2
+
2+
Fig. 5 The fluorescence intensity changes of 1/Cu (20: 20 mM) upon
Fig. 6 The fluorescence intensity changes of 1/Cu (20: 400 mM) upon
the addition of various protein in buffer solution containing serum,
indicating that the fluorescence intensity of 1 can be released after the
the addition of various protein in buffer solution, indicating that the
fluorescence intensity of 1 can be released after the coordinated Cu were
2+
2+
extracted by the His-tag protein.
coordinated Cu were extracted by the His-tag protein.
(
20 mM) the non-His-tag GST showed no fluorescence recovery.
The authors are grateful to the projects of the Natural Science
Foundation of China (Nos. 20934002, 91027027 and 20973073),
Jilin Province Natural Science Foundation (20070926–01), the
National Basic Research Program (2007CB808006), the 111
Project (B06009) and the support from State Key Laboratory of
Supramolecular Structure and Materials, Jilin University.
14
Simultaneously, the His-tagged survivin was also used to clarify
the switch-on fluorescence response of 1/Cu to the histidine-rich
protein, which show very similar result as the His-tagged GST
(
2
+
Fig. 5).
In addition, in wondering if proteins which are consist of
different number and various sequences of amino acids will affect
the fluorescence recovery, non-His-tagged BSA, lysozyme and
survivin were used as control, respectively. When BSA was added
Notes and references
2
+
into the solution containing 1/Cu complex, a strong band at 485
1
(a) N. C. Shaner, P. A. Steinbach and R. Y. Tsien, Nat. Methods, 2005,
2, 905; (b) A. V. Pakhomov and V. I. Martynov, Chem. Biol., 2008, 15,
755.
nm (data not shown), being far from 550 nm, appeared because of
15
the inclusion of dansyl moiety in the hydrophobic cavity of BSA.
2
(a) M. J. Murcia, D. E. Minner, G. M. Mustata, K. Ritchie and C.
A. Naumann, J. Am. Chem. Soc., 2008, 130, 15054; (b) X. C. Chen,
Y. L. Deng, Y. Lin, D. W. Pang, H. Qing, F. Qu and H. Y. Xie,
Nanotechnology, 2008, 19, 235105.
However, in the presence of 20 mM non-His-tagged survivin or
lysozyme, the fluorescence intensity at 550 nm did not recover.
Therefore, we could draw conclusions that the general number
and/or sequences of the amino acid in protein, except BSA, had
3
(a) C. T. Hauser and R. Y. Tsien, Proc. Natl. Acad. Sci. U. S.
A., 2007, 104, 3693; (b) W. Gao, B. Xing, Roger Y. Tsien and J.
Rao, J. Am. Chem. Soc., 2003, 125, 11146; (c) H. Ren, F. Xiao, K.
Zhan, Y.-P. Kim, H. Xie, Zu. Xia and J. Rao, Angew. Chem., Int.
Ed., 2009, 48, 6704; (d) S. Mizukami, S. Watanabe, Y. Hori and K.
Kikuchi, J. Am. Chem. Soc., 2009, 131, 5016; (e) A. Ojida, K. Honda,
D. Shinmi, S. Kiyonaka, Y. Mori and I. Hamachi, J. Am. Chem.
Soc., 2006, 128, 10452; (f) Y. Li, H.-W. Li, L.-J. Ma, Y.-Q. Dang
and Y. Wu, Chem. Commun., 2010, 46, 3768; (g) K. K. Sadhu, S.
Mizukami, S. Watanabe and K. Kikuchi, Chem. Commun., 2010, 46,
2
+
no obvious effect on the fluorescence recovery of 1/Cu . The
2
+
1
/Cu complex had specific fluorescence response to the His-
tagged proteins in buffer solutions.
2
+
Finally, in order to demonstrate that 1/Cu has a potential
application in protein detection practically, the complex was
applied to the solution containing serum to mimic the conditions
of real case. When 1 was added into buffer solution containing
standard foetal bovine serum (v/v = 1 : 1), a strong fluorescence
band appeared at 511 nm, other than 550 nm, because of the
inclusion of dansyl moiety in BSA. Similarly as the response in
7
403.
4
H. Y. Park, K. Kim, S. Hong, H. Kim, Y. Choi, J. Ryu, D. Kwon, R.
Grailhe and R. Song, Langmuir, 2010, 26, 7327.
5 K. Honda, D. Shinmi, S. Kiyonaka, Y. Mori and I. Hamachi, J. Am.
Chem. Soc., 2006, 128, 10452.
2
+
buffer solution, further addition of Cu quenched the fluores-
cence; however, identical quenching level can be achieved only
6
D.-L. Ma, W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So, T.-S. Lai,
Z.-Y. Zhou, Y.-C. Leung and K.-Y. Wong, Angew. Chem., Int. Ed., 2008,
47, 3735.
2
+
after the concentration of Cu was increased up to 400 mM, which
may relate to the competitive binding between 1 and BSA. Once
7 B. Wang, H. Li, Y. Gao, H. Zhang and Y. Wu, J. Fluoresc., JOFL1517.
8 C. Harford and B. Sarkar, Acc. Chem. Res., 1997, 30,
2
+
we increase the ratio of serum in buffer solution, more Cu will
need. In such a case, when the his-tagged survivin and GST was
added into the solution, respectively, the fluorescence of 1 could
be recovered partly either. But the non-his-tagged GST had no
obvious effect on the fluorescence recovery (Fig. 6). The results
1
23.
9
G. Bhargava, T. A. Ramanarayanan and S. L. Bernasek, Langmuir,
2009, 26, 215.
1
1
1
0 (a) J. Wang, D. Wang, E. K. Miller, D. Moses, G. C. Bazan and A. J.
Heeger, Macromolecules, 2000, 33, 5153; (b) S. M. Hoenigman and C.
E. Evans, Anal. Chem., 1996, 68, 3274.
2
+
indicated that 1/Cu could be used to detect his-tagged protein
in the real system although the sensitivity needs to be improved
further.
1 (a) Romain F. H. Viguier and Alison N. Hulme, J. Am. Chem. Soc.,
2006, 128, 11370; (b) J. Rosenthal and S. J. Lippard, J. Am. Chem. Soc.,
2
010, 132, 5536.
2 W. Lin, L. Long, B. Chen, W. Tan and W. Gao, Chem. Commun., 2010,
6, 1311.
In summary, we have developed a new luminescent switch-on
4
2
+
1
/Cu complex, which could be used to distinguishing histidine
13 C. J. Sarell, C. D. Syme, S. E. J. Rigby and J. H. Viles, Biochemistry,
2009, 48, 4388.
and cysteine from other amino acids, and further to detecting
proteins with and without His-tags either in buffer or foetal bovine
1
4 Y. Gao, H. Zhang, M. Zhang, H. Zhang, X. Yu, X. Zha and Y. Wu, J.
Phys. Chem. B, 2010, 114, 15656.
5 L.-J. Ma, Y. Li, J. Sun, C. Tian and Y. Wu, Chem. Commun., 2008,
6345.
2
+
serum solution, indicating that 1/Cu had potential application
1
in real sample.
4
034 | Org. Biomol. Chem., 2011, 9, 4032–4034
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