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

Jui-Yoa Chang (§) , Patrick Schindler , Benoit Chatrenet

From the (1) Pharmaceuticals Research Laboratories, K-121,104 Ciba-Geigy Ltd., Basel CH-4002, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Scrambled hirudins consist of a collection of equilibrated isomers and serve as essential folding intermediates during the in vitro renaturation of hirudin (Chatrenet, B., and Chang, J.-Y.(1993) J. Biol. Chem. 268, 20988-20996). Ten fractions of scrambled hirudins have been isolated. Their disulfide structures were deduced from the analysis of thermolysin-digested peptides by amino acid sequencing and mass spectrometry. The results reveal 9 fractions of pure scrambled species, and, together, 11 species of scrambled structures have been identified. About all possible disulfide isomers of hirudin have been found to exist. The three native disulfides, Cys-Cys, Cys-Cys, and Cys-Cys, are detected in five different scrambled species and constitute 18% of the total disulfide bonds found in scrambled hirudins.


INTRODUCTION

Hirudin is a thrombin specific inhibitor isolated from leech Hirudo medicinalis (Markwardt and Walsmann, 1958; Markwardt, 1970). It is the most potent thrombin inhibitor known, both natural and synthetic (Fenton, 1981; Markwardt, 1985; Stone and Hofsteenge, 1986). This high potency is a consequence of the multiple binding sites between the inhibitor and the enzyme. Hirudin contains two functional domains (Haruyama and Wuethrich, 1989; Folkers et al., 1989; Rydel et al., 1990; Gruetter et al., 1990), a compact N-terminal domain (49 amino acids) which blocks the catalytic site of the enzyme (Chang, 1990), and a disordered C-terminal domain (16 amino acids) which binds to the fibrinogen recognition site of thrombin (Chang, 1983; Krstenansky and Mao, 1987; Mao et al., 1988). The N-terminal core domain is stabilized by three native disulfides (Dodt et al., 1986) and requires proper folding to maintain its biological activity. The disulfide folding pathway of hirudin core domain has been recently elucidated (Chatrenet and Chang, 1993). The folding undergoes an initial stage of nonspecific packing (nonspecific disulfide pairing) that leads to the formation of 3-disulfide scrambled species as relevant folding intermediates. This is followed by disulfide reshuffling and consolidation of scrambled species to acquire the native disulfide structure. The process of nonspecific packing is characterized by three key observations. 1) The enormous heterogeneity of the 1- and 2-disulfide intermediates that serve as the precursor of the scrambled species. According to the number of fractions detected by both HPLC() and capillary electrophoresis, nearly all possible 1- and 2-disulfide isomers exist along the folding pathway. 2) The insensibility of 1- and 2-disulfide intermediates to denaturants. The HPLC patterns of 1- and 2-disulfide intermediates remain indistinguishable, regardless of whether folding is carried out in the absence or presence of the denaturant. 3) The possibility to manipulate the speed of the flow of 1- and 2-disulfide intermediates (and hence the level of the accumulation of scrambled species) during the folding by oxidized glutathione and cystine. Under selected folding conditions that involve no denaturant, more than 80% of the total hirudin samples could be recovered as scrambled structures (Chang, 1994).

Hirudin is not alone in displaying these properties. Scrambled species have been found to accumulate along the folding pathway of potato carboxypeptidase inhibitor (Chang et al., 1994) by a similar mechanism. In addition, they have also been observed during the productive folding of probovine pancreatic trypsin inhibitor (Weissman and Kim, 1992) and ribonuclease A (Creighton, 1979) that engaged no denaturant. These data suggest that scrambled proteins, contrary to the conventional wisdom that they represent products of abortive folding, must play a constructive role during the folding process of disulfide-containing proteins. Indeed, from the viewpoint of thermodynamics (Anfinsen, 1973; Haber and Anfinsen 1962), the existence of scrambled proteins as folding intermediates and passage to the native structure seems to be logical. They simply represent a state of more advanced packing than 2-disulfide intermediates. For these reasons, we believe that structure and function analysis of scrambled hirudins are essential. Ten fractions of scrambled hirudins have been found and isolated. Their disulfide structures were analyzed, and the results are reported here.


EXPERIMENTAL PROCEDURES

Preparation of Scrambled Hirudins

Hirudin core domain (Hir) was derived from recombinant hirudin variant 1 (HV1) by selective removal of its disordered C-terminal tail using -chymotrypsin (Chang, 1990). Scrambled hirudins were prepared by allowing reduced/denatured Hir to refold in the alkaline buffer alone. The protein (5 mg/ml) was first treated with 30 mM dithiothreitol for 90 min in the Tris-HCl buffer (0.5 M, pH 8.5) containing 5 M GdmCl. The sample was then passed through a PD-10 column (Pharmacia Biotech Inc.) and eluted by ammonium bicarbonate solution (50 mM, pH 8.5). Reduced hirudin, recovered in 1.2 ml, was immediately diluted with the same ammonium bicarbonate buffer to a final protein concentration of 0.5 mg/ml. Folding was performed at 22 °C for 24 h. At the end of folding, the sample was freeze-dried, redissolved in 0.1% trifluoroacetic acid, and subjected to HPLC purification (Fig. 1). Under these folding conditions, about 70-75% of the hirudin sample was reproducibly trapped as scrambled species, unable to convert to the native structure due to the absence of free thiol catalysts. In case that folding was performed in the presence of 5 M GdmCl, ammonium bicarbonate buffer was replaced by Tris-HCl buffer (0.1 M, pH 8.5); otherwise, conditions were identical.


Figure 1: HPLC separation of scrambled hirudins. Bottom panel, end-products of hirudin folding carried out in the buffer alone. About 70% of the starting material is recovered as scrambled species (designated a to h). They are eluted as a cluster 14 min after the native hirudin (N). Scrambled species obtained from this experiment were subsequently isolated for structural analysis. Top panel, folding of hirudin performed in the presence of 5 M GdmCl. The yield of native hirudin is 12%, and the pattern of scrambled species is also different from that performed in the absence of the denaturant (bottom panel). HPLC column is Vydac C-18 for peptides and proteins (10 mm, 3 µm). Solvent A is water containing 0.1% trifluoroacetic acid. Solvent B is acetonitrile/water (9:1, by volume) containing 0.1% trifluoroacetic acid. The gradient is 14-32% solvent B linear in 50 min. Detector wavelength was 214 nm.



Digestion of Scrambled Hirudins with Thermolysin

Isolated scrambled hirudin (30 µg) was treated with 3 µg of thermolysin (Sigma P-1512) in 100 µl of N-ethylmorpholine/acetate buffer (50 mM, pH 6.4). Digestion was carried out at 23 °C for 14 h. The samples were acidified with 20 µl of 4% trifluoroacetic acid, and peptides were then separated and isolated by HPLC. Structural analysis of thermolysin-digested peptides was performed by both amino acid sequencing and mass spectrometry.

Amino Acid Sequencing and Mass Spectrometry

Amino acid sequences were determined by using an Applied Biosystems 470A sequencer equipped with an on-line PTH analyzer (Hewlett-Packard 1090). An internal standard, 2-nitroacetophenone, which eluted in-between PTH-His and PTH-Tyr, was introduced in order to ensure precise quantitation of PTH-derivatives (Ramseier and Chang, 1994). It was predissolved in the solvent (2 µM) which transfers PTH-derivatives from the conversion flask to the HPLC. During the analysis of cysteine-containing peptides, an unique signal di-PTH-Cys appeared when both half-cystines were recovered at the same degradation cycle (Haniu et al., 1994). Di-PTH-Cys is eluted near PTH-Tyr, but can be easily distinguished from the tyrosine derivative by an additional absorbance at 313 nm.

The MALDI mass spectrometer was a home-built time of flight instrument with a nitrogen laser of 337 nm wavelength and 3-ns pulse width. The apparatus has been described in detail elsewhere (Boernsen et al., 1990). Two different matrices were used, either a solution containing (2:1, v/v) 2,6-dihydroxyacetophenone (20 mg/ml in ethanol), 0.1 M diammonium hydrogen citrate (in water) or 2,5-dihydroxybenzoic acid (30 mg/ml) in acetonitrile. An aliquot (1 µl) of the matrix was deposed onto a golden probe, and an aliquot (0.7 µl) of the sample solution (5-0.1 10M in 0.1% trifluoroacetic acid in 4:1 HO/CHCN) was added to it. Then the resulting mixture was vacuum-dried before analysis. The calibration was performed either externally or internally, by using standard proteins (hypertensin, M = 1031.19; Synacthen, M = 2934.50; and calcitonin, M = 3418.91). In the case that the calibration was performed internally, an aliquot (0.4 µl) of an aqueous solution (5-0.1 10M) of the standard proteins was mixed to the sample solution and the matrix before MALDI-MS analysis.

Anti-thrombin Activity

Anti-amidolytic activities of refolded samples were measured by their ability to inhibit human -thrombin from digesting Chromozym (Boehringer Mannheim). The reaction was carried out at 22 °C in 67 mM Tris-HCl buffer, pH 8.0, containing 133 mM NaCl and 0.13% polyethylene glycol 6000. The rate of digestion was followed at 405 nm for a period of 2 min. The concentration of substrate was 200 µM. The concentration of thrombin was adjusted in between 2.5 and 25 nM.

RESULTS

Scrambled hirudins are fully oxidized, but biologically inactive species. Their disulfide content was determined by amino acid composition analysis and mass spectrometry. Both methods confirm that scrambled hirudins contain three intact disulfides (with standard deviation of ±4%). They were separated into 10 fractions by reverse-phase HPLC (Fig. 1). During preparative separation, however, fraction b* co-eluted with fraction b, and peaks a and a* broadened and became partially overlapped. Therefore, only nine fractions of scrambled species were isolated for structural determination. Further analysis of isolated fractions by capillary electrophoresis revealed that peak c consisted of two subfractions with approximately equal concentration. Thus, there exist a minimum of 11 species of scrambled hirudins.

Elucidation of the disulfide pairings of scrambled hirudins are based upon the structural analysis of cystine-containing peptides derived from thermolysin digestion (Fig. 2). There are three non-cysteine-containing peptides, Hir (peak 8), Hir (peak 15), and Hir (peak 17), which appear constantly in the digest of all fractions. They were sequenced only once using peptides derived from fraction d, and the same non-cysteine peptides from other fractions were confirmed by mass spectrometry. All cysteine-containing peptides were characterized by both amino acid sequencing and mass spectrometry (). Several aspects of the data need to be elaborated. 1) Peaks marked with the same number do not necessarily contain the same peptide. For example, the retention times of c-7 and d-7 are indistinguishable, but peptides embodied in these two peaks contain Cys-Cys and Cys-Cys, respectively. 2) Some peaks consist of more than one peptide (e.g. b-15, d-8, and e-15 etc.). However, their structures could be unambiguously identified through the combined information of amino acid sequence and molecular mass. 3) The same disulfide pairing may generate more than one peptide due to nonspecific cleavages. One case is shown in fraction f. Peptides f-6 and f-20, both containing Cys-Cys, are linked by Hir/Hir and Hir/Hir, respectively. 4) Two different disulfide pairings may be found in the same peptide fraction as a consequence of incomplete digestion. For instance, sequence analysis reveals that peak h-12 consists of three chains (Hir, Hir, and Hir) interconnected by either Cys-Cys and Cys-Cys or Cys-Cys and Cys-Cys. For this kind of peptide, further analysis was necessary in order to determine the correct disulfide structures. This was achieved in two ways. The peptide could be digested by a second enzyme (e.g. lysine endopeptidase), followed by mass analysis. Correct disulfide linkages could also be identified by the presence of di-PTH-Cys during Edman sequencing. This unique signal appears when both half-cystines are released from the same degradation cycle. In the case of peptide h-12, di-PTH-Cys was positively detected at the second cycle, and this is consistent only with the pairings of Cys-Cys and Cys-Cys. The results and deduced disulfide structures are summarized in and Fig. 3.


Figure 2: Peptide mapping of scrambled hirudins digested by thermolysin. Panel b includes fractions b and b*. Peptides are numbered according to the order of their retention times. Those marked with the same number do not necessarily contain the same peptide. HPLC column is Vydac C-18 for peptides and proteins, 4.6 mm (inside diameter), 10 µm. The compositions of solvents are as those described in Fig. 1. The gradient is 5-22% solvent B linear in 32 min, 22-50% B linear from 32 to 45 min, staying at 50% B until 50 min and returning to the initial condition within 1 min. Peaks 8 and 20 are eluted at 19 min and 32 min, respectively. All numbered peaks were analyzed by both amino acid sequencing and mass spectrometry. The disulfide linkages found in each of those peaks are listed in Table I.




Figure 3: The disulfide structures of scrambled hirudins. Fraction c consists of two scrambled isomers. All together, 11 species of scrambled hirudins have been identified. N is the native disulfide structure.



Fractions d, e, f, g, and h all consist of single disulfide species, and their disulfide structures can be unambiguously assigned (noted that fraction e is partially contaminated by fraction d). Surprisingly, aside from fraction e, none of them admits the native disulfides (Fig. 3). Fraction c contains two scrambled species and their disulfide structures were established by puzzle-game strategies. First, all disulfide-containing peptides were characterized, and the following pieces of disulfide pairings were found: Cys-Cys (c-2, 521 pmol), Cys-Cys (c-7, 470 pmol), Cys-Cys (co-eluted in c-8, 450 pmol), Cys-Cys (co-eluted in c-8, 150 pmol) Cys-Cys and Cys-Cys (c-11, 170 pmol); Cys-Cys (c-6 and c-20, 465 pmol). Cys-Cys cannot belong to the same species that generated peptides containing Cys-Cys and Cys-Cys. Also, Cys-Cys and Cys-Cys must be derived from the same species. Therefore, one of the two species eluted in fraction c has to be Cys-Cys, Cys-Cys, and Cys-Cys. The other isomer was consequently assigned as Cys-Cys, Cys-Cys, and Cys-Cys (Fig. 3).

Fractions b and b* were isolated and analyzed together. The data show that the predominant species (b) is made of Cys-Cys (b-7), Cys-Cys (b-9 and b-10), and Cys-Cys (b-12), whereas the minor species (b*) could be either Cys-Cys, Cys-Cys, Cys-Cys, or Cys-Cys, Cys-Cys, Cys-Cys. The latter structure was assigned to b* (Fig. 3) because the former one was found in fraction a*. The results of fraction a are less conclusive due to contamination by fraction a*. Aside from the peptides observed in fraction a*, Cys-Cys was found in a-8, similar to the peptide recovered in d-8. A three-chain peptide eluted in a-13 gave sequences and molecular mass identical with that found in c-11. Since a-13 and c-11 have different retention times (about 0.6 min), they are likely to be isomers, and the only isomeric structure to c-11 is Cys-Cys, Cys-Cys. These structural data permit a tentative assignment of Cys-Cys, Cys-Cys, Cys-Cys to fraction a.

DISCUSSION

For a protein containing three disulfides, there are 15 possible 1-disulfide pairings, which permit formation of fifteen 3-disulfide isomers. Three out of the fifteen 1-disulfide pairings are native. Among 3-disulfide isomers, 1 is native and the remaining 14 are scrambled species. In the case of hirudin, the formation of one of the non-native disulfides, Cys-Cys, is probably unfavorable due to steric constraint. In the absence of Cys-Cys, there are only 14 different 1-disulfide linkages and 11 possible scrambled species. Our results have shown that all these possible structures exist in scrambled hirudins (). Aside from the isomers found in fractions b and c, all other scrambled species distribute rather evenly, with the lowest contribution of 4% to the highest of 8%. The predominant scrambled species found in fraction b consists of three disulfide bonds all bridged by neighboring cysteines [Cys-Cys; Cys-Cys; Cys-Cys] (beads form). This pattern of disulfide structure coincides with the hypothesis of Kauzmann(1959) which predicts that in a fashion of random disulfide arrangement, the pairing of adjacent cysteines has the highest probability. In a simple 2-disulfide model (conotoxin), which consists of only two possible scrambled isomers, the beads form has also been shown to be the predominant species (Zhang and Snyder, 1991). The distribution of single disulfide pairing is also presented in . The three native disulfides together constitute 17.9% of the total disulfides found in scrambled hirudins. However, two of them, Cys-Cys (2.6%) and Cys-Cys (2.7%) are under-represented. For both scrambled species and disulfide pairings, there is an apparent correlation between their contents and the size of their disulfide loops. In general, the larger the disulfide loop, the lower the concentration ().

The properties of scrambled hirudins, the mechanisms of their formation and their consolidation to attain the native structure, may provide key answers to our understanding of protein folding. One of the most intriguing properties of scrambled hirudins is that their sensitivities toward denaturants vary substantially. For instance, in the presence of 6 M GdmCl, concentrations of fractions a, e, f, g, and h as compared to that of b, c, and d decrease by nearly 5-fold (Fig. 1). This suggests that scrambled species eluted within fractions a, e, f, g, and h contain favorable structures that are partially abrogated by denaturants. Whether these structures resemble native-like structure still remains to be elucidated. Interestingly, our data have shown that three out the five denaturant-sensitive species do not even contain any native disulfide, and the two major species in fractions b and c that do contain the native disulfide (Cys-Cys) are entirely insensitive to denaturants. These findings also raise a crucial question as to whether formation of the favorable native disulfide, Cys-Cys, is guided by the native-like interactions or is merely a consequence of probability.

  
Table: Structures of the disulfide-containing peptides derived from the thermolysin-digested scrambled hirudins


  
Table: 0p4in ``Not found'' does not imply ``not exist.'' Presentation of any species lowers than 0.5% will most likely evade the detection.(119)


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.

§
To whom correspondence and reprint requests should be addressed: K-121, 104 Ciba-Geigy Ltd., Basel CH-4002, Switzerland. Tel./Fax: 41-61-696-8313.

The abbreviations used are: HPLC, high performance liquid chromatography; Hir, hirudin; PTH, phenylthiohydantoin; MALDI, matrix-assisted laser desorption ionization; GdmCl, guanidinium chloride.


ACKNOWLEDGEMENTS

We thank Ueli Ramseier for performing the sequence analysis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.