ESI-MS in the study of the activity of α-chymotrypsin in aqueous surfactant media

Article abstract of DOI:10.1039/b302931j

The catalytic activity of α-chymotrypsin on a model and a peptide substrate, in the supramolecular system enzyme-surfactant in water solution, has been studied by electrospray ionization mass spectrometry. Hydrolysis of N-succinyl-L-phenylalanine p-nitroanilide as the model compound, catalysed by α-chymotrypsin in the presence of monomeric cetyltributylammonium bromide, has been followed by UV and ESI-MS detection. Kinetic data, which are essentially identical independent of their determination techniques, show a twelve fold improvement of the enzyme catalytic efficiency when compared with the reaction carried out in the absence of the additive. Once validated, the ESI-MS technique was used to study the hydrolytic activity of the enzyme on a peptide substrate like substance P; it is worth emphasising that the spectrophotometric detection cannot be employed on peptides, where the chromophores are untouched by the hydrolytic process. Substance P hydrolyses in aqueous surfactant following dichotomic kinetics, which are initially rapid but then slow down as the reaction progress. The results presented in this paper are expected to extend studies on biocatalysis in aqueous surfactant media to a wide range of substrates, independent of their spectroscopic properties.

Full text of DOI:10.1039/b302931j

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ESI-MS in the study of the activity of ꢀ-chymotrypsin in aqueous
surfactant media†
Francesco De Angelis,*a Alessandra Di Tullio,a Piero Del Boccio,a Samantha Reale,a
Gianfranco Savellib and Nicoletta Spreti*a
a Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università dell’Aquila,
I-67100 L’Aquila, Italy
b Dipartimento di Chimica, Università di Perugia, I-60123 Perugia, Italy
Received 17th March 2003, Accepted 14th July 2003
First published as an Advance Article on the web 30th July 2003
The catalytic activity of α-chymotrypsin on a model and a peptide substrate, in the supramolecular system
“enzyme–surfactant” in water solution, has been studied by electrospray ionization mass spectrometry. Hydrolysis
of N-succinyl--phenylalanine p-nitroanilide as the model compound, catalysed by α-chymotrypsin in the presence
of monomeric cetyltributylammonium bromide, has been followed by UV and ESI-MS detection. Kinetic data,
which are essentially identical independent of their determination techniques, show a twelve fold improvement of
the enzyme catalytic efficiency when compared with the reaction carried out in the absence of the additive. Once
validated, the ESI-MS technique was used to study the hydrolytic activity of the enzyme on a peptide substrate like
substance P; it is worth emphasising that the spectrophotometric detection cannot be employed on peptides, where
the chromophores are untouched by the hydrolytic process. Substance P hydrolyses in aqueous surfactant following
dichotomic kinetics, which are initially rapid but then slow down as the reaction progress. The results presented in
this paper are expected to extend studies on biocatalysis in aqueous surfactant media to a wide range of substrates,
independent of their spectroscopic properties.
ESI-MS plays an important role in biological applications: it
Introduction
allows the primary structure determination of proteins and
other biomolecules,6,7 the elucidation of protein folding8,9 and
the detection of protein–protein interactions.10 In recent years,
ESI-MS has also been used for monitoring the kinetics of
enzymatic reactions;11–15 it is an accurate, rapid and sensitive
tool to follow, in real time, the change of the concentrations of
the substrate as well as of the products during the progress of
the hydrolytic reaction.
We will show that ESI-MS can be employed with success as
a tool to detect α-CT activity in water in the presence of a
surfactant medium. Kinetic data are totally in agreement with
those obtained by UV-Vis spectroscopy, at least in cases where
this technique is applicable. Moreover, the presumed super-
activation of the enzyme in water, promoted by the synthetic
surfactant, is for the first time partially verified in its reaction
against a natural substrate like the neuropeptide substance P.16
Enzymes have been widely used as catalysts in organic synthesis
and several methods have been developed to carry out bio-
conversions in organic media.1 However, many organic solvents
lead to inactivation of the enzyme, its stabilization being thus
an important goal in biocatalysis. In general, interactions
between an enzyme molecule and the surrounding water are
of crucial significance for enzymatic catalysis but the use of
the water as solvent in biocatalysis has always represented a
problem, because of the general low solubility of the reagents
in this reaction medium as well as of the reaction products.
On the other hand, aqueous surfactant media can provide an
aqueous phase for hydrolytic enzymes and an organic phase
for hydrophobic substrates and products. Moreover, examples
of the stabilization and/or superactivation of enzymes in water
solutions, in the presence of functionalized synthetic surfact-
ants, have recently been reported.2 Among these, the super-
activity of α-chymotrypsin (from now on referred to as α-CT)
in water, in the presence of cetyltributylammonium bromide
(CTBABr) is well documented.3,4 The reaction progress, in all
the cases reported, is followed by UV-Vis spectroscopy by using
model substrates which have been selected according to their
spectroscopic properties.
In this paper, we wish to report on the study of the catalytic
activity of α-chymotrypsin in the supramolecular system
“enzyme–surfactant”, in water solution, on substrates such as
amides and peptides, following the reaction progress by electro-
spray ionization mass spectrometry (ESI-MS). In the case of
compounds, which after enzymatic digestion preserve the
chromophore moieties unchanged, UV-Vis detection cannot be
used to register directly the reaction progress. In these cases,
spectroscopic methods require substrates modified by chromo-
genic agents. Such substrate modifications, however, often
necessitate multistep chemical syntheses and could lead to
altered enzyme kinetics.5
Results and discussion
The need for a model to validate, in an independent way, the
ESI-MS data became mandatory during early experiments. As
a preliminary study the activity of α-chymotrypsin, both with-
out and with the presence of CTBABr, was checked on a
reference substrate, the hydrolysis of which was followed by
ESI-MS as well as by UV detection.
In previous papers,3,4,17 N-glutaryl--phenylalanine p-nitro-
anilide (GPNA) was used as a model substrate and the effect
of different ionic surfactants and additives, along with their
concentration, was monitored on α-CT activity and stability.
GPNA is poorly soluble in aqueous ammonium acetate,
a volatile salt commonly used in the ESI-MS technique;
N-succinyl--phenylalanine p-nitroanilide (SPNA) was thus
selected for the MS experiments, as a more soluble model
substrate. Nevertheless, 1% CH3CN had to be added in order to
improve substrate solubility.
† Dedicated to Professor D. Spinelli on the occasion of his 70th
birthday.
SPNA is a substrate for α-CT; moreover, the anilide itself, as
well as the product of the hydrolytic reaction, gives abundant
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Table 1 Kinetic constants for α-chymotrypsin at 25.0 ЊC
Reaction medium
104 KM/M
kcat/sϪ1
(kcat/KM)/MϪ1 sϪ1
0.05 M Tris-HCl pH 8.0a
9.10
7.30
0.51
16.0 × 10Ϫ3
5.4 × 10Ϫ3
8.3 × 10Ϫ3
17.60
7.40
163.33
0.015 M Ammonium acetateb
3 × 10Ϫ5 M CTBABr in 0.015 M ammonium acetateb
a Ref. 18. b 1% CH3CN.
[M ϩ 1] ions under ϩve ESI conditions. As to its spectroscopic
characteristics there is, also, an evident bathochromic shift of
the absorbance of the aromatic moiety after hydrolysis. p-
Nitroaniline (PNA), one of the two reaction products, shows in
fact a maximum UV absorbance at 410 nm, while SPNA has its
maximum at 328 nm.
brought about a slight increase in the reaction rate, according to
the literature.3,19,20 On the contrary, in CTBABr solutions, the
SPNA hydrolysis rate dropped with a non-linear trend as salt
concentration increased: the superactivity, which occurred at
0.015 M ammonium acetate, was annihilated when a 0.2 M
value was reached. The decrease of the reaction rate with
increasing salt concentration can be attributed to interactions
between the salt itself and the surfactant.3 Ammonium acetate
could shield the positively charged surfactant head group while
the methyl group of the acetate anion could interact with the
hydrophobic region of the surfactant molecule. Therefore, the
favourable interactions between enzyme and surfactant, that
induce superactivity, were altered by the presence of a high salt
concentration.
As reported in ref. 4, α-CT superactivity is observed both in
monomeric and micellised CTBABr; positive enzyme–sur-
factant interactions taking place independent of the supra-
molecular organization of the medium. The large activity
improvement induced by CTBABr was thus explained by the
favourable interaction of the enzyme with the surfactant
monomers. α-CT activity was then investigated at 3 × 10Ϫ5
M
CTBABr, a concentration slightly lower than the cmc (cmc =
3.2 × 10Ϫ5 M), SPNA concentration being 1 × 10Ϫ4 M, as calcu-
lated by the KS definition (see experimental: KS = 3000 MϪ1).
The critical micelle concentration (cmc) and SPNA binding
constant (KS) were determined in 1% CH3CN ammonium
acetate.
α-CT activity as a function of the percentage of CH3CN
added to the reaction medium is reported in Fig. 2. Both in the
absence and in the presence of CTBABr, a decrease in reaction
rate was observed; when the percentage of CH3CN was equal to
20% no reaction occurred.
Kinetic parameters were also determined both in the pres-
ence and absence of CTBABr, and compared with the literature
data18 (Table 1). All our data were obtained by following the
reaction by UV detection (see experimental).
The data from the tris buffer and ammonium acetate experi-
ments show that the KM values are quite similar, while kcat in the
case of ammonium acetate decreases to a noticeable extent;
consequently, the enzyme efficiency (kcat/KM) was quite lowered.
This result could be explained taking into account that α-CT is
very sensitive to the buffer nature and concentration;3,19,20 the
presence of the aprotic water-miscible organic solvent can also
perturb the catalytically active conformation of the protein.
Moreover, the presence of the monomeric surfactant caused a
large increase in the α-CT–SPNA affinity (lower KM) together
with a higher kcat value: a large superactivity (22-fold) in 3 ×
10Ϫ5 M CTBABr was thus revealed.
In order to clarify the role of the salt and CH3CN on α-CT
activity, the effect of their concentrations on SPNA hydrolysis
rate in the presence and absence of CTBABr was investigated.
Fig. 1 reports the dependence of the SPNA hydrolysis rate on
ammonium acetate concentration, ranging from 0.015 M to 0.2
M. In pure ammonium acetate, an increase in its concentration
Fig. 2 Effect of CH3CN percentage on SPNA hydrolysis rate in 0.015
M ammonium acetate, pH 8 at 25.0 ЊC. [α-CT] = 0.2 mg mLϪ1, [SPNA] =
1 × 10Ϫ4 M. (᭿) Pure ammonium acetate and (᭹) in 3 × 10Ϫ5
M
CTBABr.
Kinetic parameters were then determined in 5% CH3CN
(Table 2). The comparison with the data in 1% CH3CN (Table
1) show that KM values are not greatly affected by the increase
of the added co-solvent and hence the decrease in α-CT activity
is almost entirely due to a reduction of the kcat values. The
presence of CH3CN does not modify the enzyme–substrate
interaction, since their affinity is almost unchanged; it follows
that the reaction rate decrease is probably due to a progressive
protein denaturation induced by the higher solvent concen-
tration. In fact, the effects of dipolar organic solvents on the
enzyme activity are generally attributed to alterations of vari-
ous non-covalent interactions in the protein, including sol-
vations of ionic groups and dipoles, hydrogen bonding and
hydrophobic interactions.21–23 These alterations can end up in
the modification of the tertiary structure of the protein to pro-
duce a variable degree of denaturation.
Having then set the reaction conditions for ESI-MS experi-
ments and with a full knowledge of the kinetic parameters, we
switched to mass spectrometry to follow the kinetics of the
reaction, performed in ammonium acetate alone and in the
presence of 3 × 10Ϫ5 M CTBABr both doped with 1% CH3CN.
Fig. 1 Effect of ammonium acetate concentration on SPNA hydrolysis
rate in 0.015 M ammonium acetate, pH 8 at 25.0 ЊC. [α-CT] = 0.2 mg
mLϪ1, [SPNA] = 1 × 10Ϫ4 M. (᭿) Pure ammonium acetate and (᭹) in
3 × 10Ϫ5 M CTBABr.
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Table 2 Kinetic constants for α-chymotrypsin in the presence of 5% CH3CN at 25.0 ЊC
Reaction medium
104 KM/M
kcat/sϪ1
(kcat/KM)/MϪ1 sϪ1
0.015 M Ammonium acetate
8.70
0.72
3.7 × 10Ϫ3
5.0 × 10Ϫ3
4.3
69.0
3 × 10Ϫ5 M CTBABr in 0.015 M ammonium acetate
FIA (flow injection analysis) was used for sample introduction,
and SIM (selected ion monitoring) to acquire data as mass peak
intensities. In particular, the ion corresponding to protonated
PNA was selected to quantitatively monitor the α-CT activity
since it shows an abundant peak at m/z 139.
SIM was used to generate a calibration curve for PNA (Figs.
3A, and 4A): an excellent correlation between concentration
and ion peak intensity was obtained (r2 = 0.999 in pure
ammonium acetate, and r2 = 0.996 in CTBABr solutions). Ion
intensities of the analytes, throughout the experiments, are
obviously affected by the presence of the surfactant; this
phenomenon appears to be completely controlled by building
up the calibration curves themselves, thus ensuring the com-
plete reliability of the different experiments performed. The
hydrolysis of SPNA was then performed at the same experi-
mental conditions used for the spectrophotometric analysis:
the ESI-MS results are shown in Figs. 3B and 4B. The change
in intensity of the peak relevant to the product ion was
determined as a function of time by repeated injections of the
reaction mixture in the MS instrument, at increasing hydrolysis
times. Data were then treated to give a measure of the reaction
progress under both the reaction conditions. The initial rates
were then measured and the results compared with those
obtained in parallel by UV-Vis spectroscopy, both in the
absence and in the presence of CTBABr. Data are reported in
Table 3.
Fig. 4 ESI-MS Plots; SIM of m/z 139 (protonated PNA): calibration
curve for PNA (A) and kinetic plot for the hydrolysis of SPNA (B) in
0.015 M ammonium acetate, pH 8 in the presence of 3 × 10Ϫ5
CTBABr. [α-CT] = 0.2 mg mLϪ1, [SPNA] = 1 × 10Ϫ4 M, 1% CH3CN.
M
Table 3 Comparison of the initial reaction rates obtained by UV-Vis
and ESI-MS monitoring in 0.015 M ammonium acetate alone and in
the presence of 3 × 10Ϫ5 M CTBABr
Reaction rate/µM minϪ1
UV-VIS
ESI-MS
Kinetics in ammonium acetate
Kinetics in surfactant solution
0.258
3.330
0.259
3.130
positive interactions between α-CT and CTBABr that induce
superactivity.4 Moreover, the results obtained by UV-Vis
spectroscopy and ESI-MS are practically identical; it can be
thus concluded that the ESI-MS technique can be efficiently
used to follow an enzymatic hydrolytic reaction.
The necessary extension of the ESI-MS methodology was to
study the kinetics of the enzymatic digestion of a real substrate.
We chose the neuroundecapeptide substance P, a widely studied
substrate already investigated in our laboratories by following
its partial acid hydrolysis by mass spectrometry.24,25 It is cleaved
by α-CT at the level of the aromatic amino acids. Five frag-
ments are formed, as listed in Table 4; the calculated m/z values,
relevant to single and doubly protonated molecular ions, along
with the amino acid sequence, are also reported.
The ESI mass spectrum of substance P reveals the presence
of the single and doubly protonated molecular ion at m/z
1348.7 and 674.9 respectively. Since the latter peak is more
intense than the former one, we decided to monitor the intensity
of this peak when following the kinetics of the α-CT digestion
of the peptide.
Fig. 3 ESI-MS Plots; SIM of m/z 139 (protonated PNA): calibration
curve for PNA (A) and kinetic plot for the hydrolysis of SPNA (B) in
0.015 M ammonium acetate, pH 8. [α-CT] = 0.2 mg mLϪ1, [SPNA] = 1 ×
10Ϫ4 M, 1% CH3CN.
Despite the change of the experimental conditions, i.e. the
buffer/salt (tris or ammonium acetate) and the substrate used,
and the addition of the organic solvent, we have confirmed the
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Fig. 5 ESI-Mass spectrum of the hydrolytic products of substance P.
Table 4 Hydrolytic fragments of substance P along with their single and doubly protonated molecular ions
m/z
Fragments
Sequence
(M ϩ H)ϩ
(M ϩ 2H)ϩϩ
8
9–11
8–11
1–7
1–8
Substance P
H-Phe-OH
H-Gly-Leu-Met-OH
H-Phe-Gly-Leu-Met-OH
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-OH
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-OH
166.1
320.2
467.2
900.5
1047.6
1348.7
83.6
160.6
234.1
450.8
524.3
674.9
Table 5 Comparison of the initial reaction rates obtained by ESI-MS
monitoring in ammonium acetate alone and in the presence of 3 × 10Ϫ5
M CTBABr
The hydrolysis of substance P was initially carried out both
with and without CTBABr, following the same experimental
conditions used for the model substrate. A few minutes after the
addition of the enzyme, the peak relevant to the doubly proton-
ated substrate had disappeared while the fragment peaks were
easily recognizable. The mass spectrum of the products is
reported in Fig. 5.
Reaction rate/µM minϪ1
Kinetics in ammonium acetate
Kinetics in surfactant solution
4.11
6.75a
1.25b
The high hydrolysis rate is probably due to a high enzyme–
substrate affinity; thus, to adequately follow the substrate con-
sumption, the enzyme concentration was 100-fold decreased.
A calibration curve was performed both in ammonium acetate
and in surfactant solutions (Figs. 6A and 7A); the correlation
coefficients between the substrate concentration and its ion
peak intensity were 0.996 and 0.997 respectively. Kinetics were
then performed; experiments were carried out in triplicate, in
order to ensure their reproducibility. Figs. 6B and 7B report the
change in abundance of substance P as a function of time in
pure ammonium acetate and in 3 × 10Ϫ5 M CTBABr, respect-
ively. The reaction rate values are reported in Table 5. In the
absence of surfactant, a linear decrease of the substrate con-
centration is observed; conversely, in the presence of CTBABr,
a 64% enhancement of the enzyme activity is observed only at
the very beginning of the reaction progress, while the reaction
kinetic rapidly slows down with time, becoming even lower with
respect to the reference reaction.
α-CT efficiency strongly depends on the substrate used. In
general, substrates with an extended peptide chain show a low
KM value, since the enzyme contains an extended binding site
for peptide substrates.26,27 The hydrolysis of a more sensitive
(low KM and high kcat) synthetic substrate, the tetrapeptide
succinyl--Ala--Ala--Pro--Phe-p-nitroanilide, was studied
in our laboratories to evaluate the role of the substrate on
surfactant-induced enzyme activation:28 only a 73% increase in
a 0–3 min. b 3–11 min.
kcat was observed thus showing that CTBABr did not signif-
icantly affect the high enzyme–substrate affinity. Conversely, the
α-CT efficiency observed for GPNA hydrolysis in the presence
of monomeric CTBABr was enhanced by about 20-fold.4 It can
be concluded that superactivity induced by CTBABr depends
on the substrate, the higher the enzyme–substrate affinity the
lower being the activating effect of the surfactant.
Coming back to substance P hydrolysis in the presence of
CTBABr, the increase in the enzyme activity observed at the
early stage of the reaction is very similar to that obtained with
the model tetrapeptide substrate. The subsequent abrupt
decrease in substrate hydrolysis can be explained taking into
consideration the high product concentration which could
inhibit the reaction progress. This possibility was verified by
following substance P hydrolysis by HPLC (under the same
reaction conditions), by monitoring the substrate consumption
and the lower peptide formation by UV detection, at the back-
bone absorption at 214 nm. Actually, the reaction rate slows
down after three minutes, thus exhibiting a feedback inhibition
by product formation. It is worth considering, however, that the
HPLC detection appears extremely time consuming as com-
pared to the ESI-MS method.
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Conclusions
As far as the activity of α-chymotrypsin in water is concerned at
the presence of a surface active compound, it can be concluded
that superactivity itself is not only related to the enzyme–
surfactant interaction; it strongly depends, in fact, also on the
nature of the substrate itself.
We have used SPNA as a model substrate, which can be
considered typical “organical” in nature; in this case α-CT–
surfactant cooperativeness is clearly observable. When using
a more “biological” substrate, like the neuropeptide substance
P, a much more complicate interaction amongst the three
reaction actors (substrate, α-CT and surfactant) is to be
conceived.
We also emphasize that our ESI-MS investigation on the
enzyme activity can contribute to extend the field of application
of ESI-MS to the study of biocatalysis in sustainable organic
reactions, carried out in water.
Experimental
Materials
α-Chymotrypsin (α-CT) from bovine pancreas (EC 3.4.21.1,
type II, MW 24.8 KDa, pI 8.8) and the substrates, N-succinyl-
-phenylalanine p-nitroanilide (SPNA) and substance P,
were purchased from Sigma and used without further purifi-
cations. Enzyme and substrate solutions were freshly prepared
immediately before their use. Ammonium acetate was from
Aldrich. The preparation and purification at laboratory
scale of the synthesized surfactant, cetyltributylammonium
bromide (CTBABr), have already been described.29 CTBABr
was chemically pure as tested by spectroscopic and elemental
analysis, and the absence of minima in surface tension vs.
concentration plots excluded the presence of hydrophobic
impurities.
Fig. 6 ESI-MS Plots; SIM of m/z 674.9 (doubly protonated substance
P): calibration curve for substance P (A) and kinetic plot for the
hydrolysis of substance P (B) in 0.015 M ammonium acetate, pH 8.
[α-CT] = 2 µg mLϪ1, [Substance P] = 1 × 10Ϫ4 M.
ꢀ-Chymotrypsin activity assay
The hydrolytic activity of α-CT toward SPNA was monitored
by following the increase in absorbance at 410 nm due to the
formation of p-nitroaniline (PNA). Kinetic determinations
were measured at 25.0 ЊC, using a Shimadzu UV-160A UV-Vis
spectrophotometer equipped with a thermostated cell (3 mL
volume and 1 cm pathlength). The product extinction coeffic-
ient was 10800 MϪ1 cmϪ1, either in ammonium acetate or in the
presence of CTBABr.
α-CT activity was typically assayed in 0.015 M ammonium
acetate, taken at pH 8 with a few drops of diluted ammonium
hydroxide, with
1 × M SPNA (solubilized in
10Ϫ4
ammonium acetate plus CH3CN) and 0.2 mg mLϪ1 (8 µM)
enzyme. The reaction was started by adding 60 µL of
α-chymotrypsin stock solution (10 mg mLϪ1) to a thermostated
solution of the substrate. PNA formation was recorded as a
function of time (spontaneous hydrolysis of SPNA did not
occur during the kinetic tests). The specific reaction rate, v,
defined as moles of PNA formed per unit of time and mass of
enzyme, was calculated from the slope of initial linear curve
of PNA concentration vs. time. The Michaelis–Menten
parameters (Michaelis constant, KM and rate constant, Vmax
)
were obtained by linear regression analysis of the double
reciprocal Lineweaver–Burk plots.
All sets of experiments were reproduced several times and the
differences between duplicates were always below 5%.
Determination of the critical micelle concentration (cmc)
Fig. 7 ESI-MS Plots; SIM of m/z 674.9 doubly protonated substance
P): calibration curve for substance P (A) and kinetic plot for the
hydrolysis of substance P (B) in 0.015 M ammonium acetate, pH 8 in
the presence of 3 × 10Ϫ5 M CTBABr. [α-CT] = 2 µg mLϪ1, [Substance
P] = 1 × 10Ϫ4 M.
The cmc of the surfactant was determined at 25 ЊC by
measuring the surface tension of the solution at different
surfactant concentrations. A Krüss du Nouy type tensiometer
was used.
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Determination of the binding constant (KS)
Hydrolytic activity of ꢀ-CT towards substance P
The following binding equilibrium between SPNA and the
micellised surfactant was assumed:
Calibration curves. The two calibration curves were con-
structed by injecting 1µL of the substance P solution of 10, 30,
50 and 100 µM in (i) 0.015 M ammonium acetate (Fig. 6A) and
in (ii) 0.0015 M ammonium acetate and 3 × 10Ϫ5 M CTBABr
(Fig. 7A), respectively.
where SW corresponds to the free substrate, Dn to the micellised
surfactant and SM to the substrate coordinated by the
surfactant aggregates. The determination of the binding con-
stant was conveniently performed by the spectrophotometric
method,30 at two different substrate concentrations ([ST] = 6 ×
10Ϫ5 M and 8 × 10Ϫ5 M), according to the following equation:
Kinetic analyses. α-CT activity was assayed with 100 µM
solution of substance P, 2 µg mLϪ1 of enzyme in (i) 0.015 M
ammonium acetate (Fig. 6B) and in (ii) 0.015 M ammonium
acetate and 3 × 10Ϫ5 M CTBABr (Fig. 7B). Enzymatic reactions
were monitored by injecting 1 µL of the reaction mixture every
1 minute for an overall time of 15 minutes.
Acknowledgements
The MS laboratory is part of the Italian network “Rete di
Spettrometria di Massa”, supported by funds from the
European Economic Community.
(εW and εM are the SPNA molar absorptivities in pure
ammonium acetate and in concentrated surfactant solutions,
respectively—the absorbance reaches a limit value with the
surfactant concentration) and was refined with the least square
fit. Determinations at 328 nm provided the following values: in
pure ammonium acetate εW = 10800 MϪ1 cmϪ1, in ammonium
acetate plus surfactant εM = 14500 MϪ1 cmϪ1; KS = 3000 MϪ1
(r2 = 0.97).
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15 A. J. Norris, J. P. Whitelegge, K. F. Faull and T. Toyokuni,
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ESI-MS Experiments
Mass spectral data were obtained on a QUATTRO triple-
quadrupole mass spectrometer (Micromass, Manchester, UK),
operating in the positive ion mode and equipped with a Z-spray
electrospray source. A HPLC system (Hewlett Packard Series
1100) with autosampler, without chromatographic column, was
used for sample introduction. Aliquots of sample solution were
directly injected into the ion source (flow injection analysis:
FIA), kept at 140 ЊC, via a 20 µL loop, at a flow-rate of 30 µL
minϪ1 (H2O–CH3CN–HCOOH 50 : 50 : 1 v/v/v), using nitrogen
as drying gas at 250 ЊC. The mass spectrometer operated with a
capillary voltage of 3.7 kV, and the sampling cone at 28 V for
SPNA, and at 42 V for the neuropeptide substance P. Full-scan
spectra were recorded by scanning from m/z 100 to 500 at 3 s
per scan (SPNA), and from m/z 150 to 1400 at 4 s per scan
(substance P).
The mass spectrometer was set and routinely employed
in selected ion monitoring (SIM, dwell time 0.1 s) to quanti-
tatively monitor the α-CT activity: at m/z 139 (protonated
p-nitroanilide) and m/z 674.9 (doubly protonated substrate
molecular ion) to follow hydrolysis of SPNA and substance P,
respectively.
16 S. Harrison and P. Geppetti, Int. J. Biochem. Cell Biol., 2001, 33,
555–576.
17 N. Spreti, P. Di Profio, L. Marte, S. Bufali, L. Brinchi and G. Savelli,
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1986, 236, 609–612.
Data acquisition and analysis were performed using the
software MassLynx (v. 3.5) running under Windows NT.
Hydrolytic activity of ꢀ-CT towards SPNA
Calibration curves. SIM was used to generate two calibration
curves for PNA both in ammonium acetate alone and in
surfactant solution. The curve of Fig. 3A was constructed by
injecting 1 µL of PNA solution 1, 3, 5, 7 and 10 µM in 0.015 M
ammonium acetate. The curve of Fig. 4A was generated by
injecting 20 µL of PNA solution 10, 15, 20, 25, 30 and 40 µM in
0.015 M ammonium acetate and 3 × 10Ϫ5 M CTBABr.
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623–624.
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27 K. Morihara and T. Oka, Arch. Biochem. Biophys., 1977, 178,
188–194.
Kinetic analyses. Enzymatic reactions were monitored as
follows: i) in ammonium acetate alone, 1 µL of the reaction
mixture was injected every 2 minutes (Fig. 3B) for an overall
time of 10 minutes; ii) in 3 × 10Ϫ5 M CTBABr, 20 µL of the
reaction mixture were injected every 1 minute (Fig. 4B) for an
overall time of 15 minutes. In both cases, SIM profiles were
smoothed and peak areas were used to give the kinetic plots of
peak areas versus time.
28 N. Spreti, unpublished results.
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1497–1502.
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1986, 25, 1–57.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 1 2 5 – 3 1 3 0
3130