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Analytical Utilization of Phenylfluorone as a Hydrogen Donor Substrate for Peroxidase
Microchemical Journal 61, 134 –142 (1999) Article ID mchj.1998.1679, available online at http://www.idealibrary.com on
Analytical Utilization of Phenylfluorone as a Hydrogen Donor Substrate for Peroxidase Zhong-Xian Guo, Han-Xi Shen, 1 and Li Li Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China Received September 12, 1998; accepted October 8, 1998 Phenylfluorone (PF) is used as an efficient hydrogen donor substrate for peroxidase. Based on the horseradish peroxidase (HRP)– hydrogen peroxide–PF catalytic system, the spectrophotometric methods for the measurement of HRP activity and the determination of hydrogen peroxide are proposed. The quantitative assay of HRP is sensitive to 9.2 3 10 212 mol/liter, and the calibration curve for hydrogen peroxide by a equilibrium procedure is linear in the range from 1.2 3 10 27 to 1.6 3 10 25 mol/liter. The system can be easily coupled with a glucose oxidase-catalyzed reaction, and glucose in human serum has been determined with the same results by a phenol– 4-aminoantipyine method. Phenylfluorone offers advantages over some presently used chromogenic substrates in the sense of sensitivity and simplicity of the substrate system. © 1999 Academic Press
INTRODUCTION The determination of hydrogen peroxide is considerably important in clinical chemistry and analytical biochemistry, for hydrogen peroxide is produced in stoichiometric amounts during the oxidation of biological species (e.g., glucose, cholesterol, and uric acid) by dissolved oxygen in the presence of the corresponding oxidase (1, 2). A great many procedures have been proposed to determine hydrogen peroxide. Examples include electrochemical methods (3, 4), spectrophotometric methods (5, 6), fluorescence techniques (7, 8), and chemiluminescence methods (9, 10). Nowadays, the most widely used methods for the determination of hydrogen peroxide in clinical chemistry are the spectrophotometric procedures by reaction with a chromogenic hydrogen donor substrate in the presence of peroxidase (2). Among the various hydrogen donors recommended for this aim, phenol derivatives (11–13) and aniline derivatives (14 –16) are popularly adopted. However, a coupling reagent such as 4-aminoantipyrine (4-AAP) is necessary when phenol/aniline is used. The poor sensitivity of the phenol– 4-AAP system is a common disadvantage. In addition, the aniline– 4-AAP system proceeds only in an acid medium, so its utilization is limited during the determination of hydrogen peroxide in the presence of oxidases, such as cholesterol oxidase. Trihydroxyfluorones, such as phenylfluorone (PF) (17), are xanthene reagents. Based on their complex reactions with metal ions, especially in the presence of surfactant, they are widely applied in the spectrophotometric, spectrofluorimetric, and polarographic determination of metal species (18, 19). Fujita et al. have studied the determination of hydrogen peroxide based on its effect on decoloration of trihydroxyfluorone–titanium (IV) complexes (20, 21). 1 To whom correspondence should be addressed. Fax: 186 22 23502458. E-mail: hxshen@sun. nankai.edu.cn.
134 0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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In this paper, phenylfluorone is advocated as a new hydrogen donor substrate for peroxidase. On the basis of oxidation decoloration of PF, a highly sensitive spectrophotometry for peroxidase is proposed, and a simple and practical method with a linear range from 1.2 3 10 27 to 1.6 3 10 25 mol/liter is presented for hydrogen peroxide. By coupling with a glucose oxidase (GOD)-catalyzed reaction, glucose in human serum is determined with a linear range from 4.0 3 10 27 to 1.2 3 10 25 mol/liter. MATERIALS AND METHODS Reagents Horseradish peroxidase (HRP, E.C.1.11.1.7, 150 –200 U/mg, RZ $3.0) was purchased from Sino-American Biotechnology Co., Beijing, China. Glucose oxidase (GOD, E.C. 1.1.3.4, $100 U/mg) was obtained from Baitai Biochemical Co., Chinese Academy of Science, Beijing, China. All other reagents were of analytical grade and the best grade commercially available. Water used throughout was doubly deionized. A hydrogen peroxide working solution of 9.0 3 10 25 mol/liter was freshly prepared by appropriate dilution of a stock solution, which was standardized by potassium permanganate. A glucose stock solution of 0.1 mol/liter was prepared and further diluted as required. HRP and GOD solutions were prepared to 2.3 3 10 26 mol/liter and 3.4 U/ml, respectively, and stored below 4°C. The concentration of HRP was determined at 403 nm, by using a molar absorpitivity (e) of 1.02 3 10 5 liter/(mol z cm) (22). A PF solution of 2.0 3 10 24 mol/liter was prepared by dissolving an appropriate amount of PF in ethanol acidified with 0.005 mol/liter sulfuric acid and preserved in a dark place. An ammonium buffer was prepared by mixing 1.0 mol/liter NH 4OH and 1.0 mol/liter NH 4Cl at 1:10 volume ratio and adjusted to pH 8.5. Apparatus All spectrophotometric measurements were performed with 10-mm standard cells on a Shimadzu UV-240 Spectrophotometer (Kyoto, Japan) with a Shimadzu TB-85 thermo bath. Procedure Quantitative assay of HRP. Just before use, a reagent mixture was prepared by mixing 14.0 ml PF and 10 ml buffer and diluting to 100 ml with water. In a 10-mm cell 2.40 ml mixed reagent and 50 ml of 4.5 3 10 22 mol/l H 2O 2 solution were added, and then 50 ml of appropriate concentration of HRP was added. Promptly the oxidation of PF was recorded at 508 nm against water. The initial velocity (V) was obtained from the absorbance–time curve. Determination of hydrogen peroxide. In a 10-ml colorimetric tube, 1.40 ml of PF, 1.0 ml of ammonium buffer (pH 8.5), 10 ml of 2.3 3 10 26 mol/liter HRP, and an appropriate volume of 9.0 3 10 25 mol/liter H 2O 2 standard solution were mixed and diluted to the mark with water. During the reaction for 7–12 min, the absorbance ( A) of the solution was monitored at 508 nm against water. The absorbance ( A 0 ) of the blank sample without H 2O 2 was obtained under the same conditions; thus the absorbance difference DA was obtained.
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Determination of glucose. Human serum samples were deproteinized by the zinc sulfate– barium hydroxide method (23). In a 10-ml colorimetric tube, a known volume of glucose standard solution of 1.0 3 10 24 mol/liter or deproteinized serum sample and 0.30 ml of GOD were mixed, diluted to about 5 ml, and incubated at 25°C for 15 min. Then 1.0 ml of 0.05 mol/liter EDTA (adjusted to pH 8.5), 1.40 ml of PF, 1.0 ml of ammonium buffer, and 10 ml of 2.3 3 10 26 mol/l HRP were added, and the solution was diluted to volume. With a sample without GOD run in same way, the DA was obtained by measurement as described above. RESULTS AND DISCUSSION Reaction of PF with H 2O 2 Catalyzed by HRP The oxidation of trihydroxyfluorones with H 2O 2 has been used for catalytic determination of some ions (24, 25); however, little emphasis has been placed on the reaction mechanism. In our experiments the oxidation of PF with H 2O 2 catalyzed by HRP was investigated at pH 8.5. As shown in Fig. 1, the absorption peak at 508 nm decreases immediately at room temperature, and the decrease relys on the concentration of H 2O 2. The oxidation product, even in the case of greatly excessive H 2O 2, has absorption in the range 380 – 600 nm. Nevertheless, the oxidation of PF catalyzed by HRP is feasible to utilize for the determination of H 2O 2 when the concentrations of H 2O 2 are at low levels. PF can be considered a phenol derivative, and thus the oxidation of PF and phenol by the HRP–H 2O 2 system may proceed in a similar way. PF presents mainly in H 2L 2 form at pH 8.5, and its catechol group is mostly subject to oxidize, so the initial oxidation product can be a derivative of o-benzoquinone, as shown in following scheme.
During the reaction, the conjugated and double bond system of PF is destroyed to some extent, but the planar and rigid structure may be maintained. In the case of H 2O 2 at low concentrations, the HRP-catalyzed oxidation of PF with H 2O 2 may proceed at a stoichi-
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FIG. 1. Absorption spectra of the HRP–H 2O 2–PF catalytic system in the presence of different concentrations of H 2O 2 (3 10 25 mol/liter): (a) 0; (b) 0.180; (c) 0.360; (d) 0.540; (e) 0.720; (f) 1.08; (g) 1.44; (h) 27.0. The concentrations of HRP and PF are 2.3 3 10 29 and 2.8 3 10 25 mol/l, respectively.
ometric ratio of 1:1, by considering the absorption strength of PF and the sensitivity for H 2O 2 assay. Measurement of Constant of Enzyme Reaction Steady-state catalytic velocity is closely related to enzyme and substrate concentrations. The method used to measure velocity is to make the concentration of the hydrogen donor, PF, greatly excessive and constant. Then the enzyme-catalyzed process is a single substrate reaction, and Michaelis–Menten behavior is observed. When the concentrations of PF and HRP are 2.8 3 10 25 and 1.15 3 10 29 mol/liter, respectively, an approximately
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straight line is obtained by a Lineweaver–Burk plot. Its linear regression equation is 1/V 5 359 1 3.10 3 10 23 /C H2 O2 (where the unit of V is s 21) with a correlation coefficient of 0.9990 (n 5 7). With the intercept and slope, and the molar absorptivity of PF [e 5 7.5 3 10 4 liter/(mol z cm)] (17) at pH 8.5, it can be obtained that Michaelis constant K m is 8.6 3 10 26 mol/liter, and maximum velocity V max is 3.71 3 10 28 mol/(liter z s). Since V max 5 K cat [E] 0 , where [E] 0 is the initial concentration of enzyme, the catalytic constant K cat is 323 s 21 and the specificity constant K cat/K m is 3.74 3 10 7 liter/(mol z s). Quantitative Assay of HRP When 2.8 3 10 25 mol/liter PF and 9.0 3 10 24 mol/liter H 2O 2 are used and various concentrations of HRP are present, the absorbance of the HRP–H 2O 2–PF catalytic system decreases monotonically with time. During the initial reaction period the concentrations of both hydrogen donor and acceptor are much larger than that of the enzyme, and the enzyme molecules are completely bound with substrate molecules; therefore the catalytic reaction is first-order kinetics in enzyme. The linear range for the relationship between V and the concentration of enzyme C HRP is 9.2 3 10 212–1.84 3 10 210 mol/liter HRP. The regression equation is V 5 6.69 3 10 25 1 6.89 3 10 6 C HRP, with a correlation coefficient of 0.9981 (n 5 5). The assay for HRP is much more sensitive than some common procedures, such as the tetramethylbenzidine method (26). Determination of Hydrogen Peroxide Optimization of reaction conditions. The effect of acidity on the system lies on the wavelength and strength of maximum absorption of PF and activity of HRP. At pH 5.0 – 8.0, PF exhibits two bands at approximately 475 and 500 nm with relatively weak absorption. As shown in Fig. 2, maximum and constant DA occurs in the range pH 8.3– 8.7; a pH 8.5 ammonium buffer is chosen. The result indicated that the addition of 0.5–2.0 ml of 1.0 mol/liter NH 4OH–NH 4Cl buffer is appropriate, and thus 1.0 ml is selected. For 7.2 3 10 26 mol/liter H 2O 2 in the catalytic system, DA remains maximum and constant on addition of 1.2–2.0 ml of 2.0 3 10 24 mol/liter PF solution to the final volume of 10 ml. When the concentrations of PF and HRP are 2.8 3 10 25 and 1.84 3 10 29 mol/liter, respectively, with the variation of temperature from 10 to 50°C, the oxidation of PF is completed within 8.0 to 2.0 min, but DA is constant. In the absence of HRP, the oxidation of PF with H 2O 2 proceeds at an obvious speed at 40 –50°C. Therefore room temperature (10 –30°C) is suggested for the utilization of the catalytic system. Reaction time is also related to HRP concentration. The reaction is completed within 1 min when 9.6 3 10 29 mol/liter HRP is present and completed within 13 min when 9.6 3 10 210 mol/liter enzyme is used. So 2.3 3 10 29 mol/liter HRP is applied. As shown in Fig. 3, the absorbance of the catalytic system decreases to a minimum, and then increases slowly with time. It can be considered that the oxidation product is not stable enough, so DA measurement should be performed during reaction for 7–12 min with a variation of less than 62%. Analytical characteristics. A calibration curve for H 2O 2 is constructed by the standard procedure, and it consists of two segments with different slopes and intercepts. Their regression equations are
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FIG. 2. Effect of acidity on the absorbance difference between the catalytic and blank (without H 2O 2) reaction solutions. The concentration of H 2O 2 was 1.08 3 10 25 mol/liter, and 2.3 3 10 29 mol/liter HRP and 2.8 3 10 25 mol/liter PF were present.
DA 5 0.0008 1 6.43 3 10 4 C H2O2
in the range 1.2 3 10 27 –7.2 3 10 26 mol/liter,
and DA 5 0.2376 1 3.09 3 10 4 C H2O2
in the range 7.2 3 10 26 –1.6 3 10 25 mol/liter,
with correlation coefficients of 0.9998 (n 5 5) and 0.9999 (n 5 6), respectively. Calculated from the regression equation in the range 1.2 3 10 27–7.2 3 10 26 mol/liter, the apparent molar absorptivity of the HRP–H 2O 2–PF system is 6.43 3 10 4 liter/(mol z cm), which is 3–11 times larger than that of the Trinder system (11, 12). The relative standard deviations for the determinations of 4.5 3 10 26 and 1.35 3 10 25 mol/liter H 2O 2 are 1.3% and 1.6%, respectively (n 5 7). The influence of foreign ions on the determination of 6.3 3 10 26 mol/liter H 2O 2 was examined. As shown in Table 1, anions and metal ions (in the presence of 0.005 mol/liter EDTA) at given levels cause relative errors less than 65%. Interference of protein indicates that deproteinization is necessary in the analysis of biological samples. Bilirubin in serum has no influence on the assay.
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FIG. 3. Absorbance–time curves of the HRP–H 2O 2–PF catalytic system in the presence of varied concentrations of H 2O 2 (mol/liter): a 0 (blank); (b) 7.2 3 10 26; (c) 1.08 3 10 25. The concentrations of HRP and PF are 2.3 3 10 29 and 2.8 3 10 25 mol/liter, respectively, 15°C.
Determination of Glucose The calibration curve for glucose is obtained by the proposed procedure. The linear range is 4.0 3 10 27–1.2 3 10 25 mol/liter, and the regression equation is DA 5 0.0030 1
TABLE 1 Tolerance of Foreign Ions in the Determination of 6.3 3 10 26 mol/liter H 2O 2 Cation added a
Tolerance (mg/ml)
Fe 31 Pb 21, Al 31, Cr (VI) Co 21 Cu 21, Cr 31
0.5 1.0 2.0 3.0
Cd 21, Ni 21 Mn 21, Ba 21, Se (IV) Mg 21, Zn 21, Ca 21
5.0
a
10 20
In the presence of 0.005 mol/liter EDTA.
Anion S 22 I2 Br 2 SO 322 Cl 2, SO 422, PO 432, NO 32
Tolerance (mg/ml) 0.05 0.2 1.0 3.0 500
141
PHENYLFLUORONE AS A SUBSTRATE FOR PEROXIDASE TABLE 2 Determination Results of Glucose in Human Serum (n 5 6) This method Sample No. 1 2 3
Phenol–4-AAP method
Mean (g/liter)
RSD (%)
Mean (g/liter)
RSD (%)
0.740 0.677 1.038
2.0 2.3 1.6
0.725 0.636 1.045
2.3 3.3 2.7
5.02 3 10 4 C glucose with a correlation coefficient of 0.9990 (n 5 5). The molar absorptivity for glucose is 5.02 3 10 4 liter/(mol z cm), indicating that the proposed procedure is very sensitive. Some human serum samples were analyzed, and the results were compared with that of the phenol– 4-AAP method, as listed in Table 2. No marked difference is observed. CONCLUSIONS The proposed hydrogen donor substrate for peroxidase, phenylfluorone, is better than some of the presently used chromogenic substrates in the sense of sensitivity and simplicity of the substrate system. An important potential application of the HRP– H 2O 2–PF catalytic system is that it can be coupled with oxidase-catalyzed systems to determine substances of clinical interests, such as glucose in human serum. ACKNOWLEDGMENTS The financial support of the National Natural Science Foundation of China (Grant 29875011) is gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Bowers, L. D. Anal. Chem., 1986, 58, 513A–530A. Mizoguchi, M.; Ishiyama, M.; Shiga, M.; Sasamoto, K. Bunseki Kagaku, 1996, 45, 111–124. Lin, M. S.; Jan, B. I. Electroanalysis, 1997, 9, 340 –344. Sorokina, N. V.; Urusov, Yu. I.; Petrukhin, O. M. Zh. Anal. Khim., 1996, 51, 927–930. Almuaibed, A. M.; Townshend, A. Anal. Chim. Acta, 1994, 295, 159 –163. Mori, I.; Taguchi, K.; Fujita, Y.; Matsuo, T. Anal. Lett., 1995, 28, 1261–1274. Guibault, G. G.; Brignac, P. J. Jr.; Juneau, M. Anal. Chem., 1968, 40, 1256 –1263. Kelly, T. A.; Christian, G. D. Anal. Chem., 1991, 53, 2110 –2114. Hayashi, K.; Sasaki, S.; Ikebukuro, K.; Karube, I. Anal. Chim. Acta, 1996, 329, 127–134. Ponten, E.; Appelblad, P.; Stigbrand, M.; Irgum, K.; Nakashima, K. Fresenius’ J. Anal. Chem., 1996, 356, 84 – 89. Trinder, P. Ann. Clin. Biochem., 1969, 6, 24 –27. Barham, D.; Trinder, P. Analyst, 1972, 97, 142–145. Fossati, P.; Prencipe, L.; Berti, G. Clin. Chem., 1980, 26, 227–231. Ngo, T. T.; Genhoff, H. M. Anal. Biochem., 1980, 105, 389 –397. Tamaoku, K.; Ueno, K.; Akiura, K.; Ohkura, Y. Chem. Pharm. Bull., 1982, 30, 2492–2497. Shiga, M.; Saito, M.; Kina, K. Anal. Chim. Acta, 1983, 153, 191–197. Zeng, Y. E.; Zhang, H. S.; Chen, Z. H. A Handbook of Modern Chemical Reagents, Part 4, Chromogenic Reagents for Inorganic Ions, pp. 377–379. Chemical Industry Press, Beijing, China, 1989. Shen, H. X.; Wang, L. S. Yejin Fenxi yu Ceshi (Yejin Fenxi Fence), 1984, 4(2), 49 – 60.
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Fang, G. Z.; Guo, Z. X. Huaxue Shiji, 1993, 15, 353–359. Fujita, Y.; Mori, I.; Toyoda, M. Anal. Sci., 1991, 7, 327–328. Fujita, Y.; Mori, I.; Toyoda, M.; Matsuo, T. Anal. Sci., 1994, 10, 827– 830. Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev., 1976, 19, 187–251. Henry, J. R.; Cannon, C. D.; Winkelman, W. J. Clinical Chemistry. Principles and Techniques, 2nd ed., pp. 389 – 404, Harper and Row, Hagerstown, MD, 1974. 24. Merkulov, A. I.; Skvortsova, R. I. Zh. Anal. Khim., 1981, 36, 1778 –1783. 25. Zhu, Q. Z.; Gong, G. Q.; Wang, H. G. Mikrochim. Acta, 1994, 116, 197–204. 26. Andrews, P. C.; Krinsky, N. I. Anal. Biochem., 1982, 127, 346 –350.
Analytical Utilization of Phenylfluorone as a Hydrogen Donor Substrate for Peroxidase Zhong-Xian Guo, Han-Xi Shen, 1 and Li Li Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China Received September 12, 1998; accepted October 8, 1998 Phenylfluorone (PF) is used as an efficient hydrogen donor substrate for peroxidase. Based on the horseradish peroxidase (HRP)– hydrogen peroxide–PF catalytic system, the spectrophotometric methods for the measurement of HRP activity and the determination of hydrogen peroxide are proposed. The quantitative assay of HRP is sensitive to 9.2 3 10 212 mol/liter, and the calibration curve for hydrogen peroxide by a equilibrium procedure is linear in the range from 1.2 3 10 27 to 1.6 3 10 25 mol/liter. The system can be easily coupled with a glucose oxidase-catalyzed reaction, and glucose in human serum has been determined with the same results by a phenol– 4-aminoantipyine method. Phenylfluorone offers advantages over some presently used chromogenic substrates in the sense of sensitivity and simplicity of the substrate system. © 1999 Academic Press
INTRODUCTION The determination of hydrogen peroxide is considerably important in clinical chemistry and analytical biochemistry, for hydrogen peroxide is produced in stoichiometric amounts during the oxidation of biological species (e.g., glucose, cholesterol, and uric acid) by dissolved oxygen in the presence of the corresponding oxidase (1, 2). A great many procedures have been proposed to determine hydrogen peroxide. Examples include electrochemical methods (3, 4), spectrophotometric methods (5, 6), fluorescence techniques (7, 8), and chemiluminescence methods (9, 10). Nowadays, the most widely used methods for the determination of hydrogen peroxide in clinical chemistry are the spectrophotometric procedures by reaction with a chromogenic hydrogen donor substrate in the presence of peroxidase (2). Among the various hydrogen donors recommended for this aim, phenol derivatives (11–13) and aniline derivatives (14 –16) are popularly adopted. However, a coupling reagent such as 4-aminoantipyrine (4-AAP) is necessary when phenol/aniline is used. The poor sensitivity of the phenol– 4-AAP system is a common disadvantage. In addition, the aniline– 4-AAP system proceeds only in an acid medium, so its utilization is limited during the determination of hydrogen peroxide in the presence of oxidases, such as cholesterol oxidase. Trihydroxyfluorones, such as phenylfluorone (PF) (17), are xanthene reagents. Based on their complex reactions with metal ions, especially in the presence of surfactant, they are widely applied in the spectrophotometric, spectrofluorimetric, and polarographic determination of metal species (18, 19). Fujita et al. have studied the determination of hydrogen peroxide based on its effect on decoloration of trihydroxyfluorone–titanium (IV) complexes (20, 21). 1 To whom correspondence should be addressed. Fax: 186 22 23502458. E-mail: hxshen@sun. nankai.edu.cn.
134 0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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In this paper, phenylfluorone is advocated as a new hydrogen donor substrate for peroxidase. On the basis of oxidation decoloration of PF, a highly sensitive spectrophotometry for peroxidase is proposed, and a simple and practical method with a linear range from 1.2 3 10 27 to 1.6 3 10 25 mol/liter is presented for hydrogen peroxide. By coupling with a glucose oxidase (GOD)-catalyzed reaction, glucose in human serum is determined with a linear range from 4.0 3 10 27 to 1.2 3 10 25 mol/liter. MATERIALS AND METHODS Reagents Horseradish peroxidase (HRP, E.C.1.11.1.7, 150 –200 U/mg, RZ $3.0) was purchased from Sino-American Biotechnology Co., Beijing, China. Glucose oxidase (GOD, E.C. 1.1.3.4, $100 U/mg) was obtained from Baitai Biochemical Co., Chinese Academy of Science, Beijing, China. All other reagents were of analytical grade and the best grade commercially available. Water used throughout was doubly deionized. A hydrogen peroxide working solution of 9.0 3 10 25 mol/liter was freshly prepared by appropriate dilution of a stock solution, which was standardized by potassium permanganate. A glucose stock solution of 0.1 mol/liter was prepared and further diluted as required. HRP and GOD solutions were prepared to 2.3 3 10 26 mol/liter and 3.4 U/ml, respectively, and stored below 4°C. The concentration of HRP was determined at 403 nm, by using a molar absorpitivity (e) of 1.02 3 10 5 liter/(mol z cm) (22). A PF solution of 2.0 3 10 24 mol/liter was prepared by dissolving an appropriate amount of PF in ethanol acidified with 0.005 mol/liter sulfuric acid and preserved in a dark place. An ammonium buffer was prepared by mixing 1.0 mol/liter NH 4OH and 1.0 mol/liter NH 4Cl at 1:10 volume ratio and adjusted to pH 8.5. Apparatus All spectrophotometric measurements were performed with 10-mm standard cells on a Shimadzu UV-240 Spectrophotometer (Kyoto, Japan) with a Shimadzu TB-85 thermo bath. Procedure Quantitative assay of HRP. Just before use, a reagent mixture was prepared by mixing 14.0 ml PF and 10 ml buffer and diluting to 100 ml with water. In a 10-mm cell 2.40 ml mixed reagent and 50 ml of 4.5 3 10 22 mol/l H 2O 2 solution were added, and then 50 ml of appropriate concentration of HRP was added. Promptly the oxidation of PF was recorded at 508 nm against water. The initial velocity (V) was obtained from the absorbance–time curve. Determination of hydrogen peroxide. In a 10-ml colorimetric tube, 1.40 ml of PF, 1.0 ml of ammonium buffer (pH 8.5), 10 ml of 2.3 3 10 26 mol/liter HRP, and an appropriate volume of 9.0 3 10 25 mol/liter H 2O 2 standard solution were mixed and diluted to the mark with water. During the reaction for 7–12 min, the absorbance ( A) of the solution was monitored at 508 nm against water. The absorbance ( A 0 ) of the blank sample without H 2O 2 was obtained under the same conditions; thus the absorbance difference DA was obtained.
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Determination of glucose. Human serum samples were deproteinized by the zinc sulfate– barium hydroxide method (23). In a 10-ml colorimetric tube, a known volume of glucose standard solution of 1.0 3 10 24 mol/liter or deproteinized serum sample and 0.30 ml of GOD were mixed, diluted to about 5 ml, and incubated at 25°C for 15 min. Then 1.0 ml of 0.05 mol/liter EDTA (adjusted to pH 8.5), 1.40 ml of PF, 1.0 ml of ammonium buffer, and 10 ml of 2.3 3 10 26 mol/l HRP were added, and the solution was diluted to volume. With a sample without GOD run in same way, the DA was obtained by measurement as described above. RESULTS AND DISCUSSION Reaction of PF with H 2O 2 Catalyzed by HRP The oxidation of trihydroxyfluorones with H 2O 2 has been used for catalytic determination of some ions (24, 25); however, little emphasis has been placed on the reaction mechanism. In our experiments the oxidation of PF with H 2O 2 catalyzed by HRP was investigated at pH 8.5. As shown in Fig. 1, the absorption peak at 508 nm decreases immediately at room temperature, and the decrease relys on the concentration of H 2O 2. The oxidation product, even in the case of greatly excessive H 2O 2, has absorption in the range 380 – 600 nm. Nevertheless, the oxidation of PF catalyzed by HRP is feasible to utilize for the determination of H 2O 2 when the concentrations of H 2O 2 are at low levels. PF can be considered a phenol derivative, and thus the oxidation of PF and phenol by the HRP–H 2O 2 system may proceed in a similar way. PF presents mainly in H 2L 2 form at pH 8.5, and its catechol group is mostly subject to oxidize, so the initial oxidation product can be a derivative of o-benzoquinone, as shown in following scheme.
During the reaction, the conjugated and double bond system of PF is destroyed to some extent, but the planar and rigid structure may be maintained. In the case of H 2O 2 at low concentrations, the HRP-catalyzed oxidation of PF with H 2O 2 may proceed at a stoichi-
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FIG. 1. Absorption spectra of the HRP–H 2O 2–PF catalytic system in the presence of different concentrations of H 2O 2 (3 10 25 mol/liter): (a) 0; (b) 0.180; (c) 0.360; (d) 0.540; (e) 0.720; (f) 1.08; (g) 1.44; (h) 27.0. The concentrations of HRP and PF are 2.3 3 10 29 and 2.8 3 10 25 mol/l, respectively.
ometric ratio of 1:1, by considering the absorption strength of PF and the sensitivity for H 2O 2 assay. Measurement of Constant of Enzyme Reaction Steady-state catalytic velocity is closely related to enzyme and substrate concentrations. The method used to measure velocity is to make the concentration of the hydrogen donor, PF, greatly excessive and constant. Then the enzyme-catalyzed process is a single substrate reaction, and Michaelis–Menten behavior is observed. When the concentrations of PF and HRP are 2.8 3 10 25 and 1.15 3 10 29 mol/liter, respectively, an approximately
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straight line is obtained by a Lineweaver–Burk plot. Its linear regression equation is 1/V 5 359 1 3.10 3 10 23 /C H2 O2 (where the unit of V is s 21) with a correlation coefficient of 0.9990 (n 5 7). With the intercept and slope, and the molar absorptivity of PF [e 5 7.5 3 10 4 liter/(mol z cm)] (17) at pH 8.5, it can be obtained that Michaelis constant K m is 8.6 3 10 26 mol/liter, and maximum velocity V max is 3.71 3 10 28 mol/(liter z s). Since V max 5 K cat [E] 0 , where [E] 0 is the initial concentration of enzyme, the catalytic constant K cat is 323 s 21 and the specificity constant K cat/K m is 3.74 3 10 7 liter/(mol z s). Quantitative Assay of HRP When 2.8 3 10 25 mol/liter PF and 9.0 3 10 24 mol/liter H 2O 2 are used and various concentrations of HRP are present, the absorbance of the HRP–H 2O 2–PF catalytic system decreases monotonically with time. During the initial reaction period the concentrations of both hydrogen donor and acceptor are much larger than that of the enzyme, and the enzyme molecules are completely bound with substrate molecules; therefore the catalytic reaction is first-order kinetics in enzyme. The linear range for the relationship between V and the concentration of enzyme C HRP is 9.2 3 10 212–1.84 3 10 210 mol/liter HRP. The regression equation is V 5 6.69 3 10 25 1 6.89 3 10 6 C HRP, with a correlation coefficient of 0.9981 (n 5 5). The assay for HRP is much more sensitive than some common procedures, such as the tetramethylbenzidine method (26). Determination of Hydrogen Peroxide Optimization of reaction conditions. The effect of acidity on the system lies on the wavelength and strength of maximum absorption of PF and activity of HRP. At pH 5.0 – 8.0, PF exhibits two bands at approximately 475 and 500 nm with relatively weak absorption. As shown in Fig. 2, maximum and constant DA occurs in the range pH 8.3– 8.7; a pH 8.5 ammonium buffer is chosen. The result indicated that the addition of 0.5–2.0 ml of 1.0 mol/liter NH 4OH–NH 4Cl buffer is appropriate, and thus 1.0 ml is selected. For 7.2 3 10 26 mol/liter H 2O 2 in the catalytic system, DA remains maximum and constant on addition of 1.2–2.0 ml of 2.0 3 10 24 mol/liter PF solution to the final volume of 10 ml. When the concentrations of PF and HRP are 2.8 3 10 25 and 1.84 3 10 29 mol/liter, respectively, with the variation of temperature from 10 to 50°C, the oxidation of PF is completed within 8.0 to 2.0 min, but DA is constant. In the absence of HRP, the oxidation of PF with H 2O 2 proceeds at an obvious speed at 40 –50°C. Therefore room temperature (10 –30°C) is suggested for the utilization of the catalytic system. Reaction time is also related to HRP concentration. The reaction is completed within 1 min when 9.6 3 10 29 mol/liter HRP is present and completed within 13 min when 9.6 3 10 210 mol/liter enzyme is used. So 2.3 3 10 29 mol/liter HRP is applied. As shown in Fig. 3, the absorbance of the catalytic system decreases to a minimum, and then increases slowly with time. It can be considered that the oxidation product is not stable enough, so DA measurement should be performed during reaction for 7–12 min with a variation of less than 62%. Analytical characteristics. A calibration curve for H 2O 2 is constructed by the standard procedure, and it consists of two segments with different slopes and intercepts. Their regression equations are
PHENYLFLUORONE AS A SUBSTRATE FOR PEROXIDASE
139
FIG. 2. Effect of acidity on the absorbance difference between the catalytic and blank (without H 2O 2) reaction solutions. The concentration of H 2O 2 was 1.08 3 10 25 mol/liter, and 2.3 3 10 29 mol/liter HRP and 2.8 3 10 25 mol/liter PF were present.
DA 5 0.0008 1 6.43 3 10 4 C H2O2
in the range 1.2 3 10 27 –7.2 3 10 26 mol/liter,
and DA 5 0.2376 1 3.09 3 10 4 C H2O2
in the range 7.2 3 10 26 –1.6 3 10 25 mol/liter,
with correlation coefficients of 0.9998 (n 5 5) and 0.9999 (n 5 6), respectively. Calculated from the regression equation in the range 1.2 3 10 27–7.2 3 10 26 mol/liter, the apparent molar absorptivity of the HRP–H 2O 2–PF system is 6.43 3 10 4 liter/(mol z cm), which is 3–11 times larger than that of the Trinder system (11, 12). The relative standard deviations for the determinations of 4.5 3 10 26 and 1.35 3 10 25 mol/liter H 2O 2 are 1.3% and 1.6%, respectively (n 5 7). The influence of foreign ions on the determination of 6.3 3 10 26 mol/liter H 2O 2 was examined. As shown in Table 1, anions and metal ions (in the presence of 0.005 mol/liter EDTA) at given levels cause relative errors less than 65%. Interference of protein indicates that deproteinization is necessary in the analysis of biological samples. Bilirubin in serum has no influence on the assay.
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FIG. 3. Absorbance–time curves of the HRP–H 2O 2–PF catalytic system in the presence of varied concentrations of H 2O 2 (mol/liter): a 0 (blank); (b) 7.2 3 10 26; (c) 1.08 3 10 25. The concentrations of HRP and PF are 2.3 3 10 29 and 2.8 3 10 25 mol/liter, respectively, 15°C.
Determination of Glucose The calibration curve for glucose is obtained by the proposed procedure. The linear range is 4.0 3 10 27–1.2 3 10 25 mol/liter, and the regression equation is DA 5 0.0030 1
TABLE 1 Tolerance of Foreign Ions in the Determination of 6.3 3 10 26 mol/liter H 2O 2 Cation added a
Tolerance (mg/ml)
Fe 31 Pb 21, Al 31, Cr (VI) Co 21 Cu 21, Cr 31
0.5 1.0 2.0 3.0
Cd 21, Ni 21 Mn 21, Ba 21, Se (IV) Mg 21, Zn 21, Ca 21
5.0
a
10 20
In the presence of 0.005 mol/liter EDTA.
Anion S 22 I2 Br 2 SO 322 Cl 2, SO 422, PO 432, NO 32
Tolerance (mg/ml) 0.05 0.2 1.0 3.0 500
141
PHENYLFLUORONE AS A SUBSTRATE FOR PEROXIDASE TABLE 2 Determination Results of Glucose in Human Serum (n 5 6) This method Sample No. 1 2 3
Phenol–4-AAP method
Mean (g/liter)
RSD (%)
Mean (g/liter)
RSD (%)
0.740 0.677 1.038
2.0 2.3 1.6
0.725 0.636 1.045
2.3 3.3 2.7
5.02 3 10 4 C glucose with a correlation coefficient of 0.9990 (n 5 5). The molar absorptivity for glucose is 5.02 3 10 4 liter/(mol z cm), indicating that the proposed procedure is very sensitive. Some human serum samples were analyzed, and the results were compared with that of the phenol– 4-AAP method, as listed in Table 2. No marked difference is observed. CONCLUSIONS The proposed hydrogen donor substrate for peroxidase, phenylfluorone, is better than some of the presently used chromogenic substrates in the sense of sensitivity and simplicity of the substrate system. An important potential application of the HRP– H 2O 2–PF catalytic system is that it can be coupled with oxidase-catalyzed systems to determine substances of clinical interests, such as glucose in human serum. ACKNOWLEDGMENTS The financial support of the National Natural Science Foundation of China (Grant 29875011) is gratefully acknowledged.
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