method of quantitative amino-terminal analysis. Two control proteins, the native and the reduced/carboxymethylated hirudins, were processed in parallel. In the case of native hirudin, these peptide bonds were not digestible (<2%), even after overnight incubation. With scrambled hirudins, we have found that kinetics of cleavage of these three peptide bonds are comparable with that of reduced/carboxymethylated sample.

Enzymatic susceptibility of a protein is commonly linked to the state of its conformation. Compactly folded proteins are usually resistant to enzyme digestion, and most denatured or reduced/carboxymethylated proteins are freely digestible by enzymes. The data obtained here indicate that scrambled hirudins adopt loose conformations that closely resemble the denatured state. (Dill and Shortle, 1991).


DISCUSSION

Scrambled species are landmark intermediates that permit identification of the two-stage folding mechanism for both hirudin and PCI (Chang et al., 1994). Their formation is a consequence of the nonspecific packing (disulfide pairing) that occurred during the early stage of folding. This two-stage mechanism is compatible with the folding behavior observed with RNase A (Anfinsen et al., 1961; Pigiet and Shuster, 1986). It is also consistent with a recent example of chymotrypsin inhibitor 2 (Jackson and Fresht, 1991; Jackson et al., 1993; Otzen et al., 1994). Chymotrypsin inhibitor 2 is an interesting case because it has a size similar to hirudin but contains no disulfide. A collapsed state of chymotrypsin inhibitor 2 serves as folding intermediate, and its conversion to the native structure also represents the rate-limiting step. In our opinion, the property of this collapsed state of chymotrypsin inhibitor 2 resembles that of scrambled hirudins.

In this study, it has been demonstrated that scrambled hirudins consist of a collection of equilibrated isomers possessing a similar state of free energy. They also exhibit a markedly reduced stability as compared with that of native hirudin, a property that is consistent with what has been described for the ``molten globe'' state (Ptitsyn, 1987; Kuwajima, 1989). There are, however, detectable differences of stability among scrambled hirudins. About half of scrambled hirudins (a, e, f, g, and h) are sensitive to denaturant. They are also less productive in converting to the native structure and more resistant against reduction as compared with species a*, b, b*, c, and d. All this implies that denaturant-sensitive species are stabilized by noncovalent interactions that are partially abrogated in the presence of denaturants. These noncovalent interactions do not necessarily resemble those that fix the native structure, and their precise nature remains to be elucidated by NMR. Interestingly, most of the denaturant-sensitive species (f, g, and h) do not even contain a native disulfide, and the two well-populated species (b and c) that do contain a native disulfide are entirely insensitive to denaturants.

From the viewpoint of solely kinetic analysis of disulfide formation, one can correctly argue that scrambled species are dead-end products, because conversion of scrambled species to the native structure must undergo disulfide reshuffling and, in practice, 2-disulfide or even 1-disulfide species must serve as transitory intermediates. However, this argument misses the point that formation of disulfides are consequences, not causes, of folding and that they are used as signals to trace, not to dictate, the mechanism of folding. Thermodynamically, the presence of scrambled structures as folding intermediates is perfectly logical. Scrambled species simply represent a state of more advanced packing and relatively lower free energy (DeltaG) than that of 2-disulfide species. In this case, the loss of entropy (randomness) (DeltaS) is more than compensated by the decrease of enthalpy (DeltaH), due to the reordering of the hydrophobic structural elements in the aqueous environment. This proposal is supported by two important observations: 1) the flow of 2-disulfide intermediates to the scrambled species is spontaneous and irreversible during the folding, and 2) scrambled species are eluted earlier than 2-disulfide and 1-disulfide species on reverse phase HPLC (Chatrenet and Chang, 1993). Their conversion to the native structure, although accompanied by the disulfide reshuffling, does not necessarily require unfolding of the compact structure they have already attained. Indeed, during the process of conversion from scrambled species to the native structure, 2-disulfide and 1-disulfide intermediates have not been observed (Chang, 1994); clearly they must exist in equilibrium in such a minute concentration that it is beyond detection.

The presence of scrambled species as folding intermediates features a major distinction between the folding mechanism of the BPTI (Creighton, 1978, 1990; Weissman and Kim, 1991) and hirudin (Chatrenet and Chang, 1993) models. Similar to hirudin, there are species of folding intermediates of BPTI that become trapped during the folding and are unable to convert to the native structure (Weissman and Kim, 1991, 1992). In hirudin, they are identified as 3-disulfide scrambled species, and their trapping is due to the absence of thiol reagent to catalyze their further disulfide reshuffling. In the case of BPTI, these trapped intermediates have been characterized as 2-disulfide (native) species with buried cysteines that are incapable or extremely slow to form the third native disulfide. Aside from scrambled species, some conceptual differences concerning the definition of a folding pathway also need to be clarified. The crucial question is what types of folding intermediates actually constitute the pathway, the well populated intermediates or the productive species that account for the flow of folding? Also, how are well populated intermediates quantitatively defined? Should one ignore those minor intermediates that constitute less than 10-15% of the well populated species? These are important issues because well populated species are not necessarily the productive ones when isomers of intermediates exist in a state of dynamic equilibrium as those observed in the cases of hirudin and BPTI. Should well populated intermediates be chosen to specify the pathway, then it is obvious that hirudin, PCI, and RNase (Scheraga et al., 1984, 1987) all fold via multiple pathways because of the heterogeneity of their well populated intermediates. Even in the case of BPTI, the intermediates are probably far more complicated than what has been described (Weissman and Kim, 1991). Using HPLC and mass spectrometry, we have detected more than 46 fractions of folding intermediates of BPTI, (^2)although some of the minor fractions represent only 10-20% of the predominant species. If, on the other hand, one agrees that the productive intermediates specify the pathway, then those productive species still remain to be identified in the cases of hirudin, PCI, and RNase, as well as BPTI. We suggest that one way to identify the productive intermediates is through kinetic analysis of purified, structurally defined intermediates (both major and minor) using the stop/go folding experiments (Chang, 1993). With a protein containing three disulfides, this approach is facing two formidable challenges. The first is to separate and isolate all intermediates, major or minor, that are present at the same stage of equilibrium to homogeneity. Considering the possible isomers that exist (15 1-disulfide and 45 2-disulfide species) and the apparent complexity of intermediates observed with hirudin and PCI, this will be a daunting task. The second challenge, which is even more intractable, is to ensure that all minor species have been found and isolated, including those that exist with concentrations that may be less than 1% of the well populated species.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to the author's late father, Tschenda Chang.

§
To whom correspondence should be addressed: K-121,104 Ciba-Geigy Ltd., Basel CH-4002, Switzerland. Tel./Fax: 4161-6968313.

(^1)
The abbreviations used are: PCI, potato carboxypeptidase inhibitor; BPTI, bovine pancreatic trypsin inhibitor; GdmCl, guanidinium chloride; HPLC, high performance liquid chromatography; Cys, cystine.

(^2)
P. Schindler and J.-Y. Chang, unpublished results.


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