Continuous-flow step gradient mass spectrometry based method for the determination of kinetic parameters of immobilized mushroom tyrosinase in equilibrating conditions: Comparison with free enzyme

Article abstract of DOI:10.1002/rcm.5268

A mass spectrometry (MS)-based methodology for enzymatic assay in equilibrium conditions was designed and evaluated. This on-line assay involves the introduction of a continuous-flow step gradient (CFSG) of a substrate solution in the column containing immobilized enzyme and the simultaneous tracking of the product formation. We showed that the constant concentration of substrate in the entire bioreactor for an appropriate duration ensures the equilibration of the studied enzyme (mushroom tyrosinase). Under these conditions, it was demonstrated also that the kinetic and enzymatic parameters (Michaelis-Menten constant, K_M, the maximal specific activity, SA_max) are independent of the flow rate of the mobile phase. The feasibility of the mentioned approach for inhibitory tests was also investigated. The coupling of the mass spectrometer to the bio-reactor allows the selective monitoring of the enzymatic reaction products and increases their detection level. Very high sensitivity, 500 pmol/min/column, and selective monitoring of the products of the enzymatic reaction are allowed by MS detection. The methodology developed here constitutes a sensitive analytical tool to study enzymes requiring long equilibration times. Copyright

Full text of DOI:10.1002/rcm.5268

Research Article
Received: 26 July 2011
Revised: 20 September 2011
Accepted: 20 September 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554
(wileyonlinelibrary.com) DOI: 10.1002/rcm.5268
Continuous-flow step gradient mass spectrometry based method
for the determination of kinetic parameters of immobilized
mushroom tyrosinase in equilibrating conditions: comparison
with free enzyme
*
Aleksander Salwiński, Raphaël Delépée and Benoît Maunit
Institute of Organic and Analytical Chemistry (ICOA), UMR CNRS 6005, University of Orleans, BP 6759, 45067 Orléans Cedex 2,
France
A mass spectrometry (MS)-based methodology for enzymatic assay in equilibrium conditions was designed and
evaluated. This on-line assay involves the introduction of a continuous-flow step gradient (CFSG) of a substrate solu-
tion in the column containing immobilized enzyme and the simultaneous tracking of the product formation. We
showed that the constant concentration of substrate in the entire bioreactor for an appropriate duration ensures
the equilibration of the studied enzyme (mushroom tyrosinase). Under these conditions, it was demonstrated also
that the kinetic and enzymatic parameters (Michaelis-Menten constant, KM, the maximal specific activity, SAmax
)
are independent of the flow rate of the mobile phase. The feasibility of the mentioned approach for inhibitory tests
was also investigated. The coupling of the mass spectrometer to the bio-reactor allows the selective monitoring of the
enzymatic reaction products and increases their detection level. Very high sensitivity, 500 pmol/min/column, and
selective monitoring of the products of the enzymatic reaction are allowed by MS detection. The methodology devel-
oped here constitutes a sensitive analytical tool to study enzymes requiring long equilibration times. Copyright ©
2011 John Wiley & Sons, Ltd.
Immobilized enzymes are widely used on industrial scale[1] and
on a laboratory scale, including analytical chemistry,[2,3] diag-
nostics,[4] drug screening,[5,6] etc. In many cases immobilized
enzymes are used to determine enzymatic and kinetic para-
meters: maximal enzymatic velocity, Vmax, Michaelis-Menten
constants, KM, for novel substrates or for the evaluation of the
inhibitory properties of leading drug molecules.[7,8] The on-
line kinetic assays of immobilized enzymes are normally
conducted by injection of a known amount of substrate into
an immobilized enzyme reactor (IMER) followed with detec-
tion of the product formed within the IMER.[7–11] In the single
injection mode, both apparent KM and Vmax values depend
drastically on the flow rate of the mobile phase[7–10]; in some
cases, no specified trend of this relationship can be deter-
mined, even for the same enzyme.[9,10] A decrease in the flow
rate leads to an increase in the contact time between enzyme
and substrate. For very low flow rates the full conversion of
given amount of substrate can be achieved that consequently
leads to formation of plateau of the relation between product
amount and flow rate.[11,12] Working within this range of
flow rates makes it impossible to apply the Michaelis-Menten
model (MM) to calculate kinetic parameters; thus the pre-
optimization of the single injection assay is required. In
the event of the use of long IMERs for single-injection assay
(e.g. capillaries), enzymatic conversion of each single sub-
strate portion gradually lowers its local concentration
during its migration towards the outlet of the IMER. This
affects the apparent V value for each substrate injection
and causes deviation from the MM model. Another phe-
nomenon that may influence the results of a kinetic assay
in single injection mode occurs when the studied enzyme
shows a delay (‘lag phase’) in substrate conversion.[13–15]
To circumvent these limitations, in this paper we propose
a continuous-flow step gradient method (CFSG) for on-line
kinetic tests of immobilized enzymes using mass spectro-
metry (MS) for selective detection of the product formation.
The presented method is based on stepwise increase of the
substrate concentration in continuous-flow mode and
ensures full equilibration of the bioreactor compared to
single injection experiments. The CFSG approach that is
described here for the first time combined a continuous
flow bioreactor,[16] and a frontal analysis of ligand–protein
binding methodology.[17,18] The mushroom tyrosinase,
used as a model enzyme (EC 1.14.18.1), catalyzes the
oxygen-based oxidation of monophenols to o-diphenols and
their subsequent oxidation to o-quinones.[19] Thus, we
demonstrate that the apparent Vmax measured by the CFSG
method is independent of the flow rate, show applicability
of the CFSG method to inhibition studies and compare
obtained values to free enzyme. In addition, we show a
temperature dependence on the kinetic parameters of
mushroom tyrosinase. The feasibility of this approach
for inhibition studies was presented using kojic acid as
model tyrosinase inhibitor.[20] Thanks to the application
* Correspondence to: R. Delépée, Institute of Organic and
Analytical Chemistry (ICOA), UMR CNRS 6005, University
of Orleans, BP 6759, 45067 Orléans Cedex 2, France.
E-mail: Raphael.Delepee@univ-orleans.fr
Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554
Copyright © 2011 John Wiley & Sons, Ltd.

A. Salwiński, R. Delépée and B. Maunit
of MS it became possible to monitor all products of the tyro-
sinase activity and achieve an outstanding 500 pmol/
min/column level of quantitation.
A volume of 1 mL of a solution of mushroom tyrosinase
(0.91 mg/mL) was added to a flask containing 0.121 g of
dry silica-beadsAPTES/GLA. The mixture was allowed to react
ꢁ
at 4 C for 7 h. After completion of the reaction, the mixture
was centrifuged, the supernatant was collected for assay
purposes and the beads were packed into an empty HPLC
column (30 mm length, 4.6 mm diameter). The empty space
was filled with sand. The grafting yield was 90.2%.
EXPERIMENTAL
Instrumentation
MS experiments were performed on Bruker maXis UHR-Qq-
TOF spectrometer (Bremen, Germany) with a Bruker sprayer
in positive electrospray ionisation (ESI) mode, working in
single-mass mode. The mass spectrometer was coupled in
on-line mode with a Dionex UltiMate 3000 ultra-high-
performance liquid chromatography (UHPLC) system (binary
pump, standard thermostated column compartment and DAD
detector) (Germering, Germany). For all experiments total ion
chromatograms were recorded within the mass range of 110 to
400. Profiles of ions of interest (L-Tyr, m/z 182.081 and
M + 2 isotopic peak of L-Tyr m/z 184.084; L-DOPA,
198.079 and dopachrome 194.047) were generated as
‘extracted ion chromatograms’ within the ꢀ0.01 m/z range.
Subsequently, average mass spectra were generated for the
steady-state for each step gradient and used to obtain final
values of ion responses.
Kinetic studies of the free enzyme
All kinetic tests were performed at room temperature (25 ꢁC).
Both buffers and samples were thermostated in the water
bath. In addition, the measuring cell was also temperature
controlled. Kinetic tests were based on several the ‘time
course’ measurements of L-dopaquinone formation (at 475 nm,
e475nm = 3716 Lꢂmol/cm), produced during the enzymatic
reaction.[21] The assay was conducted in a 1 mL quartz cuvette
filled with tyrosinase solution (corresponding to 2.73 mg of
enzyme) in 10 mM sodium phosphate buffer, pH 6.8. The
reaction was started by addition of L-tyrosine solution in
the same buffer (to obtain a final concentration between 70
and 600 mM). For the inhibition assay, the three following
concentrations of kojic acid were used: 0, 11.57 and
21.21 mM. Values of specific activity (SA) for each L-Tyr
concentration (Eqn. (1)) were fitted to the Michaelis-Menten
(MM) model to obtain the kinetic parameters. Contrary to
enzymatic velocity (Venz), specific activity (SA) is an inde-
pendent parameter describing enzymatic activity per unit
of enzyme quantity; therefore, it will be used later on in this
work.
Chemicals
Mushroom tyrosinase, L-tyrosine (>99%), (3-aminopropyl)
triethoxysilane (APTES) (97%), glutaraldehyde (GLA) (25%
in water) and kojic acid (KA) were purchased from Sigma
Aldrich (Isle-d’Abeau, France), ammonia (28% solution in
water) from Alfa-Aesar (Karlsruhe, Germany), formic acid
(97%) and sodium hydroxide were obtained from Fluka
(Isle-d’Abeau, France) and hydrogen peroxide (30%) from
Fisher Scientific (Elancourt, France). Silica beads (Geduran
Si 60, 40–63 mm) were purchased from Merck (Darmstadt,
Germany). All experiments were conducted in ammonium
formate buffer (11 mM, pH 6.8, verified before each
experiment).
Â
Ã
ΔAbs475nm
Δt ꢃ e475nm ꢃ Cenz
SA mmol= min=mg ¼
ꢃ D ꢃ 103
(1)
where SA is specific activity; Cenz is the concentration of
mushroom tyrosinase in the initial stock solution, mg/mL;
e is the absorption coefficient of dopaquinone at 475 nm,
L/mol/cm; ΔAbs475nm/Δt is the slope of the linear part of
the time course curve, s–1; D is the dilution factor of the
enzyme stock solution.
Kinetic studies of immobilized enzyme by the CFSG
method
Preparation of the bioreactor
Non-porous silica beads (3 g) were purified and activated by
incubation in a mixture of a 30% aqueous solution of NaOH/
30% H2O2/H2O: 1/1/4, v/v/v (48 mL) for 15 min. Then,
silica beads were washed with distilled water and then incu-
bated for 80 min at 80 ꢁC in 48 mL of the following solution:
37% HCl/30% H2O2/H2O: 1/1/4, v/v/v. Silica beads were
then washed with distilled water and dried under vacuum.
To an amount of 2 g of activated silica beads 10 mL of
toluene and 0.5 mL of APTES were ꢁadded. The mixture was
agitated under solvent reflux at 50 C for 13 h. Silica beads
were then washed with distilled water and kept in an oven
at 100 ꢁC for 1 h to promote the complete condensation
reaction with APTES. To the resulting silica beads coated with
APTES, 30 mL of glutaraldehyde solution (1.67 mL of
aqueous solution of GLA (25%) per 1 mg of derivatised silica)
prepared in sodium phosphate buffer (0.2 M, pH 7.5) was
added. The resulting mixture was allowed to react at room
temperature for 2 h. Silica-beadsAPTES/GLA were then dried
under vacuum.
The CFSG experiment requires a binary pump LC system to
form a step gradient of two mobile phases: A contains the
buffer solution and B: the substrate diluted into the buffer
solution; this mobile phase is called the ‘substrate phase’. By
changing the ratio of A to B one may modify the effective
substrate concentration in the bioreactor (Fig. 1).
Enzymatic specific activity (SA) is described as the amount
of product generated per minute and per defined amount of
enzyme (mmol/min/mg). If we consider the bioreactor as
the place of residence of the constant mass of enzyme (mg),
we can write:
Â
Ã
npr
t ꢃ m
SA mmol= min=mg ¼
(2)
where t is the time of contact between a substrate of a given
concentration and enzyme (min); npr is the amount (mmol)
of product generated within the time t; m is the the total
weight of immobilized enzyme in the bioreactor (mg).
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Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554

Continuous-flow step gradient for determination of enzyme parameters
Cpr is correlated to the amount of product of the enzymatic
reaction (npr) found in volume equal to the void volume of the
reactor (V0) and generated within the time needed for V0 to
pass through the bioreactor.
The concentration of product for each of step substrate
gradient, Cpr;i , is directly proportional to the surface area
1!4
under the product profile,Spr;i (Eqn. (5), Figs. 1 and 2):
1!4
Cpr;i ½mmol=mLꢄ ¼ bpr ꢃ Spr;i
(5)
1!4
1!4
where index ‘i’ represents a given step of gradient; b is the
response coefficient, which depends on the flow rate and
settings of the mass spectrometer
From Eqns. (4) and (5), we obtain the following relationship
describing the apparent enzyme activity for each concentra-
tion of substrate (Eqn. (6)):
Figure 1. Schematic outlook of the methodology of continu-
ous flow step gradient for analysis of enzymatic activity.
Values in round brackets represent the contribution of the
‘substrate phase’ in the mobile phase. Spr,i/Ssub,k is the surface
below the product/substrate profile for the time equivalent of
1 min; hi is the growth of L-Tyr ion response between succes-
sive levels of step gradient.
Â
Ã
bpr ꢃ Spr;i ꢃ Q
1!4
SAi
mmol= min=mg ¼
(6)
1!4
m
In respect to the continuous-flow method, in which the
product is continuously drained from the bioreactor with a
given flow rate Q (mL/min), an average time of the residence
of non-retained substrate in the bioreactor is given by the
void volume of bioreactor (mL) divided by Q (Eqn. (3)):
The b coefficient depends highly on the current settings of
the mass spectrometer such as ion transmission parameters,
flow rate of drying gas, settings of the nebulizer, etc., and
shows a linear relationship with the flow rate of the mobile
phase. Therefore, an internal standard is required for the
comparison of the subsequent analyses. If we introduce
the ratio of product to substrate response coefficients,
apr/sub (Eqn. (7)):
V0
Q
t½ minꢄ ¼
(3)
Thus, the apparent enzyme activity may be described as:
bpr
Â
Ã
npr ꢃ Q Cpr ꢃ Q
pr
sub
a
¼
(7)
SA mmol= min=mg ¼
¼
(4)
bsub
m ꢃ V0
m
and apply Eqn. (5) for the substrate, then insert it and Eqn. (7)
into Eqn. (6), we obtain the final relationship describing the
enzymatic activity in the continuous flow mode (Eqn. (8)):
where V0 is the void volume of the bioreactor (mL), Cpr is the
product concentration (mmol/mL) in the system after passing
through the bioreactor.
Figure 2. Practical profile of the substrate (L-Tyr) step gradient for exemplary assay.
(A) The profile of ion intensities of L-Tyr (blue line, m/z 184.086, M + 2 isotopic peak)
and L-DOPA (brown line, m/z 198.076, monoisotoic peak); the M + 2 isotopic ion is
used for L-Tyr because the intensity of the monoisotopic ion of L-Tyr is too high to fit
onto the same scale as L-DOPA. Mass spectra of (B) L-Tyr and (C) L-DOPA.
Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554
Copyright © 2011 John Wiley & Sons, Ltd.
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A. Salwiński, R. Delépée and B. Maunit
pr
sub
a
ꢃ Csub ꢃ Spr;i ꢃ Q
the use of L-Tyr as internal standard for L-DOPA is justified
by the structural similarity of these compounds.[22,23] Due
to linearity of the relationship of L-Tyr ion response to
L-Tyr concentration, selection of the reference ’level’ of
the step gradient (Eqn. (8)) is fully unrestricted, and in
the rest of this work will be called ’k’.
The response ratio of L-DOPA/L-Tyr (Eqn. (7)) was found
to be 1.16 ꢀ 0.07 (n = 5; measured for different ratios of
L-DOPA/L-Tyr concentrations ranging from 0.3 to 2.3).
Â
Ã
1!4
k
SAi
mmol= min=mg ¼
(8)
1!4
Ssub ꢃ m
k
where the index ’k’ represents the reference level of the
substrate gradient.
The response ratio (Eqn. (7)) may be determined experimen-
tally on the basis of Eqn. (5) (for product and, analogically, for
substrate) for mixtures of L-Tyr and L-DOPA measured
together in the same buffer as applied during experiments
with the bioreactor. The substrate can be considered as an
internal standard only if its transformation during the
enzymatic reaction is negligibly low (approximately 1–3%).
Experimental data were fitted to the MM model to obtain
kinetic parameters and displayed as Lineweaver-Burk plots.
The L-Tyr ion was monitored in the extracted ion chromatogram
(m/z 182.081, [M + H]+, Fig. 2(B)) and L-DOPA as 198.079,
[M + H]+, Fig. 2(C) (see next section). MS parameters were
optimized to minimize formation of adducts of L-tyrosine
and L-DOPA and fragmentation of these compounds.
Influence of the flow rate on the kinetic parameters of
immobilized enzyme
ꢁ
Subsequent experiments for three flow rates at 25 C (Fig. 3
and Table 1) showed lack of significant dependence of the
maximal specific activity (SAmax) and KM on the flow rate
(Q) (Student’s t-test, a = 0.05). Moreover, the error was
homogeneous (according to Cochran’s test). The non-
significant variations obtained for these parameters are
much smaller than the ones observed in the literature. For
example, for tyrosinase: deviations of KM and Vmax are
found up to 70%, 30% respectively for ΔQ = 0.1 mL/min
and for acetylcholinesterase: KM and Vmax are up to 21%,
27% respectively for ΔQ = 0.2 mL/min.[7–10] The lack of
influence of the flow rate on enzymatic parameters for the
CFSG method is the result of reaching an equilibrium state
RESULTS AND DISCUSSION
Practical description of the methodology
The experimental profile obtained from CFSG experiments
(Fig. 2(A)) is very similar to the theoretical profile (Fig. 1).
Differences between these profiles give information on
enzyme behaviour. Indeed if the observed slope for the
substrate profile can be attributed to apparatus dead volume,
the long equilibration time observed on the product is the
outcome of the complex enzyme cycle. The cycle of tyrosinase
activity includes regeneration of enzyme from the most
reduced (Edeoxy) to fully oxidized form (Eoxy). This comprises
other products like dopaquinone or dopachrome.[13,14] Both
dopaquinone (m/z 196.063, [M + H]+) and dopachrome
(m/z 194.047, [M + H]+) ions were observed during the
experiments but at negligible levels, approximately 9.5%
of the total ion response for L-DOPA, within the range
of 4–12% for the lowest step of gradient for dopachrome,
and less than 1% for dopaquinone. Such a conversion of
L-DOPA increases KM and lowers observed SAmax values
and is in all cases smaller than 10%. One should note
that, in the same way, transformation of dopaquinone
(into leukodopachrome and dopachrome) occurs also
during assays of tyrosinase activity, both immobilized
and in solution and is never taken into account during the
data analysis.[8,9,15,19,20,24] This phenomenon is absent for
enzymes catalysing one defined reaction. Full equilibration
of the enzymatic system is only achievable by the CFSG
approach. This shows the utility of the proposed metho-
dology compared to the conventional single injection
method.[7–10]
Figure 3. Influence of the flow rate on the Lineweaver-Burk
plots representing experiments at 25 ꢁC.
Table 1. Kinetic parameters of immobilized tyrosinase for
various flow rates. For each flow rate one series consisting
of four or five points (indicated in the table) was recorded.
a
a
Flow rate
[mL/min]
SAmax
KM
[nmol/min/column]
[mM]
In the present work, L-Tyr was used as internal standard.
Consumption of L-Tyr is on average at a level of 1.7%
(0.3–3.3%). This means that the relationship between the
substrate ion intensity (M + 2 isotopic peak of L-Tyr: m/z
184.084, [M + 2 + H]+) and its concentration both before
and after passing through the bioreactor is linear
(h1 = h2 = h3 = h4, Figs. 1 and 2(A)). This linearity is also
maintained for the second isotope of L-Tyr (its response is
comparable to standard product ion response). In addition,
0.25 (n = 5)
4.60 ꢀ 0.13
4.43 ꢀ 0.24
4.46 ꢀ 0.06
4.49 ꢀ 0.21
324 ꢀ 19
350 ꢀ 39
323 ꢀ 9
0.2 (n = 5)
0.15 (n = 4)
collective (n = 14)
aNon-significant difference between all values of a row
checked by Student’s t-test (a = 0.05). Deviations of SAmax
and KM are homogenous according to Cochran’s test
(a = 0.05).
331 ꢀ 32
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Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554

Continuous-flow step gradient for determination of enzyme parameters
and keeping the substrate concentration constant within
the bioreactor. For all flow rates, the apparent substrate
concentration for a given step in gradient is the same and
is not subjected to dilution or diffusion as for the single-
injection approach. An average KM constant for immobi-
lized enzyme (331 ꢀ 32 mM) is higher than for the free
enzyme (125.8 ꢀ 11.9 mM) which is consistent with the lit-
erature.[24–26] In addition this results from the steric con-
straints caused by covalent attachment of the enzyme to
the support. Day-to-day repeatability of KM and SAmax is
satisfactory and the standard deviation does not show
any statistical significance (Student’s t-test, a = 0.05). For
example, an experiment with two values of flow rate, 0.20
and 0.15 mL/min (both n = 5 points per run) conducted
one day before the experiments summarized in Table 1,
yielded collective KM and SAmax 366 ꢀ 46 mM and
4.38 ꢀ 0.28 nmol/min/column, respectively. RSD% values for
KM and SAmax are 7% and 1%, respectively; however, we
observed a gradual drop in enzyme activity, especially after
aggravating tests at higher temperatures.
To confirm these results, a single switch of ’substrate phase’
was conducted as follows: L-Tyr, ꢃmM (%): 0 (0) – 557 (100) –
0 (0) for five various flow rates. The observed growth of
product/substrate amount ratio upon the decrease in flow
rate is caused by longer contact time between substrate
and enzyme. According to expectations, due to correction
by the flow rate, SAapp (calculated by Eqn. (8) for one,
fixed concentration of L-Tyr for all values of flow rate)
remains unchanged despite the growth in relative product
amount (Fig. 4). When the speed of product drainage from
the IMER is the same as or higher than the conversion rate,
product/substrate amount ratio is expected to reach a
plateau, because the product dilution phenomenon is
eliminated after the solvent evaporation in the ion source of
the mass spectrometer.
Figure 5. Arrhenius relation SAmax(T–1), logarithmic scale
was applied. Inset: relationship between KM and reversed
temperature.
during subsequent experiments. The activation energy of
L-DOPA formation by immobilized tyrosinase calculated
on the basis of the Arrhenius equation is 110.4 kJ/mol.
The linear relationship of ln(KM) versus inversed tem-
perature (Fig. 5, inset) is consistent with the van’t Hoff
equation and shows that the Michaelis-Menten constant
may be attributed to dissociation constant of the L-Tyr from
the enzyme-substrate complex.[27]
Inhibitory studies of tyrosinase by the CFSG method
Kojic acid (KA) was selected as a model inhibitor of
tyrosinase. It was shown to be a competitive inhibitor of
monophenolase activity of mushroom tyrosinase towards
L-Tyr.[20] For CFSG experiments, the concentration of
inhibitor was maintained constant in the bioreactor during
entire gradient duration. In the case of the free enzyme assay,
the inhibition constant (KI) was found to be 3.56 ꢀ 0.44 mM,
while for the immobilized enzyme it was 3.10 ꢀ 0.05 mM
(Lineweaver-Burk plots are shown in Fig. 6).
Influence of the temperature on the kinetic parameters of
immobilized enzyme
CONCLUSIONS
The relation between SAmax and temperature of enzymatic
reaction is fully compliant with Arrhenius’ equation (Fig. 5).
The SAmax value may be used to plot the Arrhenius relation-
ship, because the total amount of enzyme is unchanged
The continuous-flow step gradient method for studying
enzyme activity in on-line mode with MS detection is a very
useful technique for bioreactors showing very low activity
(e.g. 500 pmol/min/bioreactor, which is obtained for the
highest concentration of KA recorded for this study). This is
Figure 4. Comparison of L-DOPA/L-Tyr (product/substrate
ratio (dots, ●) and apparent SA (squares, ■, calculated on the
basis of Eqn. (8)) for fixed L-Tyr concentration.
Figure 6. Lineweaver-Burk plots representing inhibitory
properties of kojic acid towards immobilized tyrosinase.
Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554
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A. Salwiński, R. Delépée and B. Maunit
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especially valuable for screening purposes, where in many
cases human enzymes have to be applied to avoid false posi-
tive or false negative results during the use of their animal or
bacterial equivalents. Amounts of human-derived enzymes
are usually very limited thus rendering the need to apply
extremely sensitive analytical methods. The CFSG
method eliminates the influence of flow rate on the kinetic
parameters (activity, KM) and enables on-line inhibition
assays. Simultaneously, application of a mass spectrometer
as the detector enables the specific and sensitive monitoring
of enzymatic products, which is often not possible in the
case of UV-VIS spectroscopy.
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The authors would like to thank Dr. Cyril Colas (ICOA,
Orleans, France) for his continuous and valuable technical
assistance in LC/MS measurements. The authors also wish
to thank Dr. Agnes Chartier for her valuable contribution to
the linguistic part of the present work and instructive discus-
sions concerning the factual content.
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Rapid Commun. Mass Spectrom. 2011, 25, 3549–3554