Oxidatively induced Cu for Mn exchange in protein phosphatase 1γ: A new method for active site analysis

Article abstract of DOI:10.1016/j.bmc.2009.10.014

Protein phosphatase 1γ, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn²⁺ in the active site when expressed in Escherichia coli in a buffer containing MnCl₂. Herein, we report on the oxidatively induced co

Full text of DOI:10.1016/j.bmc.2009.10.014

Bioorganic & Medicinal Chemistry 17 (2009) 7978–7986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oxidatively induced Cu for Mn exchange in protein phosphatase 1
c: A new
method for active site analysis
c
c
c
Atsushi Miyazaki a, Magne O. Sydnes a, Minoru Isobe b, , Hiroshi Ohinata , Motoi Miyazu , Akira Takai
*
a Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
b Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan
c Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asahikawa 078-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein phosphatase 1
c
, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn2+ in
Received 20 August 2009
Revised 6 October 2009
Accepted 7 October 2009
Available online 31 October 2009
the active site when expressed in Escherichia coli in a buffer containing MnCl2. Herein, we report on the
oxidatively induced copper for manganese exchange in protein phosphatase 1 , thus enabling firm con-
c
firmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in
metal coordination. By exchanging manganese with copper the oxidation yields for the peptides
increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation
sites within the oxidized peptides. We also found that when copper was added during the oxidation pro-
cess a new metal coordination center was formed at cysteine 39, 105, 140, and 155.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Protein phosphatase
Metallo protein
Metal exchange
Cu and Mn
Pin-point oxidation
ROS
1. Introduction
Currently as many as one-half of all proteins are metallopro-
teins and the majority of these proteins contain transition metals
as part of their structure.1 Protein phosphatase 1 (PP1), which is
a serine/threonine phosphatase,2 belongs to this group of proteins.
PP1 is found in all tissues and is involved in a range of cellular pro-
cesses such as cell cycle progression, protein synthesis, muscle
contraction, carbohydrate metabolism, transcription, neuronal sig-
naling, learning and memory.2a–c Expressed in Escherichia coli in a
buffer containing MnCl2 PP1 contains two Mn2+ in the active site.3
Although it has not been unambiguously proved, it is thought that
natural PP1 contains Fe2+ and Zn2+ in the active site based on its
sequential similarities with protein phosphatase 2B (calcineurin)
(see Fig. S1 in the electronic Supplementary data).4
Mn+
Ascorbate
OH + OH-
Mn + 1+
H2O2
R
R
OH
HN
N
HN
N
His
H OH
Amino acid residues in the vicinity of the metal center(s) in cop-
per containing metalloproteins can be selectively oxidized by addi-
tion of H2O2, which upon reaction with Cu+ generates reactive
oxygen species (ROS) via a Fenton-like reaction (a general outline
of this reaction is shown inFig. 1).5 This has been successfully dem-
onstrated for Cu,Zn-superoxide dismutase (Cu,Zn-SOD) protein by
our group6 and other groups7 using mass spectrometry for post
oxidation analysis. Although it has been known for some time that
R
R
+
HN
NH
HN
N
H+
O
H OH
ox-His
(+ 16 Da)
Figure 1. General outline of the Fenton/Fenton-like reaction (top) and an example
of how the reactive oxygen species generated from that reaction oxidizes
histidine.
* Corresponding author. Tel.: +81 52 789 4109; fax: +81 52 789 4111.
E-mail addresses: isobem@agr.nagoya-u.ac.jp, minoru@mx.nthu.edu.tw, iso
bem@nuagr1.agr.nagoya-u.ac.jp (M. Isobe).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.014

A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
7979
first-row transition metal ions other than Cu (i.e., Mn2+, Fe2+, Co2+
,
from these two peptides, no substantial oxidation was detected be-
sides the already determined background oxidation [Cys127 in
T15, methionine 183 (Met183) in T22, Met316 in T34, as well as
one oxidized amino acid residue in peptides T24 and T31 (most
likely Cys or Met)].
and Ni2+) under appropriate reaction conditions can generate ROS,8
and for this work the ability of Mn2+ to form ROS is of particular
interest.9 However, it was Vachet and co-workers who demon-
strated that these metal ions also are capable of selectively oxidiz-
ing amino acid residues engaged in metal coordination.10 This was
showcased in two metal coordinating peptides, where for example
the metal coordinating histidines (His6 and His9) in angiotensin I
were selectively oxidized upon exposure to H2O2 and ascorbate
2.2. Oxidatively induced Cu+/Cu2+ for Mn2+ exchange
When Cu2+ (CuSO4) and sodium ascorbate (ascorbate reduces
Cu2+ to Cu+)17 was added during the oxidation process several
additional oxidations took place as detected by nano-LC-ESI-Q-
TOF-MS analysis of the peptides derived from trypsin digestion
of the protein post oxidation. This finding was intriguing and opti-
mization experiments were conducted in order to find the best oxi-
dation conditions. The outcome of these experiments were
monitored by nano-LC-ESI-Q-TOF-MS by analyzing the whole oxi-
dized protein as well as analysis of the peptides derived from tryp-
tic digestion of the oxidized protein. By such means we found the
optimum concentration of oxidant to be 5 mM H2O2 in combina-
tion with 10 mM sodium ascorbate as reducing agent (see experi-
mental part for details). The optimum conditions were decided
based on the oxidation yields obtained for the various peptides.
With the optimum concentration of H2O2 and sodium ascorbate
established we conducted a range of experiments with various
concentrations of copper added during the oxidation. By using
nano-LC-ESI-Q-TOF-MS we found that the oxidation yields for pep-
tides T9, T15, T22 and T29 gradually rose when the concentration
of copper increased (0.5?3.0 equiv) (Table 1). Further analysis of
these peptides with nano-LC-ESI-IT-MS/MS enabled us to confirm
that His involved in metal coordination was oxidized selectively
in peptides T9 (His66) and T15 (His125) as evident from the MS/
MS spectra depicted in Figures 3 and 4, respectively.
Under conditions including copper the last two peptides, viz.
T22, and T29 containing a His amino acid residue (His173, and
His248, respectively) engaged in metal coordination could also
be oxidized in a maximum of 21% and 27%, respectively, when
3 equiv of Cu2+ was employed (Table 1). MS/MS analysis of peptide
T22 only enabled us to elucidate that the site of oxidation is situ-
ated somewhere on one of the following amino acid residues; isol-
uecine 169 (Ile169), phenylalanine 170 (Phe170), Cys171, Cys172,
His173, glycine 174 (Gly174), or Gly175 (Fig. 5). Ile, and Gly can be
ruled out as oxidation sites due to the fact that they are not likely
to be oxidized under these conditions.5 Cys is easily oxidized by
ROS and can therefore not automatically be ruled out as the site
of oxidation. However, based on the arguments used previously
regarding the oxidation of Cys amino acid residues16 (vide supra)
we could also rule out Cys171, and Cys172 as the oxidation site
due to the fact that the mass increase for peptide T22 was only
16 Da, thus leaving Phe170 or His173 as the potential oxidation
site. However, based on our previous experience it is most likely
that His173 is the point of oxidation.6,12 An assumption that is con-
firmed by the already reported X-ray crystal structure of PP1,
which shows that Phe170 is remote from the metal center (>10 Å
from the metal center).3
2ꢀ
or S2O8 and ascorbate. Although the oxidation yields were vari-
able when Mn2+, Fe2+, Co2+, and Ni2+ was used, and in most cases
much lower than for Cu2+ the method seemed to bear some prom-
ise for analysis of the metal binding site in non-Cu-binding metal-
loproteins.10 To the best of our knowledge there is no report, apart
from a comment in Vachet’s paper where they refer to promising
preliminary work on Fe-SOD,10 where this strategy has been ex-
tended to analysis of metalloproteins containing Mn2+, Fe2+, Co2+
,
or Ni2+ in the active site.
Our group have for some time been interested in studying
metallopeptides and metalloproteins,11 and lately we6,12 and oth-
ers13 have been involved in research aiming at elucidating the me-
tal coordination sites of metalloproteins using oxidation by ROS
generated by for example treatment with H2O2 and sodium ascor-
bate, followed by MS analysis. Another long term research project
within the group is to elucidate the binding site of tautomycin with
PP1
report on the oxidatively induced metal exchange (Mn2+?Cu+/
Cu2+) strategy for the oxidation labeling of PP1
’s active site. By
using this strategy all four His involved in metal coordination in
PP1 could be detected.
c
using various strategies.14 As a part of this work, we herein
c
c
2. Results and discussion
2.1. Background oxidation
Upon treatment of PP1c with H2O2 (5 mM) and sodium ascor-
bate (10 mM) in buffer, peptides T9, and T15 were oxidized
(+16 Da) in 3% yield (Table 1), as evident from nano-LC-ESI-Q-
TOF-MS of the trypsin digested protein. Further analysis of these
peptides in order to determine the point of oxidation utilizing
nano-LC-ESI-IT-MS/MS only resulted in poor and inconclusive data
due to the low yield of the oxidized peptides. Firm confirmation of
the oxidation site in the two peptides was therefore difficult due to
the fact that these two peptides also contain potentially readily
oxidized cysteine (Cys) amino acid residues (Fig. 2 and Fig. S2 in
the electronic Supplementary data). However, based on literature
precedent, which states that Cys-SOH is unstable and easily under-
goes further oxidation to Cys-SO2H (+32 Da) and Cys-SO3H
(+48 Da) or reacts with thiol [dithiothreitol (DTT) was used during
digestion of the protein; see experimental part for details] to yield
disulfide,15 it is most likely that the site of oxidation in the two
peptides is His66 for peptide T9 and His125 for peptide T15. Apart
Table 1
Further analysis of peptide T29 with nano-LC-ESI-IT-MS/MS
showed that the amino acid residue that had been oxidized was
either alanine 247 (Ala247), His248 or glutamine 249 (Gln249)
(see Fig. 6). From this data we can conclude that the oxidation site
is His248 since the two other amino acid residues are not likely to
be oxidized under the conditions used in this work.5
The initial oxidation, under conditions excluding copper, points
towards ROS being generated upon reaction between H2O2 and
Mn2+. However, the oxidation yields were quite low (T9 3% and
T15 3%), and for two of the four His containing peptides involved
in metal binding the oxidized peptide could not be detected. The
addition of copper greatly increased the yield of the oxidized
Oxidation yields for peptides T9, T15, T22, and T29 at various concentrations of added
Cu2+ (CuSO4)a
Peptide
0 equiv
Cu2+ (%)b (2.85
0.5 equiv Cu2+ 1.0 equiv Cu2+ 3.0 equiv Cu2+
l
M) (%)
(5.70
l
M) (%)
(17.0 lM) (%)
T9 + 16 Da
3
3
0
0
20
18
10
10
34
24
11
14
38
32
21
27
T15 + 16 Da
T22 + 16 Da
T29 + 16 Dac
a
b
c
Oxidation yields are calculated as indicated in note 16.
Background oxidation.
The oxidation yield for this peptide was estimated since one other peptide
overlaps with this peptide in the LC chromatogram.

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A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
21 27 37 44
1
7
6
MADLDK LNIDSIIQR LLEVR GSKPGK NVQLQENEIR GLCLK SR EIFLSQPILLELEAPLK
T1 T2 T3 T4 T5 T6 T7 T8
75
61
99
114
ICGDIHGQYYDLLR LFEYGGFPPESNYLFLGDYVDR GK QSLETICLLLAYK IK YPENFFLLR
T9
T10
T11
T12
T13
T14
123
133
144
151
GNHECASINR IYGFYDECK
R
R YNIK LWK TFTDCFNCLPIAAIVDEK
T15 T16
T17 T18 T19 T20
T21
169
189
212
222
IFCCHGGLSPDLQSMEQIR R IMRPTDVPDQGLLCDLLWSDPDK DVLGWGENDR GVSFTFGAEVVAK
T22 T23 T24 T25 T26
235
239
247
262
FLHK HDLDLICR AHQVVEDGYEFFAK R QLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPAEK
T27 T28 T29 T30 T31
303
315
320
K KPNATRPVTPPR GMITK QAK K
T32 T33 T34 T35 T36
Figure 2. Peptide map for trypsin digested PP1
c
. His engaged in metal coordination is marked in red (peptide T9, T15, T22, and T29).
00
Figure 3. MS/MS spectra of peptides T9 and T9 + 16 Da. Peptide T9 + 16 Da shows a mass increase of 16 Da for fragments y090 and y compared with peptide T9. The spectrum
10
of peptide T9 + 16 Da also shows that fragment y080 is unchanged compared with peptide T9, thus confirming the oxidation of His66. The oxidation of His66 is also supported
by a 16 Da increase in the mass of fragment b6 in the spectrum of peptide T9 + 16 Da compared with peptide T9.

A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
7981
Figure 4. MS/MS spectra of peptides T15 and T15 + 16 Da. Peptide T15 + 16 Da shows a mass increase of 16 Da for fragment y080 compared with peptide T15. The spectrum of
peptide T15 + 16 Da also shows that fragment y070 is unchanged compared with peptide T15, thus confirming the oxidation of His125. The oxidation of His125 is also supported
by a 16 Da increase in the mass of fragment b3 in peptide T15 + 16 Da compared with peptide T15.
peptides, thus making it possible to confirm the His amino acid res-
idues involved in metal coordination in PP1 (Fig. 7).
of PP1c with para-nitrophenyl phosphate with various concentra-
c
tions of CuSO4 in the solution. These tests showed that even
when large excess of Cu2+ (up to 1 mM, i.e., 175 equiv) added to
the reaction mixture had practically no suppressive effect on
2.3. Plausible mechanism for metal exchange
the activity of PP1
c toward para-nitrophenyl phosphate. From
We postulate that the increased oxidation yields after addition
of copper is due to exchange of metal in the active site of the pro-
tein from Mn2+/Mn3+ to Cu+/Cu2+. Currently we have no direct evi-
dence as to how the metal exchange takes place in the active site of
work by Lee and co-workers4e we know that PP1 containing
Cu2+ in the metal center has no activity. Therefore, the fact that
PP1
c
keeps its activity in the presence of Cu2+ indicates that no
metal exchange is taking place when manganese is in oxidation
state 2+. This finding indirectly supports our working hypothesis
that the metal exchange first takes place after Mn2+ has been oxi-
dized to Mn3+. This result also shows that the higher structure of
PP1c, however, it is not so likely that the metal is exchanged prior
to oxidation (vide infra). Our current working hypothesis is that
Mn2+ may rapidly be oxidized to Mn3+ by H2O2 and in the process
generating ROS. Mn3+ in the active site is then exchanged with Cu+/
Cu2+. With Cu+ in place of Mn2+ the generation of ROS becomes
much more efficient and it is possible to improve the oxidation
yields for the amino acid residues around the metal centers, thus
making it easier to be detect by LC–MS analysis after tryptic
digestion.
PP1c remains unchanged even in a solution with high excess of
copper ions.
The method reported herein might be possible to implement for
the analysis of other metalloproteins containing first-row transi-
tion metals such as Mn2+, Fe2+, Co2+, and Ni2+, however, we have
not yet tested this methodology on proteins other than PP1c.
In efforts aiming to prove the oxidation state of manganese at
the stage of metal exchange we conducted activity measurements
Therefore comments regarding the generality of this method has
to await further investigations.

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A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
Figure 5. MS/MS spectra of peptides T22 and T22 + 16 Da. Peptide T22 + 16 Da shows a mass increase of 16 Da for fragments b7 and b8 compared with peptide T22, thus
indicating that the oxidation has taken place at one of the following amino acid residues; Ile169, Phe170, Cys171, Cys172, His173, Gly174 or Gly175.
In previous experiments with bovine Cu,Zn-SOD, we found sim-
ilar metal exchange taking place, although under different condi-
tions (first treatment with EDTA, then addition of metal ions).18
In this case, Cu and Zn ions were displaced by 2Cu2+, 2Zn2+, 2Ag+,
Cys155 is the only amino acid residue that has been modified. De-
spite numerous efforts, it was not possible to obtain good quality
MS/MS data for peptides T12 and T16. However, the mass increase
of 32 Da points towards Cys amino acid residue within these two
peptides being the point of oxidation. Since these peptides are
not oxidized under experimental conditions meant to unravel
background oxidation or under conditions excluding copper, it is
clear that these peptides are oxidized due to the addition of cop-
per. It seems likely that apart from Cu+/Cu2+ exchanging with
Mn2+ in the active site that also copper is coordinated in a newly
generated metal center. It is well known that Cys amino acid res-
idues that are not involved in disulfide bonds can coordinate met-
als in metalloproteins, such as for example in metallothioein 1a.19
We therefore postulate that the Cys amino acid residues in pep-
tides T6, T12, T16, and T21 coordinates Cu2+ in a new metal center,
and that ROS generated around this metal facilitates the oxidation
of the coordinating Cys amino acid residues (Fig. 8).20 The MS/MS
analysis of peptide T21 also confirmed that only one of the two Cys
amino acid residues was oxidized in this peptide, namely Cys155.
Cys158, which is also included in peptide T21 (see Fig. 2),
remained unoxidized most likely due to the fact that Cys158 is fac-
ing away from the new metal center (see Fig. S3 in the electronic
Supplementary data).
and 2Co2+
.
2.4. Additional oxidations under conditions including Cu+/Cu2+
Addition of Cu2+ during the oxidation reaction also resulted in
successful oxidation of other peptides, which were not found to
be oxidized when Cu2+ was not added to the reaction mixture, as
evident from extensive nano-LC-ESI-Q-TOF-MS analysis (Table 2).
Peptides T6 (+32 Da, and +48 Da), T10 (+16 Da), T12 (+32 Da),
T16 (+32 Da), and T21 (+32 Da) predominantly increased in mass
by 32 Da except for peptide T10, thus indicating that the Cys ami-
no acid residue within peptides T6, T12, T16, and T21 were pre-
dominantly oxidized. This assumption was confirmed by
conducting further MS and MS/MS analysis on the respective pep-
tides, which clearly showed that Cys in peptide T6 was oxidized.
The MS/MS spectrum of peptide T21 revealed that the site of oxi-
dation was among one of the following amino acid residues; thre-
onine 151 (Thr151), Phe 152, Thr153, aspartic acid 154 (Asp154)
or Cys155. However, a mass increase of 32 Da indicates that

A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
7983
Figure 6. MS/MS spectra of peptide T29 and T29 + 16 Da. Peptide T29 + 16 Da shows a mass increase of 16 Da for fragment b3 compared with peptide T29. The spectrum of
00
peptide T29 + 16 Da also shows y unchanged compared with peptide T29, thus confirming that the oxidation has taken place at one of the following amino acid residues;
11
Ala247, His248 or Gln249.
MS/MS analysis of peptide T10, which does not contain any Cys
amino acid residue (see Fig. 2), only revealed that the site of oxida-
tion were either leucine 75 (Leu75), Phe76, glutamic acid 77
(Glu77), tyrosine 78 (Tyr78), Gly79, Gly80, Phe81, proline 82
(Pro82), Pro83, Glu84, serine 85 (Ser85), aspartic acid 86 (Asn86),
Tyr87, or Leu88. However, based on our previous experience where
we found that Pro can be oxidized under these conditions6 and the
fact that amino acid residues Leu, Glu, Gly, Ser, and Asn are not
readily oxidized under the conditions used in this work,5 we con-
cluded that the point of oxidation is most likely at one of the fol-
lowing amino acid residues; Phe76, Tyr78, Phe81, Pro82, Pro83,
or Tyr87 (Fig. 9). The oxidation of peptide T10 seems to be nonspe-
cific due to its remote distance from any of the metal centers, but
reproducible. Attempts to reduce the amount of oxidation of pep-
tide T10 by for example increasing the amount of sodium ascor-
bate, a method previously used with success by Vachet and co-
workers,7d were not successful.
3. Conclusion
Although the oxidation yields for the amino acid residues in-
volved in metal binding in PP1c are rather modest under conditions
where ROS is generated by Mn2+ it is still possible to elucidate two
out of four His amino acid residues involved in metal coordination,
thus proving that metals other than copper can be utilized for this
type of analysis in metalloproteins. By adding Cu2+ and sodium
ascorbate during the oxidation we have developed a new supple-
mentary method for analysis of the active site of PP1cby exchanging
Mn2+ with Cu+/Cu2+. The generality of this method is still unproved,
however, if this method is found to be general it opens up a new
alternative method for metal site analysis in metalloproteins con-
taining metals other than copper. The metal exchange is carried
out by adding CuSO4 to the solution of PP1c prior to addition of so-
dium ascorbate and H2O2. Under conditions including copper a new
metal center was generated by Cys39, Cys105, Cys140, and Cys155

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A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
Figure 7. The active site of PP1 with the two metals marked in orange. The distance
from the two metals to His is indicated in Å in the figure. The figure is based on the
X-ray structure (3e7b) obtained from the protein data bank.1
Table 2
Oxidation yields for peptides T6, T10, T12, T16, and T21 at various concentrations of
Cu2+a
Peptide
0 equiv
0.5 equiv Cu2+
1.0 equiv Cu2+
3.0 equiv Cu2+
(17.0 lM) (%)
Cu2+ (%)b
(2.85
l
M) (%)
(5.70
lM) (%)
T6 + 32 Da
T6 + 48 Da
T10 + 16 Da
T12 + 32 Da
T16 + 32 Da
T21 + 32 Da
0
0
0
0
0
0
4
3
7
0
7
8
5
4
9
35
11
9
6
4
11
45
13
17
Figure 9. The X-ray structure of PP1 with the active site highlighted (His66, His125,
His173, and His248), the new metal center highlighted (Cys39, Cys105, Cys140, and
Cys155), and the potential oxidation sites on peptide T10 marked in light blue. The
figure is based on the X-ray structure (3e7b) obtained from the protein data bank.3b
coordinating to Cu+/Cu2+, thus resulting in selective oxidation of the
coordinating Cys amino acid residues.
a
Oxidation yields are calculated as indicated in note 16.
Background oxidation.
b
4. Experimental section
4.1. Instrumentation
MS spectra were recorded utilizing a Q-TOF mass spectrometer
(Micromass, Manchester, UK) equipped with a Z-spray type ESI
source. Data were acquired and processed using MassLynx version
4.0. All samples were desalted and separated by non-split type pre-
packed gradient (PPG) system and appropriately adjusted nano-
HPLC system (JASCO, Tokyo, Japan) using a Develosil ODS-HG-5
column (Nomura, 150 mm ꢁ 0.3 mm i. d.) before on-line ESI-MS
analysis. The column was equilibrated with 260
lL water contain-
ing 0.025% trifluoroacetic acid at a flow rate of 10
lL/min and then
developed using a linear gradient from 0% to 100% of acetonitrile
containing 0.025% trifluoroacetic acid for 40 min at a flow rate of
5 lL/min. The column effluent was monitored at 210 nm and then
introduced into the electrospray nebulizer without splitting.
MS/MS spectra were recorded utilizing an ion trap (IT) HCT Plus
mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped
with an orthogonal ESI source. Data were acquired and processed
using Compass version 1.0 (esquireControlTM and DataAnalysisTM
version 3.2) (Bruker Daltonics), respectively. All MS experiments
were preformed in the positive ion mode.
4.2. Chemicals
H2O2 (30%) and dithiothreitol (DTT) was purchased from Naca-
lai Tesque (Kyoto, Japan); sodium ascorbate was purchased from
TCI (Tokyo, Japan). Trypsin (sequence grade) was purchased from
Figure 8. New metal center in PP1c
formed upon addition of Cu2+: Cys involved in
Cu2+ coordination are highlighted (Cys39, Cys105, Cys140, and Cys155). The figure
is based on the X-ray structure (3e7b) obtained from the protein data bank.3b

A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
7985
Roche Diagnostics (Mannheim, Germany). Acetonitrile (HPLC
grade) for nano-LC-ESI-Q-TOF-MS was purchased from Nacalai
Tesque (Kyoto, Japan) and acetonitrile (HPLC grade) for nano-LC-
ESI-IT-MS/MS was purchased from Roche (Mannheim, Germany).
to the mixture and the resulting mixture was incubated at 25 °C for
30 min. H2O2 (2.5 L of a 100 mM solution) was added to the mix-
ture and the resulting solution was incubated at 37 °C for 30 min.
l
The reaction mixture was then diluted by addition of water
(50
order to remove trace of H2O2 additional water (50
and the sample was frozen and freeze-dried (repeated twice). The
l
L) and the resulting solution was frozen and freeze-dried. In
lL) was added
5. Experimental
sample obtained by such means was diluted with water (6.7
and subjected to tryptic digestion.
lL)
5.1. Preparation and purification of PP1
c
A full-length cDNA encoding
pressed in E. coli (provided by Dr. Patricia Cohen, Dundee, U.K.),
as described previously.21 The expressed PP1
was purified by
c-isoform of PP1 (PP1c) was ex-
5.5. General procedure for oxidation of PP1
(1 equiv)
c with copper
c
sequential chromatography at 4 °C on five columns: HiPrep Q-XL,
HiPrep SP-XL, phenyl-Sepharose, Sephacryl S-200 and Mono-Q
(all purchased from Amersham-Pharmacia Biotech, Uppsala, Swe-
To a stock solution of PP1
258 pmol) was added tris–HCl buffer (16.45
solution was incubated at 25 °C for 30 min. CuSO4ꢂ5H2O (6.45
a 40
lowed by incubation at 25 °C for 30 min. Sodium ascorbate (5
a 100 mM aq solution) was then added to the mixture and the
resulting mixture was incubated at 25 °C for 30 min. H2O2 (2.5
of a 100 mM solution) was added to the mixture and the resulting
solution was incubated at 37 °C for 30 min. The reaction was then
c
(15
l
L of a 17.2
lM solution,
lL), and the resulting
l
L of
den). Buffers used for the expression and purification of PP1c con-
l
M aq solution, 258 pmol) was then added to the mixture fol-
tained MnCl2 (1 mM) Two-dimensional electrophoresis [isoelectric
focusing on an IPG gel (pH 3–10 L type; Amersham-Pharmacia)
rehydrated in urea (8 M), DTT (20 mM), ampholyte (0.5%) and
lL of
l
L
CHAPS (2%), followed by SDS/PAGE] of the purified PP1
c revealed
a single polypeptide spot at pI 6.0 and apparent molecular mass
37 kDa. The molar concentration of PP1c was determined, as de-
scribed previously,22 by a titration procedure using microcystin-
LR (provided by Dr. Ken-ichi Harada, Meijo University, Nagoya, Ja-
pan) as the standard.
quenched by addition of water (50
was frozen and freeze-dried. In order to remove trace of H2O2 addi-
tional water (50 L) was added and the sample was frozen and
freeze-dried (repeated twice). The sample obtained by such means
was diluted with water (6.7 L) and subjected to tryptic digestion.
lL) and the resulting solution
l
l
5.2. Activity assay
5.6. Tryptic digestion of PP1
c
or oxidized PP1
c
Assay of phosphatase activity were carried out at 25 °C essen-
tially by the procedure described previously.21 Briefly, pNPP phos-
phatase activity was measured by monitoring the increase in the
absorbance at 405 nm resulting from accumulation of the reaction
product p-nitrophenol, using a spectrophotometer connected to a
pen-recorder. The buffer used contained: Tris (base) (40 mM),
MgCl2 (34 mM), EDTA (free acid) (4 mM) and DL-dithiothreitol
(4 mM). No divalent cation other than Mg2+ was added. The pH
of this solution (without adjustment) was 8.4, which is optimal
for the pNPP phosphatase activities of PP1. The specific activity
The aq. solution of PP1 or oxidized PP1
c
c
was heated at ca.
90 °C for 5 min in order to denature the protein. The sample was
then cooled to room temperature and a solution of trypsin
[4.8 lL, of a 0.1 lg/lL in tris–HCl buffer, (5% w/w of PP1c)] was
then added and the resulting solution was incubated at 37 °C for
20 h. Trypsin was then deactivated by heating the solution at ca.
90 °C for 5 min. After cooling the sample to room temperature, a
solution of DTT (1 lL of a 120 mM aq solution) was added and
the solution was incubated at 37 °C for 2 h. The resulting sample
was stored at ꢀ20 °C until it was subjected to nano-LC-ESI-Q-
TOF-MS or -MS/MS analysis.
for PP1
c was 0.29 0.03 lmol-Pi liberated/min/mg protein
(n = 64), when it was measured at 15 °C using pNPP (5.0 mM) as
the substrate.
5.7. Preparation of tris (hydroxymethyl) aminomethan-HCl
(tris–HCl) buffer
5.3. Procedure for determining background oxidation
To a stock solution of PP1
258 pmol) was added tris–HCl buffer (16.45
solution was incubated at 25 °C for 30 min. H2O (6.45
added to the mixture followed by incubation at 25 °C for 30 min.
H2O (5 L) was then added to the mixture and the resulting mix-
ture was incubated at 25 °C for 30 min. H2O2 (2.5 L of a 100 mM
c
(15
l
L of a 17.2
L), and the resulting
L) was then
lM solution,
Tris-(hydroxylmethyl)aminomethane (121.0 mg, 1.0 mmol),
NaCl (117.0 mg, 2.0 mmol) and DTT (46.0 mg, 0.30 mmol) were
dissolved in distilled water (90 mL) and pH was then adjusted to
8.43 (at 25 °C) by addition of 1 M HCl (aq solution). The volume
was then adjusted to 100 mL by adding distilled water.
l
l
l
l
solution) was added to the mixture and the resulting solution
was incubated at 37 °C for 30 min. The reaction mixture was then
Acknowledgements
diluted by addition of water (50
frozen and freeze-dried. In order to remove trace of H2O2 addi-
tional water (50 L) was added and the sample was frozen and
freeze-dried (repeated twice). The sample obtained by such means
was diluted with water (6.7 L) and subjected to tryptic digestion.
lL) and the resulting solution was
Financial support from a Grant-in-Aid for Specially Promoted
l
Research [16002007 (Isobe group)] and
a
Grant-in-Aid
[19590202 (Takai group)] from the Ministry of Education, Culture,
Sports, Science and Technology Japan (MEXT) is gratefully
acknowledged. MOS is grateful for the provision of a JSPS Postdoc-
toral Fellowship for foreign researchers. We would like to thank Dr.
Naoki Tani for helpful discussions during the course of this work
and Dr. Masakuni Kurono and Aya Shiomura for conducting some
preliminary work. We also thank Professor Petricia Cohen (Univer-
sity of Dundee) and professor Ken-ichi Harada (Meijo University)
l
5.4. General procedure for oxidation of PP1c )
(Mn2+
To a stock solution of PP1
258 pmol) was added tris–HCl buffer (16.45
solution was incubated at 25 °C for 30 min. H2O (6.45
added to the mixture followed by incubation at 25 °C for 30 min.
Sodium ascorbate (5 L of a 100 mM aq solution) was then added
c
(15
l
L of a 17.2
L), and the resulting
L) was then
lM solution,
l
l
for providing samples of E. coli expressing PP1c and microsystin-
LR, respectively.
l

7986
A. Miyazaki et al. / Bioorg. Med. Chem. 17 (2009) 7978–7986
Goto, T. Biochem. Cell. Biol. 1991, 69, 115; (f) Hayashi, Y.; Isobe, M.; Mutoh, N.;
Supplementary data
Nakagawa, C. W.; Kawabata, M. Methods Enzymol. 1991, 205, 348; (g) Isobe, M.;
Hayashi, Y.; Imai, K.; Nakagawa, C. W.; Uyakul, D.; Mutoh, N.; Goto, T. In
Synthesis, Structure and Properties of Metallothioneins, Phytochelatins and Metal-
Thiolate Complexes; Stillman, M. J., Shaw, C. F., Suzuki, K. T., Eds.; VCH
Publishers: New York, 1992; pp 227–256.
Figures S1–S3 and MS/MS spectra of peptides T6, T10, and T21
are included as supplementary materials. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.10.014.
12. Isobe, M.; Kai, H.; Kurahashi, T.; Suwan, S.; Pitchayawasin-Thapphasaraphong,
S.; Franz, T.; Tani, N.; Higashi, K.; Nishida, H. ChemBioChem 2006, 7, 1590.
13. (a) Zhang, Z.; Barlow, J. N.; Baldwin, J. E.; Schofield, C. J. J. Biochem. 1997, 36,
15999; (b) Cao, W.; Barany, F. J. Biol. Chem. 1998, 273, 33002; (c) Hlavaty, J. J.;
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Hovorka, S. W.; Williams, T. D.; Schöneich, C. Anal. Biochem. 2002, 300, 206; (e)
Lim, J.; Vachet, R. W. Anal. Chem. 2003, 75, 1164; (f) Lim, J.; Vachet, R. W. Anal.
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