Utilization of real-time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

Article abstract of DOI:10.1002/rcm.6855

RATIONALE Related with its ability to degrade nucleotides, intestinal alkaline phosphatase (iAP) is an important participant in intestinal pH regulation and inflammatory processes. However, its activity has been investigated mainly by using artificial non-nucleotide substrates to enable the utilization of conventional colorimetric methods. To capture the degradation of the physiological nucleotide substrate of the enzyme along with arising intermediates and the final product, the enzymatic assay was adapted to mass spectrometric detection. Therewith, the drawbacks associated with colorimetric methods could be overcome. METHODS Enzymatic activity was comparatively investigated with a conventional colorimetric malachite green method and a single quadrupole mass spectrometer with an electrospray ionization source using the physiological nucleotide substrates ATP, ADP or AMP and three different pH-values in either methodological approach. By this means the enzymatic activity was assessed on the one hand by detecting the phosphate release spectrometrically at defined time points of enzymatic reaction or on the other by continuous monitoring with mass spectrometric detection. RESULTS Adaption of the enzymatic assay to mass spectrometric detection disclosed the entire course of all reaction components - substrate, intermediates and product - resulting from the degradation of substrate, thereby pointing out a stepwise removal of phosphate groups. By calculating enzymatic substrate conversion rates a distinctively slower degradation of AMP compared to ADP or ATP was revealed together with the finding of a substrate competition between ATP and ADP at alkaline pH. CONCLUSIONS The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity. Copyright 2014 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6855

Research Article
Received: 24 October 2013
Revised: 23 January 2014
Accepted: 24 January 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
wileyonlinelibrary.com) DOI: 10.1002/rcm.6855
(
Utilization of real-time electrospray ionization mass spectrometry
to gain further insight into the course of nucleotide degradation
by intestinal alkaline phosphatase
1
1
2
1
Christine M. Kaufmann , Johanna Graßmann , Dieter Treutter and Thomas Letzel *
1
Chair of Urban Water Systems Engineering, Technische Universität München, Am Coulombwall 8, 85748 Garching, Germany
Institute of Fruit Science, Technische Universität München, Dürnast 2, 85354 Freising, Germany
2
RATIONALE: Related with its ability to degrade nucleotides, intestinal alkaline phosphatase (iAP) is an important
participant in intestinal pH regulation and inflammatory processes. However, its activity has been investigated mainly
by using artificial non-nucleotide substrates to enable the utilization of conventional colorimetric methods. To capture
the degradation of the physiological nucleotide substrate of the enzyme along with arising intermediates and the final
product, the enzymatic assay was adapted to mass spectrometric detection. Therewith, the drawbacks associated with
colorimetric methods could be overcome.
METHODS: Enzymatic activity was comparatively investigated with a conventional colorimetric malachite green
method and a single quadrupole mass spectrometer with an electrospray ionization source using the physiological
nucleotide substrates ATP, ADP or AMP and three different pH-values in either methodological approach. By this means
the enzymatic activity was assessed on the one hand by detecting the phosphate release spectrometrically at defined time
points of enzymatic reaction or on the other by continuous monitoring with mass spectrometric detection.
RESULTS: Adaption of the enzymatic assay to mass spectrometric detection disclosed the entire course of all reaction
components – substrate, intermediates and product – resulting from the degradation of substrate, thereby pointing out
a stepwise removal of phosphate groups. By calculating enzymatic substrate conversion rates a distinctively slower
degradation of AMP compared to ADP or ATP was revealed together with the finding of a substrate competition
between ATP and ADP at alkaline pH.
CONCLUSIONS: The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and
specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-
component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different
nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity. Copyright © 2014
John Wiley & Sons, Ltd.
The sphere of the enzymatic activity of intestinal alkaline
phosphatase (iAP) is already known to be multifaceted, ranging
from adjustment of intestinal pH-value to downregulation of
the stimulation of ATP receptors on enterocytes. The conse-
quent rise in pH coincidently increases the activity of iAP,
thus inducing an enhanced ATP breakdown followed by a
[1]
[1]
lipid intestinal absorption and to effects on immunity and
reduced bicarbonate secretion.
[
2]
inflammation. The impact of iAP on immunity includes its
capability to dephosphorylate lipopolysaccharide (LPS), the
regulation of intestinal bacterial entry into the body and its
involvement in the formation of adenosine by the breakdown
of adenosine-5’-triphosphate (ATP), either possessing regu-
The investigation of enzymatic substrate degradation is
usually conducted by means of applying colorimetric methods,
which are often well established and therefore easy to handle.
However, they predominantly provide information solely about
either substrate degradation or product generation, resulting in a
picture lacking important information or that is ambiguous.
In this regard, mass spectrometry (MS) has already proved its
advantageous features, attested by the presence of a variety of
studies investigating a considerable amount of different
enzymes. Studies range from the determination of the kinetics
[2–4]
latory effects on inflammatory processes.
Besides the modulatory function of iAP in inflammation,
the enzyme takes part in intestinal pH regulation, whereby a
drop in pH-value leads to a reduction in the activity of the
enzyme and with that to an accumulation of ATP in the
intestine, in this manner provoking bicarbonate secretion by
[
5–7]
of enzymes, including the assessment of inhibitor constants,
to the elucidation of noncovalent complexes
[8,9]
and confor-
[10]
mational fluctuations associated with enzymatic catalysis.
Besides, elaborate setups like online coupled continuous flow
approaches coupled with mass spectrometric detection have
been applied for the finding of regulatory molecules in complex
*
Correspondence to: T. Letzel, Chair of Urban Water Systems
Engineering, Technische Universität München, Am
Coulombwall 8, 85748 Garching, Germany.
E-mail: t.letzel@tum.de
[11,12]
mixtures affecting enzymatic activity.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
Copyright © 2014 John Wiley & Sons, Ltd.

C. M. Kaufmann et al.
Concomitantly, MS detection provides the possibility to
entirely capture the substrate and product trace conti-
water (Milli-Q), 33.3% of a 0.081% (w/v) malachite green
oxalate solution dissolved in water (Milli-Q) and 33.3%
of water (Milli-Q).
[
13]
nuously in real-time.
Moreover, detailed information
about multiple reaction intermediates may be unveiled,
which are usually not measureable with colorimetric
methods.
Activity of iAP was investigated by means of colorimetric
detection of inorganic (free) phosphate. Due to the reaction
between malachite green molybdate and inorganic phosphate
under acidic conditions a malachite green phosphomolybdate
complex is formed that can be directly related to the
phosphate released by the enzyme’s activity.
[
14]
In fact the degradation of ATP by iAP has been shown to
include the formation of intermediates, but in an elaborate
and time-consuming procedure only monitoring single time
points of enzymatic reaction and also disregarding the
The assay was conducted in 10 mM ammonium acetate
solution (pH 6.0, 7.4 or 9.0) with either ATP, ADP or AMP
employed as enzymatic substrates. Ammonium acetate
[15]
generation of adenosine.
Because of its pH dependence, the activity of iAP has
already been investigated in a wide range of pH-values using
[
20]
was selected according to the work of Hogenboom et al.
and Dennhart and Letzel,
[16,17]
[21]
mainly the artificial substrate p-nitrophenyl phosphate.
However, it has been shown that the use of chromogenic
who have already been able
to demonstrate its applicability for the investigation of
enzymatic activity.
[18]
substrates may significantly affect enzymatic specificity.
Due to the variety of important functions associated with
the ability of iAP to degrade nucleotides and in view of the
possibility provided by MS detection to employ a non-
chromogenic substrate, enzymatic activity was tested with
physiological substrates. An MS-compatible enzymatic assay
was developed to enable the specific monitoring of all
reaction intermediates present in the course of the reaction
and to gain further insight into the enzymatic degradation
of several substrates.
For each experiment phosphate release was determined at
seven time points within 90 min. Therefore, seven indi-
vidual enzymatic assays were prepared in 10 mM
ammonium acetate solution with the respective pH-values
with 0.2U/mL (≜ 44.64nM) iAP and 40 μM of the respective
substrate (500 μL total volume). The reaction was started
simultaneously by the addition of the enzyme to all assays.
In the case of time point 0, the enzyme was added to the
assay, whereupon an aliquot was removed immediately and
the enzymatic reaction was terminated by addition of
malachite green reagent.
EXPERIMENTAL
In case of time points 15, 30, 45, 60, 75 and 90 min an
aliquot of one preliminarily prepared enzymatic assay was
withdrawn and mixed with malachite green reagent to
terminate the enzymatic reaction. The remaining assay was
discarded. The aliquot/malachite green mixture was
incubated for 20 min at room temperature and absorption
was measured at 620 nm to detect the released phosphate.
All experiments were performed six to nine times on two
different days at room temperature.
Reagents and chemicals
Ammonium heptamolybdate (#RDHA31402) was purchased
from Riedel de Häen (Seelze, Germany); malachite green
oxalate (#3076) was purchased from VWR BDH prolabo
(Darmstadt, Germany); poly(vinyl alcohol) (#363138),
ammonium acetate (#A7330), ATP (#A2383), ADP (#A2754),
AMP (#A1752), intestinal alkaline phosphatase from bovine
intestinal mucosa (Enzyme Commission (EC) number
Data evaluation in colorimetric experiments
3
.1.3.1., molecular weight (MW) ~160kDa) (#P7640), water
LC-MS CHROMASOLV(R) (39253-1L-R) were purchased
from Sigma-Aldrich (Steinheim, Germany).
Controls measured with the respective substrate in 10 mM
ammonium acetate solution pH 6.0, 7.4 or 9.0 without
enzyme revealed no increase in absorption. Therefore, an
auto-dephosphorylation of the substrates could be excluded,
whereupon a blank value correction was not implemented.
Data interpretation was conducted by comparing the slopes
of trend lines within the initial linear range of phosphate
release. The time interval for data evaluation was minutes 0 to
Instrumentation
Photometric measurements were performed with a SLT
Spectra plate reader (SLT Instruments, Crailsheim, Germany).
Mass spectrometric measurements were conducted with
a
MSQ Plus single quadrupole mass spectrometer
1
5 for enzymatic assays in pH 9.0, due to the faster reaction
and minutes 0 to 30 for enzymatic assays in pH 6.0 or 7.4
cf. Supplementary Table S1, Supporting Information).
(
Wissenschaftliche Gerätebau Dr. Ing. Herbert Knauer,
Berlin, Germany) equipped with an ESI source in the
positive ionization mode. Mass spectrometric needle voltage
was set at 3.5 kV, cone voltage at 75 V and temperature at
(
2
25°C. All samples were detected with a mass range of
Sample preparation for MS determination of enzymatic
activity
100 to 1000 m/z.
Initially, different substrate and enzyme concentrations were
tested with ATP substrate at pH 7.4. The most suitable
combination was identified as that which resulted in a complete
degradation of the substrate within 30 min and therewith in a
considerable flattening of ATP substrate trace. iAP assays were
performed in positive ionization mode, to maintain com-
parability to other enzymatic assays already established and
in this context to prospectively conduct experiments with
Sample preparation for colorimetric determination of
enzymatic activity
Reagents for malachite green assay, adapted from Henkel
[
19]
et al.
were always prepared freshly by thoroughly mixing
a proportion of 16.7% of a 5.72% (w/v) ammonium
heptamolybdate solution solved in 6 M HCl, 16.7% of a
2
.32% (w/v) poly(vinyl alcohol) solution dissolved in
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878

Studies on enzymatic activity of alkaline phosphatase
[22]
multiplex enzymatic assays. Even though higher intensities
may be anticipated in negative mode, initial intensities of
the nucleotides substrates between at least 150 000 counts
for ATP substrate in pH 9.0 and up to 400 000 counts for
AMP in pH 7.4 were obtained in positive mode.
Due to the pH-values applied and the disparity of available
phosphate groups of the substrate, intermediates and of the
product, different charge states of the assay components
might be present. Therefore, the negatively charged groups
might lead to a signal intensity underestimation of ATP and
ADP towards AMP or adenosine. Nevertheless, the intensities
of all assay components were exceedingly sufficient, thus
enabling the assessment of the substrate degradation by means
of an exponential trend line approach, also taking into account
that quantification was not intended. The activity of iAPs was
examined towards ATP, ADP or AMP in 10 mM ammonium
acetate solution at pH 6.0, 7.4 or 9.0, respectively. The pH-value
of the 10 mM ammonium actetate solution was checked daily
and was readjusted to the respective value if necessary. All
experiments were conducted 5 to 11 times on two different days
at 21°C.
Initially, 40 μM of the respective substrate was mixed with
10 mM ammonium acetate solution pH 6.0, 7.4 or 9.0. The
enzyme concentration of assays conducted at pH 6.0 or 7.4
was set to 0.2 U/mL (≜ 44.64 nM). Due to a distinct higher
enzymatic activity at pH 9.0 (see Results, Figs. 4(a) and 4(b)),
the concentration of enzyme was quartered to 0.05 U/mL
(≜ 11.16 nM) to retain the viability of the data-evaluation
procedure via the application of an exponential trend line.
Controls were measured with all possible substrate-pH assay
combinations but without enzyme to be able to exclude
phosphate release related to mass spectrometric settings.
Reactions were always started together with mass
spectrometric recording by pipetting the enzyme into the
substrate/ammonium acetate solution, followed by a thorough
and brief mixing. The assay was then drawn up into a 500 μL
glass syringe (Hamilton-Bonaduz, Switzerland), clamped into
a syringe pump (model 11 Plus, Harvard Apparatus, Hugo
Sachs Elektronik, Hugstetten, Germany) and continuously
pumped with 10 μL/min through a Peek tubing (1/16" × i.d.
0.13 mm, length 410 mm) into the source of the mass
spectrometer. This procedure caused a time delay until the first
signal could be detected and therefore the first 3 min of MS
measurements were not considered for further data evaluation.
Enzymatic reaction was recorded for 30 min.
Data evaluation in MS experiments
Data were processed using Xcalibur software 2.1.0.1139
(
Thermo Fisher Scientific Inc., Waltham, MA, USA). The
extracted ion chromatograms (EICs) of each assay
component, obtained by using a m/z range of ±0.5 Da to
the respective calculated m/z-value, were summed for the
following compounds: Adenosine with m/z 268.1 and
+
+
2
90.1, i.e. [Adenosine+H] and [Adenosine+Na] ; AMP
with m/z 348.1, 370.1, 386.0, 392.0 and 408.0; ADP with
m/z 428.0, 450.0, 466.0, 472.0 and 488.0; ATP with 508.0,
+
+
5
[
30.0, 546.0, 552.0 and 568.0, i.e. [M+H] , [M+Na] ,
+
+
+
M+K] , [M–H+2Na] and [M–H+Na+K] . The EICs were
smoothed with a Gaussian function using a 15-points
function width. Further processing was conducted with
Microsoft Office Excel 2007.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

C. M. Kaufmann et al.
At first each individual assay was normalized to 1
whereupon an exponential trend line was applied to the
respective substrate trace from minute 3 to 30 for assays at
pH 7.4 and 6.0 and minute 3 to 15 for assays at pH 9.0
contrast, acidic pH 6.0 was selected due to pH-values
present in the small intestine, that range from 5.5 to 7.5,
and might even locally decrease further during intestinal
[
24]
inflammation.
In addition, pH 7.4 was chosen to
(
Table 1) resulting in Eqn. (1). The substrate degradation
represent the standard pH of physiological relevance.
Besides the employment of three different nucleotide
substrates as well as three different pH-values, the activity
of iAP was determined using on the one hand mass
spectrometric detection and on the other a well-established
photometric method. The absorption values obtained hereby
are attributed to the overall release of phosphate. Besides its
advantageous features like being well established and easy
to handle, a main disadvantage of this procedure is the
indistinguishability of the actual origin of released
phosphate. A mass spectrometric assay was therefore
established to comprehensively examine the course of the
reaction, to obtain insight into the generation of all interme-
diates and to detect the final product adenosine. The gain in
information provided by mass spectrometric measurements
may thereby help to further elucidate pH-dependent
substrate degradation and substrate preferences of iAP in a
physiological context.
was observed until a plateau at a remaining intensity of 0.05
was reached, which reflected the termination of the reaction.
y = 0.05 was therefore set as the end point of the reaction. By
using Eqn. (1), the time (x) required for the enzyme to
completely degrade the substrate (y = 0.05) was calculated,
which was then inserted into Eqn. (2) to determine the
enzymatic conversion rate.
y ¼ a* expðbxÞ
(1)
Â
Ã
À
Â
ÃÁ
–1
conversion rate min ¼ c½substrateꢀ=c enzyme =x (2)
Statistics
Statistical analysis was performed in the same way for
photometric and mass spectrometric data sets by applying
Welch’s t-test, which takes into account the extent of the
respective standard deviations. Assays conducted with the
same substrate at different pH or assays with ATP, ADP or
AMP at the same pH-value were compared pairwise.
Statistical significance was assumed for p-values of 0.05 to
Colorimetric determination of iAP activity
The photometrical determination of enzymatic activity was
conducted by observing the phosphate released from either
0
.01 (*), very significant for p-values of 0.01 to 0.001 (**) and
[25]
ATP, ADP or AMP substrate at pH 6.0, 7.4 or 9.0.
extremely significant for p-values <0.001 (***).
By comparing the initial increases of absorption of all assay
compositions (data not shown) the most obvious differences
in slopes between the substrates used were measured at pH
9.0, where the increase is highest for ATP, followed by ADP
RESULTS AND DISCUSSION
(62% compared to ATP) and lowest for AMP (36% compared
Intestinal pH regulatory mechanisms and the enzyme’s
contribution to inflammatory processes along with a variety
of other functions are associated with the ability of iAP to
dephosphorylate nucleotides.
to ATP) (Fig. 2). Due to the fact that nearly all phosphate is
released during the first 15 min at pH 9.0, the differences in
slopes may be explained by the availability of phosphate
groups with either ATP or ADP or AMP substrate. This
hypothesis was confirmed by finding the highest final amount
of phosphate using ATP as substrate, followed by ADP and
AMP at pH 9.0 (data not shown).
At pH 7.4 the increases in phosphate are considerably
slower in case of all substrates compared to pH 9.0 (Fig. 2).
However, just as at pH 9.0, most phosphate is released
from ATP, followed by ADP and AMP. Although the
difference in phosphate increase during the first 30 min
is not significant between ATP and ADP substrate at pH
7.4, the mean value was detected to be slightly lower for
ADP (86%) compared to ATP (100%), whereas it was
Since ATP is naturally degraded to adenosine by the
[15]
stepwise removal of its three phosphate groups,
possible
intermediate products ADP and AMP were also used as
substrates (Fig. 1).
Moreover, enzymatic activity was examined at three
different pH-values (pH 6.0, 7.4 and 9.0). pH 9.0 was chosen,
since the enzyme possesses its highest activity in alkaline
[
17]
medium.
Besides, the alkaline pH was selected to
prospectively investigate regulatory compounds affecting
enzymatic activity, which was similarly done previously by
[23]
Vovk et al., who studied inhibition of iAP at pH 9.0.
In
Figure 1. Enzymatic hydrolysis and stepwise removal of inorganic phosphate
leads to the generation of the final iAP product adenosine. Nucleotide
structures are given along with the average molecular weights of the
respective neutral compounds.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878

Studies on enzymatic activity of alkaline phosphatase
Figure 2. Box-and-whisker plot of numeric values of slopes of linear trend lines
of initial phosphate increase photometrically detected; positive and negative
whisker, first quartile, median and third quartile, mean value (♦), significant
for p-values of 0.05 to 0.01 (*), very significant for p-values of 0.01 to 0.001 (**)
and extremely significant for p-values <0.001 (***), not significant for values
>0.05 (n.s.), n = 6–9.
significantly reduced with AMP substrate (42%) (Fig. 2).
That is because the amount of available phosphate groups
does not decrease markedly during the early stages of
reaction using ATP or ADP, since the breakdown of ATP
or ADP substrate again results in the generation of
ADP/AMP or AMP, respectively, which in turn serve as
Figure 3. Assay component’s signal changes within the m/z range 200 to 550 at
pH 7.4 at the beginning of enzymatic conversion (a: average intensity of time
range minute 3 to 6 corresponding to light grey area) and the end of the
measurement (b: average intensity of time range minute 27 to 30 corresponding5
to dark grey area). Spectrum intensity maximum ’100’ corresponds to 2.63 × 10
+
+
counts. Prominent m/z within spectrum: [Ado+H] : 268.0; [AMP+H] : 348.0,
+
+
+
+
[
AMP+Na] : 369.9; [ADP+H] : 428.0, [ADP+Na] : 450.0; [ATP+H] : 507.9,
+
[
ATP+Na] : 529.8.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

C. M. Kaufmann et al.
[
28]
substrate. In contrast with AMP substrate merely the final
product adenosine is generated. Therefore, with ATP or
ADP compared to AMP substrate, it is more likely for
the enzyme to encounter a substrate during the first stages
of the reaction, indicating the availability of substrate as
the rate-limiting step with AMP substrate at pH 7.4 and
ATP, ADP and AMP substrate at pH 9.0.
At pH 6.0 enzymatic activity is distinctly reduced
compared to alkaline or neutral pH, resulting in a consi-
derably slower phosphate release from ATP (7% compared
to pH 9.0), ADP (13% compared to pH 9.0) and AMP (15%
compared to pH 9.0) (Table 1).
green assay does not respond to pyrophosphate,
its
formation would lead to an underestimated phosphate
release in the case of ATP substrate. In fact, mass spectro-
metric measurements (see below) revealed a tendency for
faster degradation of ATP compared to that of ADP (83%)
at pH 6.0.
Although the application of a colorimetric method to detect
the enzymatic activity provides information about the effect
of pH on the degradation of different nucleotide substrates,
it also reveals several drawbacks. First of all a differentiation
between the phosphate released from initial substrate or
intermediate products is not achievable. Additionally, it
cannot be clarified whether the phosphate groups are
removed in a stepwise manner or if, for instance, pyrophos-
phate is released. This matter was first addressed by Moss
However, the differences in slopes (Fig. 2) between the
three substrates are significant, with ADP leading to a
faster increase in detectable phosphate (122%) than ATP
(
100%) and AMP (80% compared to ATP). At acidic pH
et al., who proved a stepwise removal from ATP resulting in
[
26,27]
[15]
the pyrophosphate synthesis by iAP is enhanced,
ADP and AMP as intermediate products.
Nevertheless,
probably leading to an excess production of pyrophosphate
with ATP compared to ADP substrate. Since the malachite
this investigation was conducted at one pH-value, i.e. pH
9.5, and solely ATP was used as substrate. So to our best
Figure 4. Mass spectrometric determination of enzymatic degradation of 40 μM
ATP substrate: (a) iAP concentration 44.64 nM, pH 9.0; (b) iAP concentration
1.16 nM, pH 9.0; (c) iAP concentration 44.64 nM, pH 7.4; (d) iAP concentration
4.64 nM, pH 6.0; m/z evaluated, summarized and displayed here: Ado with m/z
1
4
2
3
5
+
+
68.1 and 290.1, i.e. [Ado+H] and [Ado+Na] ; AMP with m/z 348.1, 370.1, 386.0,
92.0 and 408.0; ADP with m/z 428.0, 450.0, 466.0, 472.0 and 488.0; ATP with
+
+
+
+
08.0, 530.0, 546.0, 552.0 and 568.0, i.e. [M+H] , [M+Na] , [M+K] , [M–H+2Na]
+
and [M–H+Na+K] ; n = 5–11.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878

Studies on enzymatic activity of alkaline phosphatase
knowledge, no information about detailed reaction kinetics,
the degradation of ADP or AMP as substrates and also about
the activity of iAP towards these three substrates at different
pH-values is available. For these reasons, a mass spectro-
metric assay was established to obtain detailed data of the
overall course of the reaction. In this regard Fig. 3 exemplarily
shows the results of enzymatic ATP conversion at pH 7.4.
Traces of ATP substrate, intermediates ADP and AMP and
the final product adenosine are presented along with spectra
revealing the shift of m/z signals associated with the
degradation of ATP substrate (m/z 507.9, 529.8) and the
generation of ADP (m/z 428.0, 450.0), AMP (m/z 348.0, 369.9)
and adenosine (m/z 268.1).
At pH 9.0 (Fig. 4(a), high enzyme concentration) the
degradation of both ATP and ADP is completed within the first
7 to 8 min, whereas AMP slightly increases till minute 5 before
also decreasing until minute 30. Furthermore, the AMP curve
reveals a flattening, that is also reflected by the curve of the final
product adenosine, both indicating a decelerated degradation
of AMP at the end of the measurement.
By comparing the substrate degradation at pH 9.0 the
conversion rate determined by mass spectrometry is highest
for ADP (131%) and lowest for AMP (21% compared to
ATP) (Fig. 5 and Table 1). The inferior conversion rate for
ATP in comparison to ADP substrate might be associated
with an initial substrate competition between ATP and the
product ADP and AMP, eventually leading to a decreased
degradation and therewith conversion rate of ATP.
Enzymatic assays with ADP substrate revealed a similar
progress of fast enzymatic substrate degradation and
related intermediate and product generation as those
assays measured with ATP substrate (see Supplementary
Figs. S2(a)–S2(c), Supporting Information). Like ATP
substrate at pH 9.0, ADP substrate is completely degraded
within approximately 10 min, revealing a comparable
substrate preference for ATP and ADP. In contrast with
AMP substrate at pH 9.0 a continuous decrease was
observed over the whole measurement time, uncovering
AMP as a comparatively poor substrate (21% compared to
ATP, Supplementary Fig. S1(a), Supporting Information).
At pH 7.4 the degradation of ATP and simultaneously the
generation of ADP are distinctively slower compared to
assays conducted at pH 9.0 (Fig. 4(c)). After approximately
12 min a plateau is reached for ADP, i.e. formation from
ATP and degradation to AMP is of similar velocity. Contrary
to assays at pH 9.0 no decrease in AMP intensity was detected
at neutral pH. However, generation of adenosine is apparent
during the whole time of measurement, testifying to the
degradation of AMP to adenosine, which in conclusion
indicates a higher generation of AMP from ADP than
degradation of AMP to adenosine.
MS determination of iAP activity
Applying the same concentrations as in the photometric
assay, a nearly complete degradation of ATP within the first
5
min at pH 9.0 was revealed (Fig. 4(a)), that could not be
assessed with photometric detection, due to merely detecting
the overall phosphate release.
To enable the calculation of conversion rates by applying
exponential trend lines to the substrate degradation trace,
enzyme concentration at pH 9.0 was quartered to 11.16 nM,
resulting in a quartering of enzymatic activity visible by
the shift of AMP detection maximum from minute 4.18
with 44.64 nM (Fig. 4(a)) to minute 16.33 with 11.16 nM
iAP (Fig. 4(b)). Figure 4 furthermore provides an overview of
the ATP substrate degradation, the formation of ADP and
AMP intermediates and the generation of the final product
adenosine in 10 mM ammonium acetate solution at pH 9.0
(Figs. 4(a) and 4(b)), pH 7.4 (Fig. 4(c)) and pH 6.0 (Fig. 4(d)).
In contrast to the photometric approach, for which the slopes
of linear trend lines were used to obtain numeric values to
enable the comparison of phosphate releases, in the case of
MS-detected assays conversion rates were calculated and are
presented in Fig. 5.
Figure 5. Box-and-whisker plot of substrate conversion rates calculated from
exponential trend lines applied to the respective substrate traces mass
spectrometrically detected; positive and negative whisker, first quartile, median
and third quartile, mean value (♦), significant for p-values of 0.05 to 0.01 (*),
very significant for p-values of 0.01 to 0.001 (**) and extremely significant for
p-values <0.001 (***), not significant for values >0.05 (n.s.); n = 5–11.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

C. M. Kaufmann et al.
Compared to ATP substrate the conversion rate with ADP
substrate at pH 7.4 is decreased to 72% (Fig. 5), pointing out
a less pronounced substrate competition at pH 7.4 in contrast
to pH 9.0. This is attributed to a lower conversion rate of ATP
at pH 7.4 (7%, Fig. 5) compared to the conversion rate of ATP
at pH 9.0, resulting in a minor quantity of ADP generated at
pH 7.4, which is insufficient to provoke substrate competition.
Furthermore, a much more pronounced increase in AMP at
pH 9.0 was detected from the beginning of the measurement
compared to the increase in AMP at pH 7.4, which results in
a higher amount of competitive substrate AMP at pH 9.0.
At pH 6.0 the average conversion rate with ATP substrate
was calculated to be even slower (2%) than at pH 7.4 (7%)
compared to pH 9.0 (Fig. 5). Nevertheless, an increase in
products ADP, AMP and also a slight increase in adenosine
was observed (Fig. 4(d)). Average conversion rate at pH 6.0
is highest for ATP, followed by ADP (83%, not significant)
and AMP (69%), the latter significantly reduced compared
to average ATP substrate conversion rate.
AMP compared to ATP, although the substrate concentrations
of AMP and ATP resulting in maximal velocity (Vm) of
substrate conversion detected by Chappelet-Tordo et al. are
comparable to those applied in this study.
It has been demonstrated in several investigations that
diverse factors like type of buffer, ionic strength, metal
2
+
2+
ions, in particular Mg and Zn , may influence activity
[
33,34]
and specificity of iAP.
that the order of degradation of ATP, ADP and AMP is
For instance, it has been shown
[
31]
reversed by addition of magnesium.
In this work no
2
+
2+
cations like Mg or Zn were added. However, it is
unclear whether Chappelet-Tordo et al. used any additives,
which in turn impedes a final conclusion regarding the
disparity of experimental results. Anyhow, they applied
buffers containing Tris in the case of pH
8 and
ethanolamine at pH 10, both additives being good
[
35]
phosphate acceptors
which may result in a lower level
of detectable phosphate with ATP.
Moreover, as mentioned before, the ’side reactions’ of
pyrophosphate formation may also mimic a lower phosphate
release from ATP compared to AMP in the measurements of
Chappelet-Tordo et al. More probably, however, is a difference
in the enzyme used. In the study presented here iAP from
bovine intestine was employed, whereas Chappelet-Tordo
et al. used an enzyme originating from calf intestine. This
enzyme however was proven in a later study to be
With ADP substrate, conversion rate at pH 6.0 was calculated
to be only 1%, whereas at pH 7.4 it is 4% compared to the
conversion rate of ADP at pH 9.0. Conversion rates with
AMP substrate are 6% at pH 6.0 and 9% at pH 7.4 compared
to conversion rate of AMP at pH 9.0. So the relationship
between average values of conversion rates for assays at pH
6
.0, 7.4 and 9.0 with ADP and AMP substrate is comparable
to those obtained with ATP substrate (2% at pH 6.0, 7% at pH
.4 compared to pH 9.0, Table 1), suggesting a similar response
[36]
heterogenic, i.e. to consist of different forms of the enzyme.
These, moreover, possess considerable different activities
7
[37]
of the enzymatic activity to a decline in pH-value, which is
independent of the nucleotide substrate used.
towards AMP.
So it may be assumed that, although a
purification step was conducted, a heterogenic mixture of
enzymes was used, probably showing markedly different
substrate specificity.
In our opinion a lower degradation rate of AMP is more
conceivable since the proximity of both the sugar residue
and also the base of AMP may cause a steric hindrance or
even the formation of interfering hydrogen bridge linkages
between the nucleotide base and amino acids of the enzyme
active site.
By further comparing the photometric and mass
spectrometric measurements at pH 7.4 the results are similar,
both revealing the highest enzymatic activity – reflected
either by phosphate release or conversion rate – with ATP
substrate, followed by ADP substrate (not significant) and
AMP substrate (significant). AMP was again detected to be
the poorest degradable substrate at pH 6.0, pointing out the
clear substrate preference of iAP towards ATP and ADP.
Nevertheless, substrate competition between ATP and ADP
could be shown only by mass spectrometric detection, due
to the ambiguous nature of colorimetric phosphate release
detection. Mass spectrometric experiments clearly revealed a
higher degradation rate for ADP compared to ATP at pH
CONCLUSIONS
The mass spectrometric results revealed conversion rates that
are considerably lower at pH 7.4 as well as pH 6.0 compared
to pH 9.0, which is consistent with the data obtained
photometrically (Table 1) and mostly also with data from
[
31,32]
the literature.
Furthermore, these findings are in
accordance to results of Chappelet-Tordo et al. who observed
a rise in enzymatic activity and a higher dissociation constant
for the enzyme-inorganic phosphate complex with increasing
[
17]
pH-values.
Also Cocivera et al. demonstrated an increase
in activity at alkaline pH and a considerably lower rate
constant for the decomposition of the covalent enzyme-
inorganic phosphate intermediate at acidic pH-values,
thereby causing a lower availability of the enzyme’s active
site for further breakdown of substrate, indicating this step
[
29]
as rate-limiting.
Furthermore, Fernley and Walker
a
demonstrated a decrease in Ki for Pi and PPi at less alkaline
pH, suggesting a stronger inhibition of iAP by Pi and PPi at
9
.0, but not at pH 7.4 or 6.0. Furthermore, mass spectrometric
[
30]
lower pH-values.
However, in contrast to our results, Chappelet-Tordo et al.
detection allowed detailed investigation of the pH
a
[17]
dependence of iAP by elucidating the degradation of substrate,
intermediates and the generation of the final product. Increase
or decrease of the catalytic activity of iAP due to pH-value shifts
is of special interest since iAP takes part in intestinal pH
regulation and is furthermore involved in inflammatory
processes. In inflammation pH-values tend to become more
found a considerably higher activity towards AMP substrate
compared to ATP substrate at pH 10.0 and a comparable
activity at pH 8.0. This indicates a preferred degradation of
a
½Eꢀ½Iꢀ
Ki = inhibition constant (Ki =
) with [E] = concentration of
½
EIꢀ
[38]
acidic, thus causing a lowered activity of iAP and therefore
the enzyme; [I] = concentration of the inhibitor and [EI] =
concentration of the enzyme–inhibitor complex). Pi =
inorganic phosphate and PPi = inorganic pyrophosphate.
a tendency for higher ATP concentration, that would contribute
to the progression of inflammatory processes.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878

Studies on enzymatic activity of alkaline phosphatase
Interestingly, the degradation of AMP to adenosine was
demonstrated to be distinctively slower, but mainly in very
alkaline medium. Nevertheless, a reduced degradation rate
of AMP substrate compared to ATP or ADP degradation
rates was also observed at physiological pH, although the
difference was less pronounced. Physiological consequences
in the context of this disparity that may be related to a more
outstanding role for AMP in the context of inflammation or
pH regulatory processes in the intestinal tract have to be
enlightened in the future.
[11] A. R. de Boer, T. Letzel, D. A. van Elswijk, H. Lingeman,
W. M. A. Niessen, H. Irth. On-line coupling of high-
performance liquid chromatography to a continuous-flow
enzyme assay based on electrospray ionization mass
spectrometry. Anal. Chem. 2004, 76, 3155.
[
12] C. F. de Jong, R. J. E. Derks, B. Bruyneel, W. Niessen,
H. Irth. High-performance liquid chromatography-mass
spectrometry-based acetylcholinesterase assay for the
screening of inhibitors in natural extracts. J. Chromatogr. A
2
006, 1112, 303.
[13] Z. Yu, L. C. Chen, M. K. Mandal, H. Nonami, R. Erra-
Balsells, K. Hiraoka. Online electrospray ionization mass
spectrometric monitoring of protease-catalyzed reactions
in real time. J. Am. Soc. Mass Spectrom. 2012, 23, 728.
[
14] N. Dennhart, T. Fukamizo, R. Brzezinski, M. E. Lacombe-
Harvey, T. Letzel. Oligosaccharide hydrolysis by
chitosanase enzymes monitored by real-time electrospray
ionization-mass spectrometry. J. Biotechnol. 2008, 134, 253.
Acknowledgements
The authors would like to thank Vital Solutions GmbH
(
(
(
Langenfeld, Germany) and Amino Up Chemicals Co., Ltd
Sapporo, Japan) for financial support. We also thank Knauer
Wissenschaftliche Gerätebau Dr. Ing. Herbert Knauer, Berlin,
Germany) for the loan of the single-quadrupole mass
spectrometer and Stefan Bieber for his dedication and his
excellent work.
[15] D. W. Moss, A. K. Walli. Intermediates in the hydrolysis of
ATP by human alkaline phosphatase. Biochim. Biophys. Acta
1
969, 191, 476.
[
[
16] R. K. Morton. The kinetics of hydrolysis of phenyl phosphate
by alkaline phosphatases. Biochem. J. 1957, 65, 674.
17] D. Chappelet-Tordo, M. Fosset, M. Iwatsubo, C. Gache, M.
Lazdunski. Intestinal alkaline phosphatase. Catalytic pro-
perties and half of the sites reactivity. Biochemistry 1974, 13,
REFERENCES
1
788.
18] T. Letzel, E. Sahmel-Schneider, K. Skriver, T. Ohnuma,
T. Fukamizo. Chitinase-catalyzed hydrolysis of 4-
[
[
1] J. P. Lalles. Intestinal alkaline phosphatase: multiple
biological roles in maintenance of intestinal homeostasis
and modulation by diet. Nutr. Rev. 2010, 68, 323.
2] M. J. Bours, E. L. Swennen, F. Di Virgilio, B. N. Cronstein,
P. C. Dagnelie. Adenosine 5’-triphosphate and adenosine
as endogenous signaling molecules in immunity and
inflammation. Pharmacol. Ther. 2006, 112, 358.
nitrophenyl penta-N-acetyl-beta-chitopentaoside as deter-
mined by real-time ESIMS: The 4-nitrophenyl moiety of the
substrate interacts with the enzyme binding site. Carbohydr.
Res. 2011, 346, 863.
[
[19] R. D. Henkel, J. L. VandeBerg, R. A. Walsh. A microassay for
ATPase. Anal. Biochem. 1988, 169, 312.
[
3] A. la Sala, D. Ferrari, F. Di Virgilio, M. Idzko, J. Norgauer,
G. Girolomoni. Alerting and tuning the immune response
by extracellular nucleotides. J. Leukocyte Biol. 2003, 73, 339.
4] L. Antonioli, M. Fornai, R. Colucci, N. Ghisu, F. Da Settimo,
G. Natale, O. Kastsiuchenka, E. Duranti, A. Virdis,
C. Vassalle, et al. Inhibition of adenosine deaminase
attenuates inflammation in experimental colitis. J. Pharmacol.
Exp. Ther. 2007, 322, 435.
[20] A. C. Hogenboom, A. R. de Boer, R. J. Derks, H. Irth.
Continuous-flow, on-line monitoring of biospecific interactions
using electrospray mass spectrometry. Anal. Chem. 2001, 73,
3816.
[21] N. Dennhart, T. Letzel. Mass spectrometric real-time moni-
toring of enzymatic glycosidic hydrolysis, enzymatic
inhibition and enzyme complexes. Anal. Bioanal. Chem. 2006,
386, 689.
[
[
[
[
5] X. Ge, T. L. Sirich, M. K. Beyer, H. Desaire, J. A. Leary. A
strategy for the determination of enzyme kinetics using
electrospray ionization with an ion trap mass spectrometer.
Anal. Chem. 2001, 73, 5078.
6] A. J. Norris, J. P. Whitelegge, K. F. Faull, T. Toyokuni.
Analysis of enzyme kinetics using electrospray ionization
mass spectrometry and multiple reaction monitoring:
Fucosyltransferase V. Biochemistry 2001, 40, 3774.
7] D. J. Wilson, L. Konermann. Mechanistic studies on
enzymatic reactions by electrospray ionization MS using a
capillary mixer with adjustable reaction chamber volume
for time-resolved measurements. Anal. Chem. 2004, 76,
[22] R. K. Scheerle, J. Grassmann, T. Letzel. Real-time ESI-MS of
enzymatic conversion: Impact of organic solvents and
multiplexing. Anal. Sci. 2012, 28, 607.
[23] A. I. Vovk, V. I. Kalchenko, S. A. Cherenok, V. P. Kukhar,
O. V. Muzychka, M. O. Lozynsky. Calix 4 arene methylene-
bisphosphonic acids as calf intestine alkaline phosphatase
inhibitors. Org. Biomol. Chem. 2004, 2, 3162.
[24] S. G. Nugent, D. Kumar, D. S. Rampton, D. F. Evans.
Intestinal luminal pH in inflammatory bowel disease:
possible determinants and implications for therapy with
aminosalicylates and other drugs. Gut 2001, 48, 571.
[25] C. H. Fiske. The colorimetric determination of phosphorus.
J. Biol. Chem. 1925, 66.
2
537.
[
8] B. Ganem, Y. T. Li, J. D. Henion. Observation of
noncovalent enzyme substrate and enzyme product
complexes by ion-spray mass-spectrometry. J. Am. Chem.
Soc. 1991, 113, 7818.
[26] N. Yoza, S. Onoue, Y. Kuwahara. Catalytic ability of
alkaline phosphatase to promote P-O-P bond hydrolyses
of inorganic diphosphate and triphosphate. Chem. Lett.
1997, 491.
[
9] N. Dennhart, L. M. M. Weigang, M. Fujiwara, T. Fukamizo,
K. Skriver, T. Letzel. 26 kDa endochitinase from barley
seeds: Real-time monitoring of the enzymatic reaction and
substrate binding experiments using electrospray ionization
mass spectrometry. J. Biotechnol. 2009, 143, 274.
[27] R. V. Nayudu, L. Demeis. Energy transduction at the catalytic
site of enzymes – Hydrolysis of phosphoester bonds and
synthesis of pyrophosphate by alkaline-phosphatase. FEBS
Lett. 1989, 255, 163.
[28] A. A. Baykov, O. A. Evtushenko, S. M. Avaeva. A malachite
green procedure for ortho-phosphate determination and its
use in alkaline phosphatase-based enzyme-immunoassay.
Anal. Biochem. 1988, 171, 266.
[
10] P. Liuni, A. Jeganathan, D. J. Wilson. Conformer selection
and intensified dynamics during catalytic turnover in
chymotrypsin. Angew. Chem. Int. Ed. Engl. 2012, 51, 9666.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

C. M. Kaufmann et al.
[
29] M. Cocivera, J. McManaman, I. B. Wilson. Phosphorylated
intermediate of alkaline phosphatase. Biochemistry 1980,19, 2901.
30] H. N. Fernley, P. G. Walker. Studies on alkaline
phosphatase. Inhibition by phosphate derivatives and the
substrate specificity. Biochem. J. 1967, 104, 1011.
31] R. H. Eaton, D. W. Moss. Inhibition of the orthophosphatase
and pyrophosphatase activities of human alkaline-
phosphatase preparations. Biochem. J. 1967, 102, 917.
32] M. H. Jensen. Dephosphorylation of purine mononucleotides
by alkaline phosphatases. Substrate specificity and
inhibition patterns. Biochim. Biophys. Acta 1979, 571, 55.
33] A. Bannister, R. L. Foster. Buffer-induced activation of calf
intestinal alkaline phosphatase. Eur. J. Biochem. 1980, 113, 199.
34] D. Hogarth, D. Jung, T. Murooka, S. Pun, M. Szeto. A
decrease in the bovine intestinal mucosa alkaline
phosphatase activity in the presence of inorganic phosphate.
J. Exp. Microbiol. Immunol. 2002, 2, 6.
[35] J. Dayan, I. B. Wilson. The phosphorylation of Tris by
alkaline phosphatase. Biochim. Biophys. Acta 1964, 81, 620.
[36] M. Besman, J. E. Coleman. Isozymes of bovine intestinal
alkaline phosphatase. J. Biol. Chem. 1985, 260, 11190.
[37] F. J. Behal, M. Center. Heterogeneity of calf intestinal
alkaline phosphatase. Arch. Biochem. Biophys. 1965,
110, 500.
[38] Z. Gerevich, Z. S. Zadori, L. Koles, L. Kopp, D. Milius,
K. Wirkner, K. Gyires, P. Illes. Dual effect of acid pH on
purinergic P2X(3) receptors depends on the histidine 206
residue. J. Biol. Chem. 2007, 282, 33949.
[
[
[
[
[
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher’s website.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 869–878