Selective protein modification by the hydroperoxide intermediate in a photoprotein, aequorin

Article abstract of DOI:10.1016/j.bmc.2009.03.033

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three -S-SCH₂CHOHCHOHCH₂SH groups to -

Full text of DOI:10.1016/j.bmc.2009.03.033

Bioorganic & Medicinal Chemistry 17 (2009) 3399–3404
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective protein modification by the hydroperoxide intermediate
in a photoprotein, aequorin
Issei Doi a, Masaki Kuse b, Toshio Nishikawa a, Minoru Isobe c,d,
*
a Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
b Chemical Instrument Division, Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
c Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan
d Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
During the course of protein modification program, we employed a recombinant aequorin, the apo-pro-
tein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three –
S–SCH2CHOHCHOHCH2SH groups to –S–SCH2CHOHCH@CH–S@O)H or –S–SCH2CHOHCH@CH–S(@O)OH
of terminal DTT connected to cysteine residues of the C145, C152 and C180, which turned out to locate near
the chromophore.
Received 10 February 2009
Revised 18 March 2009
Accepted 19 March 2009
Available online 24 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Aequorin
Coelenterazine
Hydroperoxide
Photo irradiation
Luminescence
1. Introduction
HO
3
2
O
O
O
N
N
Exposure of proteins to the reactive oxygen species (ROS) such
as hydroxyl radical causes gross structural modification.1 We have
proven that pin-point oxidation by hydroxyl radical took place
selectively on the amino acid residues such as histidine, leucine,
proline, which were located within limited distance (ca. 7 Å) from
Cu atom of the Zn,Cu-SOD (super-oxide dismutase).2 In order to
generate the ROS, we pointed out photo irradiation of hydroperox-
OH
N
N
N
OH
6
N
H
HO
HO
1
2
Figure 1. Coelenterazine 1 and the chromophore 2 in a photoprotein, aequorin.
ide. In 1976, Ogata reported that the a-hydroperoxy ketones gen-
erates the ROS by photo irradiation.3 The photolysis of hydrogen
peroxide provides some ROS and the resulting ROS must selec-
tively oxidize some amino acid residues. Therefore we have
planned photooxidative modification of photoprotein, aequorin,
containing hydroperoxide as a substrate.
Aequorin is a well known calcium-sensitive photoprotein,
found in a luminous jellyfish Aequorea aequorea.4 It contains coel-
enterazine (8-benzyl-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)
imidazo[1,2a]pyrazine-3(7H)-one, 1, Fig. 1) in the form of (2S)-
hydroperoxide (chromophore (2), Fig. 1) as a chromophore re-
ported by X-ray crystallography studies by Head et al. (Fig. 2).5
Apoaequorin consists of 189 amino acid residues with four EF-
hand domains; three of them can bind calcium ions. Reconstitution
of apoaequorin with coelenterazine affords active aequorin by
incubation under air atmosphere with a buffer containing EDTA,
2-mercaptoethanol (2ME) or dithiothreitol (DTT).6 In the absence
of 2ME or DTT, aequorin gradually loses its activity down to 30%
compared with the original luminescence. At pH 7.5 calcium ions
work as a trigger for emitting blue light (470 nm).
In 1996, we have reported photochemical oxidation using dou-
bly 13C labeled coelenterazine analog 3 to yield the peroxide inter-
mediates 4: (2S)-hydroperoxide as well as 5, a dioxetanone;
peroxy-b-lactone as shown in Figure 3.7 These two compounds
were dissolved and photolyzed in a mixture of CF3CD2OD and
CD3OD (7:3) solvents at À78 °C with bubbling oxygen and ana-
lyzed by low-temperature NMR. The half life time of 4 was ca.
2 min at 0 °C in this solvent. Solvent effect in this low temperature
photooxidation was critically important to give these peroxidic
products, which further decomposed by elongating photolysis
* Corresponding author. Tel.: +81 52 788 6226; fax: +81 52 789 1064.
E-mail
addresses:
minoru@mx.nthu.edu.tw,
isobem@agr.nagoya-u.ac.jp
(M. Isobe).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.033

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I. Doi et al. / Bioorg. Med. Chem. 17 (2009) 3399–3404
2. Results and discussion
2.1. Modified amino acid analysis by photo irradiated aequorin
by means of LC–MS
Aequorin was reconstituted from apoaequorin by incubation
with 1 in Tris buffer (pH 7.5) for 2 h at 0 °C,8 and then the resulting
aequorin was subjected to photolysis by using a high-pressure
mercury lamp (365 nm) for 20 min at room temperature under
air. Sample was digested with trypsin in Tris buffer (pH 7.5) for
12 h at 37 °C. The resulting tryptic peptides were then analyzed
by LC–MS.
Apoaequorin has three cysteine residues; thus, C145, C152 and
C
180; where C145 and C152 forms a disulfide bond and C180 exists
as free SH form.9 Under DTT-containing condition where the disul-
fide bond cleaves, trypsin digests of aequorin yielded all the 23
peptide fragments except for T19 in the mass spectra. When LC–
MS data were analyzed with tryptic peptides by comparing with
and without photo irradiation, we found molecular weight changes
in the three peptides (T20, T21 and T22) out of 23 peptides. With-
out photo irradiation these three peptides appeared as T20 886.4 u,
T21 962.9 u, and T22 904.9 u as all data being +2 charged ions.
With photo irradiation all these three peptides appeared as T20
961.5 u and 969.5 u; T21 1037.9 u and 1045.9 u; T22 979.9 u
and 987.9 u, as shown in Figure 4 by LC–MS/MS of ESI-ion-trap.
The respective molecular weights increased in 150 or 166 u, which
were assigned to a dehydrated mono/di oxidized DTT. In the chro-
matogram of T21 after irradiation, a noticeable peak (962.9 u) was
detected, but it was proved to be an isotope peak of modified T20
(961.5 u). We also detected two kinds of peaks in the Figure 4 chro-
matograms of modified T21 (1045.9 u) and modified T22 (987.9 u),
respectively. Left peak identified as an adduct of mono-oxidized
DTT on cysteine (150 u) and oxidized methionine (16 u), while
right peak did as an adduct of di-oxidized DTT on cysteine (166
u) of their peptides.
Figure 2. Conformation of
crystallography.
2 in an aequorin analyzed by using X-ray
time.7 However, it is noteworthy that similar hydroperoxide is kept
stable in aequorin due to sharing the peroxidic proton with tyro-
sine residue in this protein. By using this stability we have em-
barked upon photooxidative modification of aequorin with the
hydroperoxide 2, and implemented photo irradiation. In the resul-
tant photolyzed solutions, we found oxidation products in both of
aequorin solution with 1. It was interesting to learn what kind of
protein modification occurred as the results through subsequent
analyses after tryptic digestion into peptides. We became inter-
ested in tracing the details of structural modification because of
the following reasons as: (1) this is the only luminescent interme-
diate, which has a long lifetime, in nature, (2) following the fate of
this hydroperoxide without luminescence, and (3) a case study of
high yield protein modification.
HO
O
13C
N
O
N
O
N
N
O
O
photo irradiation
13C
N
O
O2
+
N
N
NH
CF3CD2OD-CD3OD
-78 ºC
N
H
MeO
MeO
MeO
5
4
3
dioxetanone
2-hydroperoxide
half life time ca. 40 min at -78 ºC
half life time ca. 2 min at 0 ºC
r.t.
HO
O
O
R
ν
h
CO ,
470 nm
2
O
Apoaequorin
O
N
N
NH
N
N
N
N
N
O2
OH
OH
Ca2+
pH7.5
N
H
R'O
HO
HO
7
2
1
coelenteramide
O
2-hydroperoxide in aequorin
stable in aequorin at 0 ºC
O
photo irradiation
(365 nm)
N
N
N
OH
+
OH
HO
8
Figure 3. A chemiluminescent intermediate 4 as a photoproduct and a bioluminescent intermediate 2 in aequorin. Photolysis of 2 in aequorin gave active oxygen species 7
and hydroxyl radical.

I. Doi et al. / Bioorg. Med. Chem. 17 (2009) 3399–3404
3401
A
B
C
D
T20
886.4
T20
886.4
T20 + 150 u
961.5
T20 + 150 u
961.5
T20 + 166 u
969.5
T20 + 166 u
969.5
15
15
15
20
20
20
25
25
25
30
30
30
35
35
35
40 [min]
15
20
25
30
35
40 [min]
T21
962.9
E
F
T21
962.9
modified T20
isotope
T21 + 150 u
1037.9
T21 + 150 u
1037.9
T21 + 166 u
1045.9
T21 + 166 u
1045.9
15
20
25
30
35
40 [min]
40 [min]
T22
904.9
T22
904.9
T22 + 150 u
979.9
T22 + 150 u
979.9
T22 + 166 u
987.9
T22 + 166 u
987.9
15
20
25
30
35
40 [min]
40 [min]
Figure 4. Ion chromatogram of T20 and T21, T22 peptides before and after photo irradiation, respectively, eluted with the PPG system. (A) Ion chromatogram of T20 and
modified T20 peptides before photo irradiation. (B) Ion chromatogram of T21 and modified T21 peptides before photo irradiation. (C) Ion chromatogram of T22 and modified
T22 peptides before photo irradiation. (D) Ion chromatogram of T20 and modified T20 peptides after photo irradiation. (E) Ion chromatogram of T21 and modified T21
peptides after photo irradiation. (F) Ion chromatogram of T22 and modified T22 peptides after photo irradiation (middle figure: DTT adduct of cysteine residue was converted
into mono-oxidized DTT (+150). below figure: DTT adduct of cysteine residue was converted into mono-oxidized DTT (+150) and methionine residue was oxidized to give
sulfoxide (+16) or DTT adduct of cysteine residue was converted into di-oxidized DTT (+166), respectively.)
The results of mass spectrometric analysis are summarized in
Table 1. The predicted mass numbers and the observed mass num-
bers of the digests are shown concerning to the effect of photo irra-
diation time. The modified points were proved to be selective on
the three cysteine residues. As an example, two spectra of T20 with
(A) and without (B) photo irradiation are shown in Figure 5. It is
clear that C145 have selectively modified; and the ion peak was as-
signed as adducts of oxidized DTT with cysteine. We found the
same results with C152 in T21 and C180 in T22. Some methionine
residues in T21 and T22 were also oxidized only after the photo
irradiation.
were located within limited distance (ca. 7 Å) from Cu atom of
the Zn, Cu-SOD.
We have applied this selective oxidation by ROS obtained from
the photolysis of hydroperoxide (2). In this oxidation reaction, only
terminal sulfur atoms of DTT bound to cysteine were oxidized. Par-
tial oxidations were also occurred at methionine residues, which
were assumed to be oxidized to sulfoxide. On the other hand, oxi-
dation of H169, which locates at the distance of 3.75 Å from coelen-
terazine hydroperoxide, did not occur at all during this photo
irradiation.
The photolysis of 2 might provide some ROS as 7 and the result-
ing ROS must selectively oxidize sulfur atoms in aequorin. This is
contrastive to our previous results2; we reported that hydroxyl
radical, which was produced by the reaction of Zn, Cu-SOD with
H2O2, selectively oxidized histidine, leucine, and proline, which
2.2. Effects of photo irradiation to the luminescence ability and
to oxidative modification
During the analysis of LC–MS data obtained for tryptic peptides
comparing photolyzed apoaequorin with photolyzed aequorin, we
Table 1
Predicted and observed mass numbers of trypsin-digested peptides containing cysteine residues of aequorin and apoaequorin before and after photo irradiation
N.D.; not detected.
a
See note.13

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I. Doi et al. / Bioorg. Med. Chem. 17 (2009) 3399–3404
[Absy. Int. * 1000]
T
E
E
D
E
S
S
Q
I
I
G A
A
B
C
16
14
12
10
8
T20: SAGIIQSSEDC145EETFR
784.3
552.3
6
681.3
4
2
0
200
400
600
800
1000
1200
1400
1600
1800
m/z
[Abs. Int. * 1000]
T
E
E
D
E
S
S
Q
I
I
G
A
C*
y
9.0
C145 + 150 u
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
T20: SAGIIQSSEDDTTC145EETFR
934.3
552.3
681.3
0.0
200
400
600
800
1000
1200
1400
1600
1800
m/z
Figure 5. Tandem mass spectra of tryptic peptide T20 before (A) and after (B) photo-irradiation of aequorin reducted with DTT.
noticed the molecular ion peaks of T20, T21 and T22 peptides only
changed in photolyzed aequorin but not in apoaequorin. The chro-
mophore is indispensable for the oxidation.
T21+11 (+12), and T22+11 (+12) were increased in time dependent
manner of photolysis by reacting with ROS.
Thus, we confirmed that the oxidation of these T20–T22, in
which contained cysteine residues, were dependent for the con-
sumption of chromophore 2 by photolysis.
In order to confirm the fact, we measured each luminescent
activity of aequorin by addition of Ca2+, after photolysis every min-
utes. Because we thought the chromophore 2 should be consumed
by photolysis in order to produce ROS, therefore the luminescent
activity of aequorin should be decreased along with duration of
photolysis. Actually, the luminescent activity gradually decreased
in accordance with photo irradiation time, and it completely disap-
peared in 7 min (Fig. 6, bar graph). Furthermore, we performed LC–
MS analysis of trypsin digest of aequorin, which were irradiated for
5, 10, 15 and 20 min, by using a nano-HPLC-ESI-IT-MS, -MS/MS and
PPG solvent solution program. As shown in Figure 6, T20, T21, and
T20 were decreased in accordance with photolysis and totally dis-
appeared in 15 min, and on the other hand, modified T20+11 (+12),
2.3. Photooxidative modifications of methionine residues
Some methionine residues were also found to be oxidized and
these modifications of methionine seemed to be related to the
photo irradiation. We compared the rate of oxidation on methio-
nine before and after photo irradiation for 20 min (Fig. 7). Aequorin
has five methionine residues M19, M36, M71, M165 and M176 which
were partially oxidized to form sulfoxide before the photo irradia-
tion. The amount of the oxidation increased after irradiation, and it
especially modified M165 and M176 obtained from only irradiated
aequorin. Although these modifications seemed to be unrelated
to the distances between methionine residues and the chromo-
phore 2, we thus concluded that these modifications were derived
from photo irradiated chromophore.
100
100
80
100
80
67
50
60
40
60
40
47
43
40
35
13
20
0
20
0
30
3
1
0
21
20
0
5
10
15
20
14
13
12
Photo Irradiation Time (min)
9
10
0
6
3
Figure 6. Bar graph: The bioluminescent activity (rlu%) of aequorin depending on
photo irradiation time. Line graph: The ratio of three modified peptide fragments
T20, T21 and T22 based on mass peak intensity. T20 (black dotted line) and T21
(blue dotted line) and T22 (red dotted line), T20 + 11 (black dashed line) and T21 +
11 (blue dashed line) and T22 + 11 (red dashed line), T20 + 12 (black line) and T21 +
12 (blue line) and T22 + 12 (red line). 11: sulfenic acid, 12: sulfinic acid.
0
M19 (T5)
M36 (T6)
M71 (T10)
M165 (T21) M176 (T22)
Figure 7. The ratio of photooxidation on methionine residues before (white) and
after (black) photo irradiation based on mass peak intensity.

I. Doi et al. / Bioorg. Med. Chem. 17 (2009) 3399–3404
3403
Table 2
Predicted and observed masses of peptides in trypsin proteolytic digests of apoaequorin and aequorin before and after photo irradiation reduced with 2ME (78 u) and DTT (154 u)
2.4. Confirmation of adduct on cysteine residues by using 2ME
to conclude that only aequorin after photo irradiation could yield
the photooxidative modifications.
In order to confirm the structure of adduct on cysteine residues,
we used 2ME instead of DTT for reconstitution of aequorin. Under
2ME containing condition where the disulfide bond cleaves, diges-
tion of aequorin with trypsin also afforded all the 23 peptide frag-
ments except for T19 as separated peaks in LC–MS. We found
different changes of the molecular weight took place in the three
peptides (T20, T21 and T22) out of 23 peptides; thus, T20 886.4
u to 924.5 u, T21 962.9 u to 1000.9 and 1008.9 u, and T22 904.9
u to 942.9 and 950.9 u; all data being +2 charged ions).
We noticed the molecular ion peaks of T20, T21 and T22 pep-
tides completely disappeared in mass spectra after photo irradia-
tion (Table 2). The respective molecular weights increased in 76
and/or 92 u, which were assigned as adducts of 2ME with cysteines
and oxidized methionines from molecular weight. However, these
adducts of 2ME were not oxidized at all.
2.5. Consideration of the photooxidation mechanism of
aequorin
We supposed that sulfhydryl groups of DTT attached to these
cysteine residues were selectively oxidized and then dehydrated
to give both sulfenic acid (11; + 150 u) and sulfinic acid (12; +
166 u) as outlined in Figure 8. On the basis of relative MS peak
intensities, we calculated the ratio of 11 and 12 to be 1:1.5–5,
respectively (Fig. 6). The distance between sulfhydryl groups of
these cysteine residues and the hydroxyl group of peroxide was
estimated to be shorter than 13 Å by using
a MacroModel
(OPLS_2001, PDB file of aequorin: 1EJ3).10 Such modification was
never found in apoaequorin being photo irradiated in the same
way, because it does not absorb at 365 nm. It happened specifically
to the bound substrate hydroperoxide (2) in the aequorin that is
the origin of the photolytic reaction. These results lead us to con-
clude that photolysis of reconstituted aequorin generated a reac-
tive oxidant, which converted DTT-cysteine residues locating
nearby the peroxide into 11 and 12 derivatives.
We considered that ROS generated from the chromophore
selectively oxidized sulfhydryl group. Although sulfhydryl group
of DTT added to cysteine could cleave the own disulfide bond be-
tween cysteine and DTT, photooxidized sulfhydryl group of DTT
on cysteine lost the ability to cleave them. These results lead us
C145 (T20)
C152 (T21)
O
O
H
N
O
H
N
H
N
oxidative
addition of DTT
S
S
OH
+
C180 (T22)
S
S
OH
S
S
OH
+154 u
O
SH
oxidation (+16 u)
-2 u
S
OH
S
OH
10
OH
dehydration (-18 u)
OH
molecular weight (M + 152 u)
9.8 Å
sulfinic acid (12)
molecular weight
(M+166 u)
sulfenic acid (11)
molecular weight
(M+150 u)
HO
HO
9.9 Å
OH
O
12.8 Å
Photo irradiation
(365 nm)
O
O
O
further oxidation (+16 u)
N
N
N
N
N
N
Coelenterazine
HO
HO
8
Aequorin chromophore (2)
Figure 8. The mechanism of oxidative modification of DTT-cysteine residues caused by ROS such as hydroxy radical generated from photolysis of coelenterazine
hydroperoxide. The distance between sulfhydryl groups of these cysteine residues and the hydroxyl group of peroxide was estimated to be shorter than 13 Å by using a
MacroModel (OPLS_2001, PDB file of aequorin: 1EJ3).10

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I. Doi et al. / Bioorg. Med. Chem. 17 (2009) 3399–3404
enterazine (methanolic solution) at 0 °C and then was incubated
3. Conclusions
for 2 h. The light emission of the reconstituted aequorin solution
was measured by the addition of 3 mL of 10 mM calcium chloride
for 1 min.
As conclusion, the photo oxidative modifications described
herein are the first report of its kind of photoprotein. This paper
also describes the first example where luminescent intermediate
works as a new device for labeling around the active site of aequo-
rin. This method would be applicable in order to study other
photoproteins whether they form a stable hydroperoxide interme-
diate as a bioluminescent intermediate through the selective oxi-
dation and analysis by means of LC-mass spectrometry.11
4.4. Photo irradiation
Coelenterazine (2
to the solution of 18
EDTA, 10 mM DTT or 20 mM 2ME, pH 7.5) and 20
quorin solution (SeaLite Sciences, Inc., 20
l
l
L of 2 mM) dissolved in methanol was added
L of Tris buffer (10 mM Tris–HCl and 10 mM
L of the apoae-
M, 10 mM Tris–HCl and
l
l
4. Experimental
10 mM EDTA, 5 mM DTT, pH 7.5) in ice bath. The mixture was
loaded into any desired position in the glass capillary (ringcapsÒ
4.1. General method and chemicals
50 lL) and then sealed with gas burner. The sample was incubated
for 2 h at 0 °C in the glass capillary and then was irradiated light
with a high-pressure mercury lamp (100-W high pressure Hg
lamp, 365 nm) for 20 min at room temperature with air. These
aequorin solutions were digested by using trypsin dissolved in Tris
buffer (20% w/w, 10 mM Tris–HCl and 10 mM EDTA, 5 mM DTT, pH
7.5) and were incubated for 12 h at 37 °C. The resultant solution
Photo irradiation was performed using an Eiko-sha PIH-100
high-pressure mercury lamp (365 nm). Luminescent activity was
measured on a lumiphotometer, AB-2200-R (ATTO, Tokyo, Japan).
Coelenterazine was prepared according to Goto’s mothod.12
Apoaequorin solution was prepared from SeaLite Sciences (SeaLite
Sciences, Inc., 20 lM, 10 mM Tris–HCl and 10 mM EDTA, 5 mM
(10 lL) were injected into nano-HPLC-ESI-IT-MS, -MS/MS.
DTT, pH 7.5). Ethylenediaminetetraacetate dehydrate (EDTA) and
dithiothreitol (DTT), trifluoroacetic acid (TFA) were purchased
from Nacalai Tesque (Kyoto, Japan). Trypsin (sequencing grade)
was purchased from Roche Diagnostic (Mannheim, Germany). 2-
Mercaptoethanol (2ME) was purchased from Wako Chemicals
(Osaka, Japan). PDB file of aequorin (1EJ3) was downloaded for
MacroModel calculation via the Internet at http://www.pdb.org.
Acknowledgements
Financial support from a Grant-in-Aid for Specially Promoted Re-
search (16002007) from MEXT is gratefully acknowledged. The
authors are also grateful for Grant-in-Aid for Encouragement of
YoungScientist (B) (19780087)from MEXT (M.K.), and financial sup-
port from the Global COE program (I.D.) and Ono Pharmaceutical CO.
LTD, (M.I., I.D.). Generous gift of apoaequorin from SeaLite Sciences
Inc. is also thankful. Thispaper is dedicatedto Dr. Osamu Shimomura
on his occasion in receiving Nobel Prize in Chemistry 2008.
4.2. Nano-HPLC-ESI-IT-MS and –MS/MS
Whole LC–MS and –MS/MS experiments were conducted utiliz-
ing the house assembled HPLC system (JASCO Co., Ltd. Tokyo, Japan)
using Develosil ODS-HG-5 (Nomura Co., Ltd. Aichi, Japan,
150 Â 0.3 mm i.d.) columns and measured utilizing an ion trap mass
spectrometer Bruker Daltonics HCT Plus (Bruker Daltonics, Bremen,
Germany) equipped with an orthogonal ESI source. The columns
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l
l
The column effluent was monitored by UV at 210 nm and then intro-
duced into the electrospray nebulizer without splitting. MS and MS/
MS settings were as follows: the voltage of capillary, 4000 V; drying
gas(N2)flowrate, 5 L/min, drytemperature, 350 °C. Aniontrapscan-
ning was performed in the ultra scan mode and the range of mass
was m/z 50–2000 (target mass was m/z 1500). MS/MS scanning
was also performed in the ultra scan mode and the range of mass
was m/z 50–3000. All MS experiments were preformed in positive
ion mode. Data were acquired and processed using Compass 1.2 (es-
quireControlTM and DataAnalysisTM version 3.2) (Bruker Daltonics,
Bremen, Germany).
6. Shimomura, O.; Johnson, F. H. Nature 1975, 256, 236.
7. (a) Usami, K.; Isobe, M. Tetrahedron Lett. 1995, 36, 8613; (b) Usami, K.; Isobe, M.
Tetrahedron 1996, 52, 12061.
8. Inoue, S.; Sugiura, S.; Kakoi, H.; Hashizume, K. Chem. Lett. 1975, 141.
9. (a) Kurose, K.; Inouye, S.; Sakaki, Y.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 80; (b) Tsuji, F. I.; Inouye, S.; Goto, T.; Sakaki, Y. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 8107; (c) Ohmiya, Y.; Kurono, S.; Ohashi, M.; Fagan, T. F.; Tsuji, F. I.
FEBS Lett. 1993, 332, 226.
10. (a) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118,
11225; (b) Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. J.
Phys. Chem. B 2001, 105, 6474; (c) The 3-dimensional structure determined by
Head et al. (1E3J). The sequence of 1E3J differs by 17 residues from that of
SeaLite aequorin used here. This procedure appears valid on the basis of Deng
et al. (1SL8, 2005), concluded that their structure 1SL8 having a sequence
almost the same as SeaLite protein, did not differ significantly from 1EJ3.
11. Sydnes, M. O.; Kuse, M.; Kurono, M.; Shimomura, A.; Ohinata, H.; Takai, A.;
Isobe, M. Bioorg. Med. Chem. 2008, 16, 1747.
4.3. Incorporation and bioluminescence measurements of
coelenterazine into apoaequorin
12. Inoue, S.; Sugiura, S.; Kakoi, H.; Hashizume, K.; Goto, T.; Iio, H. Chem. Lett. 1975,
4, 141.
13. Tryptic peptide T22 (913.4 u) was not observed in the tandem mass spectra.
We found the modified T22 peptide (at 904.9 u) from the trace of fragment ion.
Incorporation reaction was started by mixing 20
aequorin solution (SeaLite Sciences, Inc., 20 M, 10 mM Tris–HCl
and 10 mM EDTA, 5 mM DTT, pH 7.5), 18 L of the buffer solution
(10 mM Tris–HCl/NaOH buffer, pH 7.5, containing 10 mM EDTA
and 10 mM DTT or 20 mM 2ME) and saturated 2 L of 2 mM coel-
lL of the apo-
l
l
This modified T22 lost 17
u from the sequence of Q168H169, and we
considered that imidazole of H169 was react to amide of Q168 during trypsin
proteolytic digest. These modifications were not related to photo irradiation.
l