Quantitative analysis, using MALDI-TOF mass spectrometry, of the
N-terminal hydrolysis and cyclization reactions of the activation
process of onconase
2
Marc Ribo
´
1
, Montserrat Bosch
1
, Gerard Torrent
1
, Antoni Benito
1
, Bruno Beaumelle
2
and Maria Vilanova
1
1
Laboratori d’Enginyeria de Proteı
¨
nes, Departament de Biologia, Facultat de Cie
`
ncies, Universitat de Girona, Girona, Spain;
2
UMR 5539 CNRS, Department Biologie-Sante
´
, Universite
´
Montpellier II, Montpellier, France
Onconase, a member of the ribonuclease superfamily, is a
potent cytotoxic agent that is undergoing phase II/III human

N- or C-terminal modifications constitute post-translational
modifications that can modulate a peptide activity and/or
resistance to degradation, as is the case with acetylation,
pyroglutamyl formation or C-terminal amidation. Many
proteins and bioactive peptides exhibit an N-terminal
pyroglutamyl, which subsequently minimizes their suscep-
tibility to degradation by aminopeptidases, although it may
also play a crucial role at the functional level [1]. This
residue is also a frequent determinant of overall peptide
function, as has been shown by the hypothalamic releasing
factor binding to its receptor [2], or by the amyloid b-peptide
and the implications in senile plaque formation and
pathogenesis in Alzheimer’s disease [3].
Onconase (ONC) is a ribonuclease that is present in the
oocytes and early embryos of the frog, Rana pipiens [4].
ONC, discovered as a result of its potent anticancer activity
[5], is now in Phase III human clinical trials for the
treatment of several types of cancer [6]. The enzyme, isolated
from frog oocytes, has an N-terminal pyroglutamyl residue
that contributes to the structure of its active site [7] and also
to its stability [8]. This N-terminal pyroglutamyl residue
is produced in vivo by the cyclization of the N-terminal
glutamyl residue. Pyroglutamyl N-termini have been found
in other frog ribonucleases that also display interesting
cytotoxic and antitumoral properties [9]. It has been
reported that non-natural N-terminal residues correlate
with a decrease in the catalytic activity and cytotoxicity of
these enzymes [10]. The interest in ONC as a therapeutic
agent has led to the expression of ONC recombinants,
created using site-specific mutagenesis, in order to study the

-galactoside; (Met1)-ONC
(M23L), onconase variant with a methionine preceding Gln1;
ONC, Onconase; (Pyr)-ONC (M23L), onconase variant with a
pyroglutamyl residue at position 1 and leucine replacing methionine
at position 23; rONC, wild-type recombinant onconase.
(Received 10 December 2003, revised 28 January 2004,
accepted 3 February 2004)
Eur. J. Biochem. 271, 1163–1171 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04020.x
variant, with the change M23L, was designed as a strategy
for obtaining recombinant ONC upon removal of (Met1) by
treatment with cyanogen bromide (CNBr) [13]. Notomista
and co-workers [12] showed that M23L substitution
increased the catalytic activity of this variant relative to the
native enzyme, but did not modify its potential as a
cytotoxin. Alternatively, the exopeptidase Aeromonas amino-
proteolytica aminopeptidase (AAP) [14] has proved to be
useful in removing Met1 from other ribonucleases, such as
angiogenin [15] and bovine seminal ribonuclease [16]. When
using (Met1)-ONC (M23L) as a substrate, it was found
necessary to completely denature and reduce the protein
in order to efficiently remove the methionyl residue [12].
Although, unlike CNBr treatment, the procedure did not
generate secondary products, it was very time consuming.
6
In this work we have characterized, using MALDI-TOF
MS, the two reactions involved in the activation process of
(Met1)-ONC (M23L) and established a rapid procedure to
activate the enzyme, without the need for reducing it once
purified. By using the purified recombinant protein, the
conversion of reagents and products of the exopeptidase-

VA, USA). Dulbecco’s modified Eagle’s medium (DMEM)
and fetal bovine serum were provided by Invitrogen
(Carlsbad, CA, USA).
Protein expression
BL21(DE3) cells, transformed either with plasmid pET11d-
(Met1)-ONC (M23L) encoding (Met1)-Gln1-ONC with
a Met in position 23 substituted by Leu, or with plasmid
pONC encoding Gln1-ONC, were grown to an attenuance
at 550 nm (D
550
)of 1.0–1.5 in LB (Luria–Bertani)
medium containing 100 lgÆmL
)1
ampicillin. Protein
expression was induced by the addition of isopropyl thio-b-
D
-galactoside (IPTG) to a final concentration of 1.0 m
M
.
After 3 h, the cells were collected by centrifugation, and the
cell pellets were stored at )20 °C.
Protein purification
Both (Met1)-ONC (M23L) and rONC were purified
according to a previously described procedure [18]. Briefly,
frozen pellets from 1 L of induced culture were resuspended
in 30 mL of 50 m
M
Tris/HCl, 10 m
M
EDTA, pH 8.0; the

M
Tris-acetate,
pH 8.5, and then incubated at 10 °Cforatleast24h.To
prevent refolding, the pH was adjusted to 5.0 with acetic
acid. The sample was then concentrated by ultrafiltration
and dialyzed against 50 m
M
sodium acetate, pH 5.0. Preci-
pitated or insoluble material was eliminated by centrifu-
gation (12 000 g,10min,4°C). The refolded sample
was then loaded onto a Mono-S HR 5/5 FPLC column,
and recombinant onconases were eluted with a linear
gradient of 0–600 m
M
NaCl in 30 min. Fractions containing
the pure (Met1)-ONC (M23L) or rONC were dialyzed
against ultrapure water, lyophilized and stored at )20 °C.
Hydrolysis and cyclization as a function of guanidinium
chloride concentration and pH
(Met1)-ONC (M23L) (0.1 m
M
) was prepared in 0.2
M
potassium phosphate buffer containing 0.5 m
M
ZnSO
4
with varying guanidinium chloride concentrations (0, 2, 3,
4.5 and 6
M

M
guanidi-
nium chloride, pH 8.0, was equilibrated for 30 min at
37 °C. The hydrolysis reaction was started, as described
1164 M. Ribo
´
et al. (Eur. J. Biochem. 271) Ó FEBS 2004
above, and allowed to proceed for 1 h. Once completed, the
sample was split into different aliquots, the pH was adjusted
to 2.5, 8.0 and 11.5, and aliquots of each pH value were
incubated at 37 °C, 50 °Cand70°C, respectively. From
these, aliquots of 2 lL were taken at 0, 1, 2, 4, 6 and 24 h,
and prepared for MS analysis as described above
9
.
MALDI-TOF MS
All mass spectra were acquired on a Bruker ULTRAFLEX
TOF mass spectrometer, at the Servei de Proteo
`
mica i
Bioinforma
`
tica (Universitat Auto
`
noma de Barcelona,
Barcelona, Spain), equipped with a nitrogen laser with an
emission wavelength of 337 nm. Spectra were obtained in
the linear positive mode at an accelerating voltage of 25 kV,
and deflection of the low mass ions m/z<5000 U was used
to enhance the target protein signal. Fifty spectra were

M
potassium
phosphate, pH 7.2. Finally, the protein was purified by ion-
exchange chromatography in a Mono S HR 5/5 FPLC
column, using a linear gradient of 0–200 m
M
NaCl in
20 m
M
potassium phosphate, pH 7.2, for 25 min. Pyr-ONC
(M23L), eluted as a single peak, was dialyzed against ultra
pure water to remove salts, then lyophilized.
Determination of conformational stability
The conformational stability of (Met1)-ONC (M23L) and
Pyr-ONC (M23L) was determined using UV spectroscopy
to measure the change in environment of the aromatic
residues during protein thermal unfolding. The proteins
were dissolved at 0.5 mgÆmL
)1
in a buffer of 50 m
M
glycine/
HCl, pH 2.0, and the UV absorbance was monitored
at 278 nm. The temperature was raised from 35 to 80 °C
in 2 °C increments. The decrease in UV absorbance was
registered after a 5 min equilibration at each temperature.
Reversibility was verified by decreasing the temperature in
10 °C decrements, equilibrating the sample for 10 min and
monitoring the absorbance at 278 nm. Unfolding transi-
tions curves induced by temperature were fitted to a two-

4
cellsÆmL
)1
. A431 cells were allowed to
attach for 2 h before the addition of ONC. After 3 days,
[
35
S]methionine was added (500 000 c.p.m. per well).
Twenty-four hours later, the cells were lysed with NaOH
and the protein precipitated with 15% trichloroacetic acid
(w/v). These proteins were collected onto glass fiber filters
using a cell harvester, washed with 5% trichloroacetic acid
(w/v), and radioactivity was counted using a liquid scintil-
lation counter. Background levels were obtained from cells
treatedwith1m
M
cycloheximide [21]. The results are
expressed as a percentage of control values obtained from
samples without ONC. The data for a single experiment
was the average of four determinations and experiments
were repeated three times. The IC
50
values represent the
ONC concentration that inhibited the cell protein
synthesis by 50%.
Other methods
ONC concentration was determined by UV spectroscopy,
using an extinction coefficient of 10 280
M
)1

single species of identical relative mass when assayed by
SDS/PAGE (data not shown). For recombinant (Met1)-
ONC (M23L), the yield was typically 15–20 mgÆL
)1
of
culture, which was, on average, double the yield observed
Ó FEBS 2004 MS characterization of onconase activation
1
(Eur. J. Biochem. 271) 1165
for rONC. This correlates well with the observation of
an induction band only in the SDS/PAGE analysis of
crude extracts of BL21(DE3)-pET11d-(Met1)-ONC (M23L)
induced cells. These yields were comparable to those
obtained by using other methods for the production and
purification of the enzyme [13]. The molecular mass of each
purified product, as measured by MALDI-TOF MS, was
11 947.87±2 for (Met1)-ONC (M23L) and 11 838.21±2
for rONC, which conforms well to the expected values:
11 949.90 for (Met1)-ONC (M23L) and 11 836.82 for
rONC with a glutaminyl residue at the N-terminus.
Reaction monitoring by MALDI-TOF MS
To determine the optimal technical conditions for acquiring
the spectra, we ran experiments in which samples were taken
at a specific time and placed on a probe spot. As soon as the
samples were dry, the probe was transferred into the mass
spectrometer so that spectra could be acquired immediately.
The mass spectra for the same samples were acquired again,
24 h and 72 h later. Comparing the peaks of the different
spectra revealed that they were indistinguishable in relative
intensity. Probably dehydration, together with acidification

for the first 8 h are shown. Within this period of time, and
under specific conditions, the hydrolysis reaction is com-
plete. When analyzing at the effect of guanidinium chloride,
it is notable that hydrolysis is favored by the increase in
guanidinium chloride concentration, being completed in as
little as 1–2 h at pH 7.2 or pH 8.0, when the guanidinium
chloride concentration is ‡ 3
M
. At pH 6.2, significant
differences can be observed in terms of guanidinium
chloride concentrations and the extent of the hydrolysis
reaction, which does not reach completion, in any case,
before the first 8 h. At 3
M
guanidinium chloride, the
reaction is complete within 24 h; however, it is still only
partially complete after 30 h at 0
M
(60%) and 2
M
(80%)
guanidinium chloride.
At pH 7.2, the differences in terms of guanidinium
chloride concentrations and Met1 removal are also clear.
In this case, the reaction is favored by higher guanidinium
chloride concentrations, being completed in 1 h at 4.5
M
and 6
M
guanidinium chloride (data not shown), in 2 h at

M
guanidinium chloride.
1166 M. Ribo
´
et al. (Eur. J. Biochem. 271) Ó FEBS 2004
between the relative intensities [Pyr-ONC (M23L)/Gln-
ONC (M23L) + Pyr-ONC (M23L)]. This value is prefer-
able to measuring only the appearance of Pyr-ONC
(M23L), as this would not be a true estimate of the
cyclization because this second reaction depends on the
hydrolysis reaction. When analyzing this set of data, we did
not find such a strong dependence of cyclization on pH
or guanidinium chloride, as was the case for hydrolysis.
However, when considering only the first 6 h of the
reaction, it was found that the rate of cyclization is almost
linear and can be obtained by calculating the slope of the
curves of Pyr-ONC (M23L) vs. time. From the rates of
cyclizationshowninTable1,itcanbeinferredthatthe
formation of the N-terminal pyroglutamyl is not dependent
on pH in the range 6.2–8.0. Interestingly, however, the
reaction is slowed down when the guanidinium chloride
concentration increases. These results concur with the
observation that the rate of conversion of L-Gln to Pyr in
aqueous solution is minimum at near neutral pH values [26].
To further characterize and establish the optimum
productive procedure for obtaining Pyr-ONC (M23L),
once the optimal hydrolysis conditions had been estab-
lished, we focused our attention on the cyclization reaction.
To evaluate the contribution of both pH and temperature
to the cyclization of the N-terminal glutaminyl to pyroglu-

protein was also observed after 6 h of reaction.
Analysis of the data as a function of pH (Fig. 3), showed
that cyclization at pH 2.5 is faster when the temperature
is increased, although for all the temperatures used in the
assay, the reactions were not complete within the first 6 h.
Fig. 2. Comparison of the hydrolysis reaction as a function of denaturing
agent and pH. (A), (B) and (C): (d), (s)and(.) correspond to
guanidinium chloride concentrations of 0 , 2 and 3
M
, respectively.
Hydrolysis was calculated as described in the Results.
Table 1. Rate of (Pyr1)-ONC (M23L) (an onconase variant with a
pyroglutamyl residue at position 1 and leucine replacing methionine at
position 23) formation as a function of guanidinium chloride concentra-
tion and pH. The rate of (Pyr1)-ONC (M23L) formation was
calculated as the slope of the curves generated by representing the
cyclized fraction, defined as [(Pyr1)-ONC (M23L)/(Gln1)-ONC
(M23L) + (Pyr1)-ONC (M23L)] during the first 6 h of the reaction.
pH
Guanidinium chloride concentration
0
M
2
M
3
M
6.2 0.1155 0.0728 0.067
7.2 0.1269 0.0734 0.066
8.0 0.1133 0.0834 0.067
Ó FEBS 2004 MS characterization of onconase activation

MALDI-TOF MS. The measured molecular mass of the
purified product was 11 799.70 ± 2, which concurs closely
with activated (Pyr)-ONC (M23L), which has a theoretical
molecular mass of 11 801.60.
Contribution of Met1 hydrolysis and pyroglutamyl
formation to the conformational stability of active
onconase
In order to evaluate the contribution of the (Met1)
hydrolysis and pyroglutamyl formation to the global
conformational stability of the protein, the thermal dena-
turation curves of (Met1)-ONC (M23L), Pyr-ONC (M23L)
and rONC were determined at pH 2 in 100 m
M
glycine/HCl
buffer by monitoring the UV absorbance at 278 nm. The
temperature unfolding transitions were reversible and fitted,
as previously described [19], to a two-state thermodynamic
model. In Fig. 4, the normalized curves are shown. The
fitted thermodynamic parameters for thermal transitions are
listed in Table 2. A comparison of the experimental data
collected for the three proteins makes it possible to estimate
the contribution of Met1, and Met23 substituted for Leu,
Fig. 3. Comparison of the cyclization reaction as a function of tem-
perature and pH. (A), (B) and (C): (d), (s)and(.) correspond to
pH 2.5, 8.0 and 11.5, respectively. Cyclization was calculated as des-
cribed in the Results.
Fig. 4. Normalized temperature unfolding curves. Samples at pH 2 in
100 m
M
glycine/HCl buffer were subjected to temperature increase

described in this work for the activation of ONC has no
detrimental effect on the conformational stability of the
enzyme.
Cytotoxic activity
The effect of (Met1) removal on the cytotoxic properties of
ONC was examined on K562 human erythroleukemic cells
and A431 human epidermoid carcinoma cells by measuring
the incorporation of [
35
S]methionine into newly synthesized
proteins after 96 h of incubation with an increasing
concentration of either (Met1)-ONC (M23L) or the activa-
ted Pyr-ONC (M23L). The results are shown in Fig. 5. For
both cell lines, activated Pyr-ONC (M23L) inhibited cell
proliferation, with an 50% inhibitory concentration (IC
50
)
value of 0.3–1 l
M
, several orders of magnitude lower than
those measured for (Met1)-ONC (M23L). IC
50
values in the
l
M
range for ONC have previously been described [27].
These data confirmed that the procedure, described in this
work, used to generate activated Pyr-ONC (M23L), resulted
in a protein that retains its full cytotoxic potential.
Discussion

tions and/or pH. It was found that the optimum pH is
pH 8.0. This result is in accordance with the fact that the
maximum stability and activity of the AAP occurs in the
pH range 8.0–8.5 [23]. When evaluating the influence of
Table 2. Thermodynamic parameters of the thermal denaturaturation of the different onconase (ONC) variants at 25 °C and pH 2.0. (C
Gdm/HCl
)
½
13
,
midpoint of guanidinium chloride denaturation curve; DG
U
14;15;16
, free energy of unfolding; DH
Tm
14;15;16
, enthalpy of unfolding calculated at T
m
; T
m
14;15;16
,midpoint
of the thermal denaturation curve. (Met1)-ONC (M23L), an onconase variant with a methionine preceding Gln1; (Pyr1)-ONC (M23L), an
onconase variant with a pyroglutamyl residue at position 1 and leucine replacing methionine at position 23; rONC, wild-type recombinant
onconase. Gdm, guanidinium.
T
m
a
(°C)
DH

35
S]methionine into newly synthesized protein
after 96 h of incubation with each onconase:
(d) (Met1)-ONC (M23L) (an onconase vari-
ant with a methionine preceding Gln1) and
(s) (Pyr)-ONC (M23L) (an onconase variant
with a pyroglutamyl residue at position 1 and
leucine replacing methionine at position 23).
The results represent the average of three
experiments.
Ó FEBS 2004 MS characterization of onconase activation
1
(Eur. J. Biochem. 271) 1169
guanidinium chloride on the reaction, the reaction rate
increases with increasing concentrations of guanidinium
chloride.
Enzymatic removal of the N-terminal methionyl residue
from other recombinant ribonucleases, such as angiogenin
[15] and BS-RNase [16], was performed using the AAP [14]
without the need for denaturing agents. However, when the
same approach was attempted with onconase, it was found
that it was previously necessary to reduce and denature the
protein to permit removal of the (Met1) [12], which resulted
in a time-consuming procedure.
AAP is a very stable enzyme, as it tolerates exposure to a
temperature of 70 °C for several hours and is only partially
inactivated in 8
M
urea. The active site, located at the
surface of the protein, is fairly open to the bulk solvent and

the N-terminal a-helix of ONC more accessible to the active
site of AAP and, thus, the hydrolysis reaction is favored.
High concentrations of guanidinium chloride do not have
a negative effect on the rate of the reaction because, as
mentioned previously, AAP remains active, even at high
concentrations of the denaturing agent.
Cyclization reaction
In contrast to other ribonuclease family members, the
N-terminal residue of ONC (Pyr1) forms part of the active
site [32]. The N-terminal glutamyl residue (Glu1) of ONC
cyclizes to form a pyroglutamyl (Pyr) residue, which folds
back against the N-terminal a-helix (a1) and forms a
hydrogen bond with the CO group of Val96 in the
C-terminal b-sheet. Pyr1 is also hydrogen bonded to the
e-amino group of the Lys9 side-chain, which simultaneously
interacts with the main phosphate of the substrate [32].
The closer positioning of the N-terminus to the main
protein body is essential for the activity and, together with
the 87–104 disulfide bridge, contributes to the high stability
of ONC against temperature [8,31]. Taking advantage of
this property, the cyclization reaction from Gln to Pyr was
evaluated as a function of temperature, up to 70 °C, by
using MALDI-TOF MS. Similarly to many reactions, it
was observed that temperature accelerates the reaction rate.
However, because the substrate is a polypeptide, care was
required with the pH of the reaction. Combining high or
low pH with high temperature resulted in the degradation,
by partial nonspecific hydrolysis, of the polypeptide chain.
When investigating the effect of pH on the cyclization
reaction, we expected that an acidic pH might enhance

a higher stability to ONC. Optimal conditions for activation
were pH 8.0, 3
M
guanidinium chloride and a temperature
of 37 °C, to permit the complete release of Met1, followed
by a 3 h incubation at 70° to allow the complete cyclization
of Gln1 to pyroglutamyl. The activation conditions defined
in this work significantly simplify the protocol and shorten
the time needed to obtain a fully active ONC compared with
other procedures described in the literature. The application
of MALDI-TOF MS to the characterization of enzymatic
and nonenzymatic reactions could be extended to other
simple systems in order to optimize the conditions under
which a reaction provides a higher product yield. Of
particular interest is the applicability of this strategy to
recombinant proteins where the presence of non-wild type,
N-terminal residues might have critical consequences on the
protein’s activity and its special biological functions, such as
cytotoxicity, stability or immunogenicity.
Acknowledgements
The authors thank Professor Ron Raines for the generous gift of the
genes encoding onconase and the variant. We are also indebted to
Dr Francesc Canals for his help in the mass spectra acquisition and
for the discussion of the results. This work was supported by grants
from the Ministerio de Ciencia y Tecnologia (BMC2000-0138-CO2-02,
1170 M. Ribo
´
et al. (Eur. J. Biochem. 271) Ó FEBS 2004
HF2000-0017), and from the Generalitat de Catalunya (SGR2000-64
and SGR2001-00196). M. B. gratefully acknowledges a predoctoral

8. Notomista, E., Catanzano, F., Graziano, G., Di Gaetano, S.,
Barone,G.&DiDonato,A.(2001)Contributionofchaintermini
to the conformational stability and biological activity of onconase.
Biochemistry 40, 9097–9103.
9. Okabe, Y., Katayama, N., Iwama, M., Watanabe, H., Ohgi, K.,
Irie, M., Nitta, K., Kawauchi, H., Takayanagi, Y. & Oyama, F.
(1991) Comparative base specificity, stability, and lectin activity of
two lectins from eggs of Rana catesbeiana and R. japonica and liver
ribonuclease from R. catesbeiana. J. Biochem. (Tokyo) 109,
786–790.
10. Leu, Y.J., Chern, S.S., Wang, S.C., Hsiao, Y.Y., Amiraslanov, I.,
Liaw, Y.C. & Liao, Y.D. (2003) Residues involved in the catalysis,
base specificity, and cytotoxicity of ribonuclease from Rana
catesbeiana based upon mutagenesis and X-ray crystallography.
J. Biol. Chem. 278, 7300–7309.
11. Mikulski, S.M., Grossman, A.M., Carter, P.W., Shogen, K. &
Kostanzi, J.J. (1993)
12
Phase I human clinical trial of ONCON-
ASEÒ (p-30 protein) administered intravenously on a weekly sch-
edule in cancer patients with solid tumors. Int. J. Oncol. 3, 57–64.
12. Notomista, E., Cafaro, V., Fusiello, R., Bracale, A., D’Alessio, G.
& Di Donato, A. (1999) Effective expression and purification of
recombinant onconase, an antitumor protein. FEBS Lett. 463,
211–215.
13. Boix, E., Wu, Y., Vasandani, V.M., Saxena, S.K., Ardelt, W.,
Ladner, J. & Youle, R.J. (1996) Role of the N terminus in RNase
A homologues: differences in catalytic activity, ribonuclease
inhibitor interaction and cytotoxicity. J. Mol. Biol. 257, 992–1007.
14. Wagner, F.W., Wilkes, S.H. & Prescott, J.M. (1972) Specificity of

23. Prescott, J.M. & Wilkes, S.H. (1976) Aeromonas aminopeptidase.
Methods Enzymol. 45, 530–543.
24. Laemmli, U.K. (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680–685.
25. Khandke, K.M., Fairwell, T., Chait, B.T. & Manjula, B.N. (1989)
Influence of ions on cyclization of the amino terminal glutamine
residues of tryptic peptides of streptococcal PepM49 protein.
Resolution of cyclized peptides by HPLC and characterization by
mass spectrometry. Int. J. Pept. Protein Res. 34, 118–123.
26. Arii, K., Kobayashi, H., Kai, T. & Kokuba, Y. (1999) Degrada-
tion kinetics of
L
-glutamine in aqueous solution. Eur. J. Pharm.
Sci. 9, 75–78.
27. Leland, P.A., Staniszewski, K.E., Kim, B.M. & Raines, R.T.
(2001) Endowing human pancreatic ribonuclease with toxicity for
cancer cells. J. Biol. Chem. 276, 43095–43102.
28. Cohen, S.L. & Chait, B.T. (1996) Influence of matrix solution
conditions on the MALDI-MS analysis of peptides and proteins.
Anal. Chem. 68, 31–37.
29. Krause, E., Wenschuh, H. & Jungblut, P.R. (1999) The domi-
nance of arginine-containing peptides in MALDI-derived tryptic
mass fingerprints of proteins. Anal. Chem. 71, 4160–4165.
30.Chevrier,B.,Schalk,C.,D’Orchymont,H.,Rondeau,J.M.,
Moras, D. & Tarnus, C. (1994) Crystal structure of Aeromonas
proteolytica aminopeptidase: a prototypical member of the
co-catalytic zinc enzyme family. Structure 2, 283–291.
31. Leland, P.A., Staniszewski, K.E., Kim, B. & Raines, R.T. (2000) A
synapomorphic disulfide bond is critical for the conformational