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Junichi O, Kozue M (Née M), Eri S, Yoko H, and Atsushi S
Junichi O, Kozue M (Née M), Eri S, Yoko H, and Atsushi S
20 707
2004 The Japan Society for Analytical Chemistry
The peroxidase-like catalytic activity of metal complexes of thiacalix[4]arenetetrasulfonate (TCAS[4]) on a modified anion-
exchanger (Men+-TCAS[4]A-500; Men+ = H2, Fe3+, Fe2+, Mn3+, Co3+, Co2+, Cu2+, Zn2+, Ni2+) for the oxidation of p-
hydroxyphenyl derivatives to produce fluorescent substances in the presence of hydrogen peroxide has been investigated.
Among the Men+-TCAS[4]A-500 tested, Fe3+-TCAS[4]A-500 exhibited the highest level of catalytic activity for the oxidation
of p-acetoamidophenol in a carbonate buffer solution of pH 10. The catalytic activity of Fe 3+-TCAS[4]A-500 was then used
for the spectrofluorometric determination of hydrogen peroxide. The calibration curve for the Fe3+-TCAS[4]A-500 method
was linear over a range spanning from 0.1 to 5.0 g of hydrogen peroxide in a 1.0 ml sample solution.
In clinical analyses, vital compounds such as glucose and uric TCAS[4]A-500, and applied them to measurements of glucose and
acid are routinely measured using enzymatic methods.1 With uric acid.14,15 It has been demonstrated that methods employing
such methods, the amount of hydrogen peroxide produced by Fe3+-TCAS[4]A-500 are effective for determining trace amounts
the oxidase corresponding to the compound (for example, of vital compounds in clinical analyses.
glucoseoxidase and uricase for glucose and uric acid, However, in clinical analyses the amount of vital samples,
respectively) is indirectly measured based on a color reaction especially blood, should be minimized as much as possible,
with the aid of peroxidase.1 Thus, hydrogen peroxide is an making it necessary to develop more sensitive methods.
important target in the determination of vital compounds in Accordingly, the peroxidase-like catalytic activity of Men+-
clinical analyses. Accordingly, it is necessary to develop a TCAS[4]A-500 for the oxidation of p-hydroxyphenyl derivatives
more sensitive method for measuring hydrogen peroxide. In in the presence of hydrogen peroxide was investigated in this
order to determine a trace amount of hydrogen peroxide, various study.
fluorometric methods in which peroxidase was used as an
enzyme have been developed.26 However, even if peroxidase
was immobilized on a carrier, such as chitosan and glass Experimental
beads,7,8 peroxidase in these methods has still some
disadvantages regarding stability, handling and storage. Thus, Reagents and apparatus
the development of an effective artificial mimesis with high Sodium thiacalix[4]arenetetrasulfonate (TCAS[4], Fig. 1) was
peroxidase-like activity has been desired to overcome these kindly provided by Cosmo Oil Co. DEAE cellulofine A-500
advantages. (an anion-exchanger of cellulose-type with diethylaminoethyl
Recently, thiacalix[n]arene derivatives have been developed.9 groups), purchased from Seikagaku Kogyo Co., was washed
Thiacalix[n]arenes have a specific structure in which the with water several times and dried over P 2O5 under reduced
methylene units of the parent calix[n]arenes have been replaced pressure. p-Acetoamidophenol from Nacalai Tesque Co. was
by S atom linkages, as shown in Fig. 1. Much attention has recrystallized from hot water several times. Peroxidase (POD,
been focused on the characteristics of thiacalix[n]arenes for from horseradish) was purchased from Wako Pure Chemical
forming stable metal complexes without modifying their upper- Industries Co. All other chemicals were of analytical or reagent
and/or lower-rims with functional groups.1013 We have recently grade and used without further purification.
found that Fe3+-thiacalix[4]arenetetrasulfonate on the modified The anion-exchangers modified with Men+-TCAS[4] (Men+-
anion-exchanger (Fe3+-TCAS[4]A-500) exhibited very strong TCAS[4]A-500, Men+: H2, Fe3+, Fe2+, Co3+, Co2+, Cu2+, Zn2+, Ni2+),
peroxidase-like catalytic activity, and was useful as a except for Mn3+-TCAS[4]A-500, were prepared according to
peroxidase mimetics.14 Moreover, we have developed new method A in the literature.14 H2- and Co3+-TCAS[4]A-500 were
methods for the spectrophotometric determination of hydrogen prepared by using TCAS[4] and K3[Co(CN)6], respectively.
peroxide using the peroxidase-like catalytic activity of Fe3+- The modified anion-exchanger (Men+-TCAS[4]A-500) contained
100 mol of Men+-TCAS[4] per gram of dry anion-exchanger.
To whom correspondence should be addressed. The proposed structure of Fe3+-TCAS[4]A-500 is shown in Fig. 1.
708 ANALYTICAL SCIENCES APRIL 2004, VOL. 20
Exa Ema
HO R R
(maximum, nm) (maximum, nm)
a. Excitation (Ex) and emission (Em) spectra were observed for the mixture after the treatment with peroxidase for p-hydroxyphenyl
derivatives in the presence of H2O2 at pH 10.
Mn3+-TCAS[4]A-500 was prepared as follows. First, 50 ml of a and buffer solution (3.0 ml); the mixture was incubated at 40C
1 mM ethanol solution of Mn(CH3COO)32H2O was added to for 20 min. After Fe3+-TCAS[4]A-500 was filtered off, the
TCAS[4] (100 mol) suspended in 50 ml of ethanol while fluorescence intensity of the supernatant was measured. A
stirring; this mixture was then refluxed at 70 75C for an blank in the absence of H2O2 was also measured under the same
additional 2 h. The produced Mn3+-TCAS[4] was filtered off, conditions.
washed with ethanol and dried over P 2O5 under reduced
pressure. Next, 50 ml of a 1 mM Mn3+-TCAS[4] solution was
added to DEAE cellulofine A-500 (500 mg) suspended in 20 ml
of water while stirring; this mixture was then stirred at room
temperature for an additional 1 h. The Mn 3+-TCAS[4]A-500
produced was filtered off, washed with water and dried over
P2O5 under reduced pressure. Mn3+-TCAS[4] was completely
adsorbed on the anion-exchanger because it was not found in
the filtrate. Oxidative reaction (1)
The fluorescence spectra and fluorescence intensities were
recorded with a quartz cell (1 cm 1 cm cross-section) on a
Shimadzu RF-5300 spectrofluorometer equipped with a xenon POD method as a reference. A mixture of a sample solution
lamp and a dual monochromator. The bandwidths for both (1.0 ml), a POD solution (1.0 ml, 10 U/ml), a 40 mM p-
excitation and emission were set at 3 nm. acetoamidophenol solution (2.0 ml) and a buffer solution (2.0
ml) was incubated at room temperature for 20 min. The
Procedures for determining H2O2 fluorescence intensity of this reaction mixture was measured
Fe3+-TCAS[4]A-500 method. The determination of H2O2 using under the same conditions as with the Fe3+-TCAS[4]A-500
this method was carried out by measuring the fluorescence method.
intensity of a fluorescent substance produced through an The buffer solutions used were 0.1 M KH2PO40.05 M
oxidative reaction (1). The fluorescence intensity was measured Na2B4O7 or 0.05 M Na2B4O7(0.2 M H3BO3 + 0.05 M NaCl) for
with excitation and emission wavelengths of 330 and 430 nm, pH 6 9, 0.1 M NH4Cl0.1 N NH4OH for pH 8 11, 0.05 M
respectively. Fe3+-TCAS[4]A-500 (20 mg, 100 mol/g) was Na2B4O70.05 M Na2CO3 for pH 10 12.4, and 0.05 M Na2CO3
added to a mixture containing the sample solution (1.0 ml, 0.1 0.1 M NaHCO3 for pH 9 11.
6.0 g of H2O2), 40 mM p-acetoamidophenol solution (2.0 ml)
ANALYTICAL SCIENCES APRIL 2004, VOL. 20 709
HO R Fluorescence
intensitya
p-Acetoamidophenol 195
p-Cresol 10
p-Ethylphenol 12
p-Hydroxyphenylacetic acid ~0
p-Hydroxyphenethylalcohol ~0
3-(4-Hydroxyphenyl)propionic acid ~0
4-(2-Aminoethyl)phenolHCl ~0
p-Hydroxyphenyl-1-propanol 15
Fig. 2 Excitation and emission spectra of a fluorescence substance a. Observed at the corresponding Ex and Em indicated in Table 1.
produced by the oxidation of p-AP catalyzed by POD at pH 10. The
spectra (A and B) show the respective blank fluorescence.
Table 3 Effects of Men+-TCAS[4]A-500 on the oxidative activity
for p-AP at pH 10 in the presence of 4.0 g of H2O2
Fluorescence
Results and Discussion Men+-TCAS[4]A-500 intensity
Added/ Added/
Substance g Error, %a Substance g Error, %a
40C, and the calibration curve was a straight line at 40C the
incubation temperature was set at 40C. amount of H2O2.
Calibration curve for the determination of H2O2 1. M. Kanai, H. Okabe, M. Sekiguchi, S. Nomoto, M.
Under the optimal conditions described above, a linear Kameko, M. Isobe, M. Tozuka, H. Hidaka, M. Oguchi, and
calibration curve for the Fe3+-TCAS[4]A-500 method was S. Kawa, Rinsyo-Kensaho-Teiyo (in Japanese), ed. I.
obtained between 0.1 and 6.0 g of H2O2 in the sample solution Kanai and M. Kanai, 1998, Chap. 6, Kinbara Press, Tokyo.
(1.0 ml). The fluorescence intensity using the Fe3+-TCAS[4]A-500 2. G. G. Guilbault, D. N. Kram, and E. Hackley, Anal. Chem.,
method was about 70% of that for the corresponding reaction 1967, 39, 271.
using the POD method as a reference. The correlation 3. A. L. Lazrus, G. L. Kok, N. Gitlin, J. A. Lind, and S. E.
coefficient and the relative standard deviation (N = 4) were McLaren, Anal. Chem., 1985, 57, 917.
greater than 0.989 and 5.5% for 4.0 g of H2O2, respectively. 4. K. Zaitsu and Y. Ohkura, Anal. Biochem., 1980, 109, 109.
For the determination of H2O2, the Fe3+-TCAS[4]A-500 method is 5. M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P.
more sensitive by a factor of 10 100 than the previous Haugland, Anal. Biochem., 1997, 253, 162.
methods, in which 4-AAP-phenol and MBTH-HALPS systems 6. T. Ohta, Y. Yamauchi, and S. Takitani, Fresenius J. Anal.
were employed as a chromogen.14,15 Chem., 1992, 343, 550.
7. A. Sakuragawa, T. Taniai, and T. Okutani, Anal. Chim.
Effect of foreign substances on the determination of H2O2 Acta, 1998, 374, 191.
The influence on the Fe3+-TCAS[4]A-500 method by the 8. Y. Hayashi, K. Zaitsu, and Y. Ohkura, Anal. Sci., 1985, 1,
presence of various foreign substances was investigated. As 65.
shown in Table 4, human serum albumin (HSA) and other 9. H. Kumagai, M. Hasegawa, S. Miyanari, Y. Sugawa, Y.
substances caused almost no interference, while ascorbic acid Sato, T. Hori, S. Ueda, H. Kamiyama, and S. Miyano,
had an appreciable effect. With the previous method using the Tetrahedron Lett., 1997, 38, 3971.
MBTH-HALPS system,8 the trend of interference by HSA and 10. N. Iki, F. Narumi, T. Fujimoto, N. Morohashi, and S.
ascorbic acid was the same as that for the present method. If the Miyano, J. Chem. Soc., Perkin Trans. 2, 1998, 2745.
sample solution contains ascorbic acid, the addition of ascorbic 11. N Iki, N. Morohashi, F. Narumi, and S. Miyano, Bull.
acid oxidase should minimize the interference from ascorbic Chem. Soc. Jpn., 1998, 71, 1597.
acid.1 12. M. Narita, Y. Higuchi, F. Hamada, and H. Kumagai,
In conclusion, Fe3+-TCAS[4]A-500 showed the strongest Tetrahedron Lett., 1998, 39, 8687.
peroxidase-like catalytic activity for the oxidation of p- 13. N. Iki, N. Morohashi, C. Kabuto, and S. Miyano, Chem.
acetoamidophenol in a carbonate buffer solution in the presence Lett., 1999, 219.
of H2O2 at pH 10. The sensitivity of the present method for the 14. J. Odo, N. Kawahara, Y. Inomata, A. Inoue, H. Takeya, S.
determination of H2O2 was about 10 100 times higher than that Miyanari, and H. Kumagai, Anal. Sci., 2000, 16, 963.
of previous methods using 4-AAP-phenol and MBTH-HALPS 15. J. Odo, Y. Inomata, H. Takeya, S. Miyanari, and H.
systems. The catalytic activity of Fe3+-TCAS[4]A-500 was Kumagai, Anal. Sci., 2001, 17, 1425.
effective for the spectrofluorometric determination of a trace