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Stopped-flow spectrophotometric determination of hydrogen peroxide with hemoglobin as catalyst
Talanta 51 (2000) 179 – 186 www.elsevier.com/locate/talanta
Stopped-flow spectrophotometric determination of hydrogen peroxide with hemoglobin as catalyst Ke Zhang, Luyuan Mao, Ruxiu Cai * Department of Chemistry, Wuhan Uni6ersity, Wuhan 430072, PR China Received 13 July 1999; received in revised form 7 September 1999; accepted 8 September 1999
Abstract A rapid and sensitive method was proposed for the determination of hydrogen peroxide based on the catalytic effect of hemoglobin using o-phenylenediamine as the substrate. Stopped-flow spectrophotometric method was used to study the kinetic behavior of the oxidation reaction. The catalytic effectiveness of hemoglobin was compared with other four kinds of catalysts. The initial rate of the formation of the reaction product 2,3-diaminophenazine at the wavelength of 425 nm was monitored, permitting a detection limit of 9.2× 10 − 9 mol/l H2O2. A linear calibration graph was obtained over the H2O2 concentration range 5.0 ×10 − 8 – 3.5×10 − 6 mol/l, and the relative standard deviation at a H2O2 concentration of 5.0 × 10 − 7 mol/l was 2.08%. Satisfied results were obtained in the determination of H2O2 in real samples by this method. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Stopped-flow spectrophotometry; Hydrogen peroxide; Hemoglobin
1. Introduction Hydrogen peroxide is a very important intermediate in clinical biochemistry [1,2] and environmental work [3,4]. Hydrogen peroxide is produced in stoichiometric amounts during the oxidation of substrates of clinical interest by dissolved oxygen in the presence of the corresponding oxidase enzyme. Presently, H2O2 is also an analyte of environmental interest for its involving in catalyzed and uncatalyzed oxidation of organic species. Therefore, a rapid, sensitive and inexpensive * Corresponding author. Tel.: + 86-27-87684184; fax: +8627-87647617. E-mail address: cai – [email protected] (R. Cai)
method is required for the determination of hydrogen peroxide at levels below submicromolar limit of determination. Numerous studies on the determination of hydrogen peroxide using polarography, amperometry, fluorimetry, spectrophotometry and chemiluminescence, etc., have been published [5–9]. Horseradish peroxidase (HRP), in which the heme iron acts as the activity center, is the most commonly used enzyme in H2O2 detection. However, HRP has its shortcomings. For example, it is expensive and unstable in solution, and has strict requirements for the experimental conditions. Therefore, the search for a replacement for HRP has been an important work. Hemin [2] and hematin [10] have been reported as a peroxidase
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K. Zhang et al. / Talanta 51 (2000) 179–186
substitute in the detection of H2O2. Some metalloporphyrins show the similar catalytic characteristics as that of peroxidase, so the peroxidase mimesis is one of the interesting trends in enzymatic analysis [11]. Saito et al. synthesized Mn (III)-tetrakis (sulfophenyl) porphine and found it can be immobilized on anion exchange resins and mimicked the behavior of immobilized HRP [12,13]. The porphyrin complexes of Mn, Co, Fe [14,15] and Mo [16] all exhibited peroxidase activity in aqueous phase. However, simple natural or synthetic metalloporphyrins do not show satisfactory activity and selectivity because they lack the spatial structure of the natural enzyme, which is essential for the special inclusion behavior between the enzyme and the substrate. A number of polymers and receptor compounds derived from b-CD are widely used in analytical chemistry and biomimetic chemistry based on their supramolecular features. [7,17 – 19]. Hemoglobin (Hb), a necessary vehicle for oxygen carriage, has the natural quaternary structure. It contains four subunits of polypeptide and each polypeptide chain contains a heme group that serves as the active center. Encouraged by reports on the studies of peroxidase substitutes and the natural spatial structure of Hb, as well as its cheaper price, we expected Hb be an excellent enzyme for catalyzing the reaction between ophenylenediamine (OPDA, i.e. 1,2-diaminobenzene) and hydrogen peroxide to form 2,3-diaminophenazine (DAPN). The reaction is as follows:
scanning UV/Vis spectrophotometer and controlled by a microcomputer for operation and data processing. The catalytic reaction was studied by measuring the increasing absorbance DAPN at 425 nm to obtain the initial reaction rate. It was found that the initial reaction rate was proportional to the concentration of H2O2 over the range 5.0×10 − 8 –3.5×10 − 6 mol/l. The catalytic activity of Hb has been compared with HRP, bCD-hemin, hemin and MnTCPP, and it was found that the catalytic effectiveness of Hb is much higher than bCD-hemin, hemin and MnTCPP, and even a little higher than HRP in the conditions of this work. The method is rapid, simple, sensitive and reliable for the determination of H2O2.
Stopped-flow method has been widely recognized as a powerful technique to monitor rapid reactions. The coupling of the stopped-flow technique with rapid scanning UV/Vis spectrophotometer is effective for investigating the mechanism of rapid reaction [20]. In the present work, the stopped-flow technique was coupled with a rapid
with buffer to the desired volume. Hydrogen peroxide solutions were prepared by appropriate dilution of the 30% solution with water (standardized by titration with KMnO4). b-cyclodextrin (bCD) was purchased from Sigma and tetra (p-canboxyphenyl) porphine (TCPP) was purchased from Aldrich. bCD-hemin and
2. Experimental
2.1. Reagents Unless indicated otherwise, all dilutions were made in 0.2 mol /l Na2HPO4 –citric acid buffer (pH=5.0). Hemoglobin (bovine erythrocytes) solution was prepared by dissolving certain amount of Hb (Shanghai Institute of Biochem., Shanghai, China) in deionized water and stored below 4°C. O-phenylenediamine (Shanghai Chem. Agent, Inc., Shanghai, China) was sublimed before use. 0.05 mol/l of o-phenylenediamine stock solution was freshly prepared by dissolving sublimed OPDA in buffer solution, and the working solution was prepared by diluting the stock solution
K. Zhang et al. / Talanta 51 (2000) 179–186
MnTCPP were synthesized using the reported method [7,21]. HRP (E.C. 1.11.1.7, 250 IU mg − 1, RZ 3, purchased from Sino-Amer. Biotech. Co.) was prepared in water solution. Distilled, de-ionized water was used throughout. All other chemicals were of analytical-reagent grade.
2.2. Apparatus SFA-12 HI-TECH scientific kinetic stoppedflow accessory (HI-TECH, Japan) was fitted to UVIKON-941 spectrophotometer (Kontron Instrument). A circulation water-bath was used to control the temperature in the stopped-flow module and the cell compartment. A microcomputer is applied to control the operation of the instrument and to collect and process the data.
2.3. Procedure A solution of Hb and a sample solution containing H2O2 and OPDA were filled into two separate reservoir syringes. At each determina-
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tion, two flow streams were merged and stopped in the 1-cm flow-through absorption cell and the oxidation reaction was monitored by the absorption at 425 nm. The initial reaction rate was calculated using the data collected in the first 0.2 min of the reaction. Because of the two-fold dilution during the mixing all concentrations reported are twice as much as the actual initial concentrations after mixing.
3. Result and discussion
3.1. Spectra characteristics of the reaction system Fig. 1 shows the rapid-scanning stopped-flow spectra during the reaction process of OPDA and H2O2 to produce DAPN. Due to formation of the reaction product, DAPN, an absorption band appeared at 425 nm and increased continuously during the process scans from 1 to 8. The insert picture of Fig. 1 is the kinetic curve monitored at 425 nm in the first minute of the reaction. The
Fig. 1. Rapid-scanning stopped-flow spectra for the reaction of OPDA and H2O2 catalyzed by Hb. Time interval between successive scans is 18 s. The insert picture is the kinetic curve (A t) of the reaction monitored at 425 nm. [Hb]=1.0 × 10 − 6 mol/l, [H2O2] =2.0 ×10 − 3 mol/l, [OPDA] = 4.0× 10 − 3 mol/l, 48°C.
K. Zhang et al. / Talanta 51 (2000) 179–186
182
Fig. 2. The spectra change of the intermediate in the reaction. Time interval between successive scans is 12 s. [Hb] = 1.0 × 10 − 5 mol/l, [H2O2] =1.0× 10 − 3 mol/l, [OPDA] = 4.0× 10 − 3 mol/l; 48°C. Table 1 Effectiveness of different catalysis studied, 48°Ca Catalyst Hb HRP bCD-hemin Hemin MnTCPP a
Concentration used (mol/l) 1×10−5 1×10−5 1×10−5 1×10−5 1×10−5
Product lmax (nm)
Intermediate lmax (nm)
Initial rate (min−1)
425 410 416 424 418
711 711 711
2.3045 1.8422 0.2231 0.0905 0.1569
/ /
[H2O2] =6×10−4 mol/l; [OPDA] =4×10−3 mol/l; pH = 5.0.
reaction rate in the first 0.2 min was the maximum and good linear relationship (r = 0.9999, between absorbance and time) was obtained. Hence, the initial rate was measured using the data recorded in the first 0.2 min. A weak absorption increased first and then decreased can be detected around 711 nm, which can be seen clearly in Fig. 2, in the presence of a higher concentration of Hb. It indicated that there existed an intermediate in the reaction process. It may be due to the combination between enzyme (E) and substrate (S) to form a complex ES, which can further dissociate to give E and the product of reaction.
3.2. Effecti6eness of different catalyst Hb, HRP, bCD-hemin, hemin and MnTCPP showed different degrees of peroxidatic activity on the OPDA–H2O2 system. The catalytic characteristics are presented in Table 1. If the catalytic effectiveness of Hb is expressed on a molar basis rather than in terms of unit weight, it has a higher catalytic activity than the HRP. As is evidence from the date of Table 1, the catalytic activity sequence of these iron-porphyrin complexes is: HbbCD-hemin hemin. Since the active centers of these three catalysts are the same iron-porphyrin, their differences in activity are mainly
K. Zhang et al. / Talanta 51 (2000) 179–186
dependent on their geometric control and conformation. The high catalytic effectiveness of Hb is determined by its specific structure. Hb has the natural quaternary structure with four subunits. Each subunit appears to be so arranged that its hydrophobic residues are buried in the interior of the folded structure, and the heme prosthetic group orientated in this hydrophobic pocket, correspondingly, the bulk of the hydrophilic residues are on the outside. This makes the assembly aqueous and also makes possible the existence of a large number of nonpolar bonds in the interior and these are probably the main factors in facilitating the oxidation rate of OPDA by H2O2 to form DAPN. Although per mole Hb contains four moles of heme is an important factor that contributes to the higher catalytic activity of Hb than HRP, if the rates are figured on per mol of heme, the average (1/4 Hb) catalytic activity is still much higher than that of Hemin and bCD-Hemin. While bCDhemin has three-dimensional structure and appropriate hydrophobic cavity, which can be taken as supermolecular receptor and coreceptors, it can bind substrate and catalyze the reaction more efficiently than its prosthetic group model hemin. However, as lacking the natural spatial structure as that of Hb, its including and recognizing effect is less than that of Hb. The importance of the conformation can also be seen from the produce of intermediate in the system. HRP, Hb and bCD-hemin all has specific spatial structures. These structures make the inclusion and recognition of the substrates successfully, so intermediates can be observed in the reaction processes. As for hemin or MnTCPP, they are simple natural or synthetic metalloporphyrins, has no spatial structure, so no intermediate can be observed in the stated conditions. In addition, it is worth to mention that the price of Hb is 500 times cheaper than that of HRP. There is little doubt that hemoglobin can be effectively substituted for HRP in the determination of H2O2 under the stated experimental conditions.
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3.3. Reaction order and reaction equation of hydrogen peroxide According to the principle of the initial rate method [22], with [OPDA] in 20 times more than that of [H2O2], changing the concentration of H2O2, different values of the initial rate r can be got. From the equation r=k [H2O2]a[OPDA]b, the reaction order a=1.01 was got. By the same method, we got b= 0.013, which can approximately be taken as 0th order reaction. That is, under the catalytic effect of Hb, reagents of the system showed a pseudo-first order reaction dependence, for which the following kinetic equation is proposed: r= k%[H2O2] where r is the initial rate of formation of DAPN and k% is the conditional rate constant.
3.4. Effect of 6ariables Peroxidatic activity of some pseudoperoxidases are dependent not only on the pH but also on the specific buffer used [10,23]. Although it is not widely recognized, this behavior is also common to HRP [24]. The dependence of the Hb-catalytic reaction kinetics on the buffer system is shown in Fig. 3. The differences are obviously. It may be due to the different ligation abilities of oxygenous ligands of acetate, citrate and phosphate, which affect the coordination of the substrate with the heme-ion to form the enzyme–substrate intermediate, and further affect the formation rate of the product DAPN. Na2HPO4 –citric acid buffer gave the largest initial rate among the buffers tested. The pH dependence of the system was studied over the range 3.8–7.0 in the presence of Na2HPO4 – citric acid buffer. The results are shown in Fig. 4. The optimum pH was found to between 4.8 and 5.3. The effect of the Na2HPO4 –citric acid buffer concentration was also tested keeping the same pH. The initial rate increased with increasing buffer capacity, reached the maximum after 0.15 mol/l, and had no significant change between 0.15 and 0.30 mol/l. So, a 0.2 mol/l, pH 5.0 Na2HPO4 –citric acid buffer was chosen for subsequent experiments.
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K. Zhang et al. / Talanta 51 (2000) 179–186
We studied the temperature effect at the condition of 1.5×10 − 5 mol/l Hb, 1.0×10 − 6 mol/l H2O2 and 2.0×10 − 3 mol/l OPDA. The higher the temperature is, the faster the final value can be attainted. The initial rate increases with the increasing temperature from 31 to 60°C. From the kinetic curve, a series of rate constant k of different temperature can be gotten. According to
Arrhenius equation Ln k= − Ea/RT + Ln A, plotting Ln k versus 1/T, a straight line was got and the calibration graph was: Ln k= (− 15919 38)/T + (5.929 0.12)(n = 7, r= 0.9988) The activation energy Ea = (13.239 0.22) kJ mol − 1 was got from it. Because a too high tem-
Fig. 3. The dependence of reaction kinetics on the buffer system used. pH = 5.0, [Hb] =1.5 ×10 − 6 mol/l, [H2O2]= 1.2 ×10 − 4 mol/l, [OPDA] =2.0× 10 − 3 mol/l.
Fig. 4. System dependence on pH, using 5.0 ×10 − 6 mol/l Hb, 1.0 ×10 − 6 mol/l H2O2 and 2.0× 10 − 3 mol/l OPDA; 48°C.
K. Zhang et al. / Talanta 51 (2000) 179–186 Table 2 Effect of foreign species on the determination of 2 mmol/l H2O2a Foreign species
Tolerated ratio
− BSA, SO2− 4 , NO3 2+ 2+ 3+ Ca , Mg , Al , Cu2+, Glucose, caffeine, theophylline, uric acid SO2− 3 NO− 2
2000 300 200 20 2.5
a
[Hb]=1.2×10−5 mol/l, [OPDA] = 4×10−3 mol/l, pH =
5.0.
perature would denature Hb and resulted in a poor precision of the determination, in this work, 48°C was chosen as the experimental temperature. With 4.0 × 10 − 6 Hb, 3.0 ×10 − 5 mol/l H2O2, the initial rate had no obvious change with the OPDA concentration increased from 1.0× 10 − 4 to 5.0× 10 − 3 mol/l, and 2.0 ×10 − 3 mol/l was chosen for the assay of hydrogen peroxide. The effect on the initial rate of Hb concentration between 1.0× 10 − 7 and 5.0 ×10 − 4 mol/l was investigated. The initial rate increased with increasing Hb concentrations, but at a too high concentration, the reproducibility of the signal is not ideal owning to the interference of the blank absorption from the high concentration of Hb. In order to ensure the sensitivity as well as the reproducibility, 1.2×10 − 5 mol/l Hb was recommended in the determination. Some inorganic ions and several organic compounds were investigated for their interference in this Hb-catalytic spectrophotometric method for H2O2 determination at a concentration of 2 mmol/ l. If the initial rate changed by more than 5%, the species was considered to have interference. The results are shown in Table 2.
3.5. System performance The calibration data for 5.0×10 − 8 –3.5× 10 − 6mol/l H2O2 are well described by the equation: Signal (initial rate) = (0.128392.28 ×10 − 3) +0.031[H2O2/10 − 7] (n =10, r = 0.9994)
185
The detection limit, defined as (3× SD of blank)/ (slope of analytical calibration) [25], was found to be 9.2× 10 − 8 mol/l. The reproducibility of the system, tested by repeated measurements on a 5.0× 10 − 7 mol/l sample (n= 11), was 2.08%.
3.6. Analytical application The applicability of this method was assessed by the determination of H2O2 in rainwater samples. Rainwater samples were collected at three different places, and were determined immediately after collecting. The values determined by this method of the three samples were: 3.3×10 − 6, 5.2× 10 − 6 and 9.5 × 10 − 6 mol/l, respectively. Percentage recoveries were measured after adding different amounts of H2O2 into the rainwater samples. Recoveries were given from 94.1 to 110.5%, indicating that the rainwater matrix did not significantly affect the detection.
Acknowledgements Authors acknowledge the financial support from National Science Foundation of China.
References [1] L.D. Bowers, Anal. Chem. 58 (1986) 513A. [2] Q.Z. Zhu, Q.G. Li, J.Z. Lu, J.G. Xu, Anal. Lett. 29 (1996) 1729. [3] B.J. Finlayson-Pitts, J.N. Pitts, Atmospheric Chemistry: Fundamentals and Atmospheric Techniques, Wiley, New York, 1986, p. 668. [4] P.A. Tanner, A.Y.S. Wong, Anal. Chim. Acta 370 (1998) 279. [5] D. Suznjevic, S. Blagojevic, D. Vucelic, P. Zuman, Electroanalysis 9 (1997) 861. [6] K. Schachl, H. Alemu, K. Kalcher, J. Jezkova, I. Svancara, K. Vytras, Analyst 122 (1997) 985. [7] Z.H. Liu, R.X. Cai, L.Y. Mao, H.P. Huang, W.H. Ma, Analyst 124 (1999) 173. [8] F. Elessi, A. Abuzuhri, S. Alkhalil, M. Abdellatif, Talanta 44 (1997) 2051. [9] M.L. Feng, Z. Li, J.R. Lu, H.J. Jiang, Mikrochim. Acta 126 (1997) 73. [10] G. Zhang, P.K. Dasgupta, Anal. Chem. 64 (1992) 517. [11] M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Chem. Rev. 96 (1996) 2841.
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[12] Y. Saito, S. Nakashima, M. Mifune, J. Odo, Y. Tanaka, M. Chikuma, H. Tanaka, Anal. Chim. Acta 172 (1985) 285. [13] Y. Saito, M. Mifune, S. Nakashima, J. Odo, Y. Tanaka, M. Chikuma, H. Tanaka, Talanta 34 (1987) 667. [14] Y.X. Ci, F. Wang, Mikrochim. Acta 1 (1990) 63. [15] Y.X. Ci, F. Wang, Talanta 37 (1990) 1133. [16] X.M. Huang, M. Zhu, L.Y. Mao, H.X. Shen, Anal. Sci. 13 (1997) 145. [17] L. Martin, M.A. Martin, B. Castilo, Analyst 122 (1997) 45. [18] T. Jing, D.S. Lawrence, J. Am. Chem. Soc. 117 (1995) 1857.
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[19] R. Breslow, Acc. Chem. Res. 28 (1995) 146. [20] K. Zhang, R.X. Cai, H.P. Huang, Z.H. Liu, Anal. Lett. 31 (1998) 1337. [21] L.Y. Mao, M. Zhu, H.X. Shen, Chem. J. Chin. Univ. 18 (1997) 1611. [22] J.W. Moor, R.G. Pearson, Kinetics and Mechanism, 3rd edn, Wiley, New York, 1981, p. 77. [23] Y. Saito, M. satouchi, M. Mifune, T. Tai, J. Odo, Y. Tanaka, M. Chikuma, H. Tanaka, Bull. Chem. Soc. Jpn. 60 (1987) 2227. [24] H. Hwang, P.K. Dasgupta, Anal. Chim. Acta 170 (1985) 347. [25] J.D. Winefordner, G.L. Long, Anal. Chem. 55 (1983) 712A.
Stopped-flow spectrophotometric determination of hydrogen peroxide with hemoglobin as catalyst Ke Zhang, Luyuan Mao, Ruxiu Cai * Department of Chemistry, Wuhan Uni6ersity, Wuhan 430072, PR China Received 13 July 1999; received in revised form 7 September 1999; accepted 8 September 1999
Abstract A rapid and sensitive method was proposed for the determination of hydrogen peroxide based on the catalytic effect of hemoglobin using o-phenylenediamine as the substrate. Stopped-flow spectrophotometric method was used to study the kinetic behavior of the oxidation reaction. The catalytic effectiveness of hemoglobin was compared with other four kinds of catalysts. The initial rate of the formation of the reaction product 2,3-diaminophenazine at the wavelength of 425 nm was monitored, permitting a detection limit of 9.2× 10 − 9 mol/l H2O2. A linear calibration graph was obtained over the H2O2 concentration range 5.0 ×10 − 8 – 3.5×10 − 6 mol/l, and the relative standard deviation at a H2O2 concentration of 5.0 × 10 − 7 mol/l was 2.08%. Satisfied results were obtained in the determination of H2O2 in real samples by this method. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Stopped-flow spectrophotometry; Hydrogen peroxide; Hemoglobin
1. Introduction Hydrogen peroxide is a very important intermediate in clinical biochemistry [1,2] and environmental work [3,4]. Hydrogen peroxide is produced in stoichiometric amounts during the oxidation of substrates of clinical interest by dissolved oxygen in the presence of the corresponding oxidase enzyme. Presently, H2O2 is also an analyte of environmental interest for its involving in catalyzed and uncatalyzed oxidation of organic species. Therefore, a rapid, sensitive and inexpensive * Corresponding author. Tel.: + 86-27-87684184; fax: +8627-87647617. E-mail address: cai – [email protected] (R. Cai)
method is required for the determination of hydrogen peroxide at levels below submicromolar limit of determination. Numerous studies on the determination of hydrogen peroxide using polarography, amperometry, fluorimetry, spectrophotometry and chemiluminescence, etc., have been published [5–9]. Horseradish peroxidase (HRP), in which the heme iron acts as the activity center, is the most commonly used enzyme in H2O2 detection. However, HRP has its shortcomings. For example, it is expensive and unstable in solution, and has strict requirements for the experimental conditions. Therefore, the search for a replacement for HRP has been an important work. Hemin [2] and hematin [10] have been reported as a peroxidase
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K. Zhang et al. / Talanta 51 (2000) 179–186
substitute in the detection of H2O2. Some metalloporphyrins show the similar catalytic characteristics as that of peroxidase, so the peroxidase mimesis is one of the interesting trends in enzymatic analysis [11]. Saito et al. synthesized Mn (III)-tetrakis (sulfophenyl) porphine and found it can be immobilized on anion exchange resins and mimicked the behavior of immobilized HRP [12,13]. The porphyrin complexes of Mn, Co, Fe [14,15] and Mo [16] all exhibited peroxidase activity in aqueous phase. However, simple natural or synthetic metalloporphyrins do not show satisfactory activity and selectivity because they lack the spatial structure of the natural enzyme, which is essential for the special inclusion behavior between the enzyme and the substrate. A number of polymers and receptor compounds derived from b-CD are widely used in analytical chemistry and biomimetic chemistry based on their supramolecular features. [7,17 – 19]. Hemoglobin (Hb), a necessary vehicle for oxygen carriage, has the natural quaternary structure. It contains four subunits of polypeptide and each polypeptide chain contains a heme group that serves as the active center. Encouraged by reports on the studies of peroxidase substitutes and the natural spatial structure of Hb, as well as its cheaper price, we expected Hb be an excellent enzyme for catalyzing the reaction between ophenylenediamine (OPDA, i.e. 1,2-diaminobenzene) and hydrogen peroxide to form 2,3-diaminophenazine (DAPN). The reaction is as follows:
scanning UV/Vis spectrophotometer and controlled by a microcomputer for operation and data processing. The catalytic reaction was studied by measuring the increasing absorbance DAPN at 425 nm to obtain the initial reaction rate. It was found that the initial reaction rate was proportional to the concentration of H2O2 over the range 5.0×10 − 8 –3.5×10 − 6 mol/l. The catalytic activity of Hb has been compared with HRP, bCD-hemin, hemin and MnTCPP, and it was found that the catalytic effectiveness of Hb is much higher than bCD-hemin, hemin and MnTCPP, and even a little higher than HRP in the conditions of this work. The method is rapid, simple, sensitive and reliable for the determination of H2O2.
Stopped-flow method has been widely recognized as a powerful technique to monitor rapid reactions. The coupling of the stopped-flow technique with rapid scanning UV/Vis spectrophotometer is effective for investigating the mechanism of rapid reaction [20]. In the present work, the stopped-flow technique was coupled with a rapid
with buffer to the desired volume. Hydrogen peroxide solutions were prepared by appropriate dilution of the 30% solution with water (standardized by titration with KMnO4). b-cyclodextrin (bCD) was purchased from Sigma and tetra (p-canboxyphenyl) porphine (TCPP) was purchased from Aldrich. bCD-hemin and
2. Experimental
2.1. Reagents Unless indicated otherwise, all dilutions were made in 0.2 mol /l Na2HPO4 –citric acid buffer (pH=5.0). Hemoglobin (bovine erythrocytes) solution was prepared by dissolving certain amount of Hb (Shanghai Institute of Biochem., Shanghai, China) in deionized water and stored below 4°C. O-phenylenediamine (Shanghai Chem. Agent, Inc., Shanghai, China) was sublimed before use. 0.05 mol/l of o-phenylenediamine stock solution was freshly prepared by dissolving sublimed OPDA in buffer solution, and the working solution was prepared by diluting the stock solution
K. Zhang et al. / Talanta 51 (2000) 179–186
MnTCPP were synthesized using the reported method [7,21]. HRP (E.C. 1.11.1.7, 250 IU mg − 1, RZ 3, purchased from Sino-Amer. Biotech. Co.) was prepared in water solution. Distilled, de-ionized water was used throughout. All other chemicals were of analytical-reagent grade.
2.2. Apparatus SFA-12 HI-TECH scientific kinetic stoppedflow accessory (HI-TECH, Japan) was fitted to UVIKON-941 spectrophotometer (Kontron Instrument). A circulation water-bath was used to control the temperature in the stopped-flow module and the cell compartment. A microcomputer is applied to control the operation of the instrument and to collect and process the data.
2.3. Procedure A solution of Hb and a sample solution containing H2O2 and OPDA were filled into two separate reservoir syringes. At each determina-
181
tion, two flow streams were merged and stopped in the 1-cm flow-through absorption cell and the oxidation reaction was monitored by the absorption at 425 nm. The initial reaction rate was calculated using the data collected in the first 0.2 min of the reaction. Because of the two-fold dilution during the mixing all concentrations reported are twice as much as the actual initial concentrations after mixing.
3. Result and discussion
3.1. Spectra characteristics of the reaction system Fig. 1 shows the rapid-scanning stopped-flow spectra during the reaction process of OPDA and H2O2 to produce DAPN. Due to formation of the reaction product, DAPN, an absorption band appeared at 425 nm and increased continuously during the process scans from 1 to 8. The insert picture of Fig. 1 is the kinetic curve monitored at 425 nm in the first minute of the reaction. The
Fig. 1. Rapid-scanning stopped-flow spectra for the reaction of OPDA and H2O2 catalyzed by Hb. Time interval between successive scans is 18 s. The insert picture is the kinetic curve (A t) of the reaction monitored at 425 nm. [Hb]=1.0 × 10 − 6 mol/l, [H2O2] =2.0 ×10 − 3 mol/l, [OPDA] = 4.0× 10 − 3 mol/l, 48°C.
K. Zhang et al. / Talanta 51 (2000) 179–186
182
Fig. 2. The spectra change of the intermediate in the reaction. Time interval between successive scans is 12 s. [Hb] = 1.0 × 10 − 5 mol/l, [H2O2] =1.0× 10 − 3 mol/l, [OPDA] = 4.0× 10 − 3 mol/l; 48°C. Table 1 Effectiveness of different catalysis studied, 48°Ca Catalyst Hb HRP bCD-hemin Hemin MnTCPP a
Concentration used (mol/l) 1×10−5 1×10−5 1×10−5 1×10−5 1×10−5
Product lmax (nm)
Intermediate lmax (nm)
Initial rate (min−1)
425 410 416 424 418
711 711 711
2.3045 1.8422 0.2231 0.0905 0.1569
/ /
[H2O2] =6×10−4 mol/l; [OPDA] =4×10−3 mol/l; pH = 5.0.
reaction rate in the first 0.2 min was the maximum and good linear relationship (r = 0.9999, between absorbance and time) was obtained. Hence, the initial rate was measured using the data recorded in the first 0.2 min. A weak absorption increased first and then decreased can be detected around 711 nm, which can be seen clearly in Fig. 2, in the presence of a higher concentration of Hb. It indicated that there existed an intermediate in the reaction process. It may be due to the combination between enzyme (E) and substrate (S) to form a complex ES, which can further dissociate to give E and the product of reaction.
3.2. Effecti6eness of different catalyst Hb, HRP, bCD-hemin, hemin and MnTCPP showed different degrees of peroxidatic activity on the OPDA–H2O2 system. The catalytic characteristics are presented in Table 1. If the catalytic effectiveness of Hb is expressed on a molar basis rather than in terms of unit weight, it has a higher catalytic activity than the HRP. As is evidence from the date of Table 1, the catalytic activity sequence of these iron-porphyrin complexes is: HbbCD-hemin hemin. Since the active centers of these three catalysts are the same iron-porphyrin, their differences in activity are mainly
K. Zhang et al. / Talanta 51 (2000) 179–186
dependent on their geometric control and conformation. The high catalytic effectiveness of Hb is determined by its specific structure. Hb has the natural quaternary structure with four subunits. Each subunit appears to be so arranged that its hydrophobic residues are buried in the interior of the folded structure, and the heme prosthetic group orientated in this hydrophobic pocket, correspondingly, the bulk of the hydrophilic residues are on the outside. This makes the assembly aqueous and also makes possible the existence of a large number of nonpolar bonds in the interior and these are probably the main factors in facilitating the oxidation rate of OPDA by H2O2 to form DAPN. Although per mole Hb contains four moles of heme is an important factor that contributes to the higher catalytic activity of Hb than HRP, if the rates are figured on per mol of heme, the average (1/4 Hb) catalytic activity is still much higher than that of Hemin and bCD-Hemin. While bCDhemin has three-dimensional structure and appropriate hydrophobic cavity, which can be taken as supermolecular receptor and coreceptors, it can bind substrate and catalyze the reaction more efficiently than its prosthetic group model hemin. However, as lacking the natural spatial structure as that of Hb, its including and recognizing effect is less than that of Hb. The importance of the conformation can also be seen from the produce of intermediate in the system. HRP, Hb and bCD-hemin all has specific spatial structures. These structures make the inclusion and recognition of the substrates successfully, so intermediates can be observed in the reaction processes. As for hemin or MnTCPP, they are simple natural or synthetic metalloporphyrins, has no spatial structure, so no intermediate can be observed in the stated conditions. In addition, it is worth to mention that the price of Hb is 500 times cheaper than that of HRP. There is little doubt that hemoglobin can be effectively substituted for HRP in the determination of H2O2 under the stated experimental conditions.
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3.3. Reaction order and reaction equation of hydrogen peroxide According to the principle of the initial rate method [22], with [OPDA] in 20 times more than that of [H2O2], changing the concentration of H2O2, different values of the initial rate r can be got. From the equation r=k [H2O2]a[OPDA]b, the reaction order a=1.01 was got. By the same method, we got b= 0.013, which can approximately be taken as 0th order reaction. That is, under the catalytic effect of Hb, reagents of the system showed a pseudo-first order reaction dependence, for which the following kinetic equation is proposed: r= k%[H2O2] where r is the initial rate of formation of DAPN and k% is the conditional rate constant.
3.4. Effect of 6ariables Peroxidatic activity of some pseudoperoxidases are dependent not only on the pH but also on the specific buffer used [10,23]. Although it is not widely recognized, this behavior is also common to HRP [24]. The dependence of the Hb-catalytic reaction kinetics on the buffer system is shown in Fig. 3. The differences are obviously. It may be due to the different ligation abilities of oxygenous ligands of acetate, citrate and phosphate, which affect the coordination of the substrate with the heme-ion to form the enzyme–substrate intermediate, and further affect the formation rate of the product DAPN. Na2HPO4 –citric acid buffer gave the largest initial rate among the buffers tested. The pH dependence of the system was studied over the range 3.8–7.0 in the presence of Na2HPO4 – citric acid buffer. The results are shown in Fig. 4. The optimum pH was found to between 4.8 and 5.3. The effect of the Na2HPO4 –citric acid buffer concentration was also tested keeping the same pH. The initial rate increased with increasing buffer capacity, reached the maximum after 0.15 mol/l, and had no significant change between 0.15 and 0.30 mol/l. So, a 0.2 mol/l, pH 5.0 Na2HPO4 –citric acid buffer was chosen for subsequent experiments.
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We studied the temperature effect at the condition of 1.5×10 − 5 mol/l Hb, 1.0×10 − 6 mol/l H2O2 and 2.0×10 − 3 mol/l OPDA. The higher the temperature is, the faster the final value can be attainted. The initial rate increases with the increasing temperature from 31 to 60°C. From the kinetic curve, a series of rate constant k of different temperature can be gotten. According to
Arrhenius equation Ln k= − Ea/RT + Ln A, plotting Ln k versus 1/T, a straight line was got and the calibration graph was: Ln k= (− 15919 38)/T + (5.929 0.12)(n = 7, r= 0.9988) The activation energy Ea = (13.239 0.22) kJ mol − 1 was got from it. Because a too high tem-
Fig. 3. The dependence of reaction kinetics on the buffer system used. pH = 5.0, [Hb] =1.5 ×10 − 6 mol/l, [H2O2]= 1.2 ×10 − 4 mol/l, [OPDA] =2.0× 10 − 3 mol/l.
Fig. 4. System dependence on pH, using 5.0 ×10 − 6 mol/l Hb, 1.0 ×10 − 6 mol/l H2O2 and 2.0× 10 − 3 mol/l OPDA; 48°C.
K. Zhang et al. / Talanta 51 (2000) 179–186 Table 2 Effect of foreign species on the determination of 2 mmol/l H2O2a Foreign species
Tolerated ratio
− BSA, SO2− 4 , NO3 2+ 2+ 3+ Ca , Mg , Al , Cu2+, Glucose, caffeine, theophylline, uric acid SO2− 3 NO− 2
2000 300 200 20 2.5
a
[Hb]=1.2×10−5 mol/l, [OPDA] = 4×10−3 mol/l, pH =
5.0.
perature would denature Hb and resulted in a poor precision of the determination, in this work, 48°C was chosen as the experimental temperature. With 4.0 × 10 − 6 Hb, 3.0 ×10 − 5 mol/l H2O2, the initial rate had no obvious change with the OPDA concentration increased from 1.0× 10 − 4 to 5.0× 10 − 3 mol/l, and 2.0 ×10 − 3 mol/l was chosen for the assay of hydrogen peroxide. The effect on the initial rate of Hb concentration between 1.0× 10 − 7 and 5.0 ×10 − 4 mol/l was investigated. The initial rate increased with increasing Hb concentrations, but at a too high concentration, the reproducibility of the signal is not ideal owning to the interference of the blank absorption from the high concentration of Hb. In order to ensure the sensitivity as well as the reproducibility, 1.2×10 − 5 mol/l Hb was recommended in the determination. Some inorganic ions and several organic compounds were investigated for their interference in this Hb-catalytic spectrophotometric method for H2O2 determination at a concentration of 2 mmol/ l. If the initial rate changed by more than 5%, the species was considered to have interference. The results are shown in Table 2.
3.5. System performance The calibration data for 5.0×10 − 8 –3.5× 10 − 6mol/l H2O2 are well described by the equation: Signal (initial rate) = (0.128392.28 ×10 − 3) +0.031[H2O2/10 − 7] (n =10, r = 0.9994)
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The detection limit, defined as (3× SD of blank)/ (slope of analytical calibration) [25], was found to be 9.2× 10 − 8 mol/l. The reproducibility of the system, tested by repeated measurements on a 5.0× 10 − 7 mol/l sample (n= 11), was 2.08%.
3.6. Analytical application The applicability of this method was assessed by the determination of H2O2 in rainwater samples. Rainwater samples were collected at three different places, and were determined immediately after collecting. The values determined by this method of the three samples were: 3.3×10 − 6, 5.2× 10 − 6 and 9.5 × 10 − 6 mol/l, respectively. Percentage recoveries were measured after adding different amounts of H2O2 into the rainwater samples. Recoveries were given from 94.1 to 110.5%, indicating that the rainwater matrix did not significantly affect the detection.
Acknowledgements Authors acknowledge the financial support from National Science Foundation of China.
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