From the
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
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
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.
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
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
Fractions b and b* were isolated
and analyzed together. The data show that the predominant species
(b) is made of Cys
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
We thank Ueli Ramseier for performing the sequence
analysis.
-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.
(
)
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).
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.
10
M in 0.1%
trifluoroacetic acid in 4:1 H
O/CH
CN) 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
10
M)
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.
(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).
-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 ().
-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)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.