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Continuous-flow potentiometric determination of horseradish peroxidase with a fluoride-selective electrode
Analytica Chimica Acta, 208 (1988) 173-181 Elsevier Science Publishers B.V.. Amsterdam -
173 Printed in The Netherlands
CONTINUOUS-FLOW POTENTIOMETRIC DETERMINATION OF HORSERADISH PEROXIDASE WITH A FLUORIDE-SELECTIVE ELECTRODE
PETER W. ALEXANDER*
and CARMELITA MAITRA
School of Chemistry, University of New South Wales, P.O. Box 1, Kensington, New South Wales (Australia) (Received 9th September 1987)
SUMMARY A continuous-flow system is reported for the determination of horseradish peroxidase (HRP) with a fluoride-selective electrode. Reaction conditions are optimized for the HRP-catalysed oxidation ofp-fluoroaniline (0.104 M) with hydrogen peroxide (4.0 mM) as the oxidant in 0.16 M acetate buffer at pH 4.6. HRP is determined in the concentration range 0.016-0.12 U ml-’ in the flow system at a sampling rate of 24 h-i. Interfering effects caused by known HRP inhibitors, including metal ions, cyanide and sulphide, are reported for the range 1 PM-1 mM. Applications of the system for determination of the enzyme in turnip extract and milk are described.
The use of the fluoride-selective electrode for the detection of horseradish peroxidase (HRP) with p-fluoroaniline as the hydrogen donor for enzyme immunoassay was reported by Alexander and Maitra [ 11. Since then, several publications and patents have appeared in which the fluoride electrode was used in conjunction with various hydrogen donors [2], including p-fluorophenol, for the determination of glucose, cholesterol and glucose oxidase [ 2-41, and in enzyme immunoassays [ 5,6]. However, the experimental conditions affecting the rate of the HRP-catalysed reaction, and inhibition factors, have not been studied in detail. Determinations of HRP are usually based on the spectrophotometric or fluorimetric measurement of the initial increase in absorbance or fluorescence of the oxidized donor. Many donors have been used. Other techniques include potentiometric, enthalpimetric and potentiostatic methods [ 71. An immunospecific enzyme assay involving the use of an anti-HRP antibody has also been reported [ 81. Interference effects, sensitivity, precision and accuracy with these methods depend mainly on the choice of hydrogen donor. With some hydrogen donors, such as pyrogallol, guiaicol and p-phenylenediamine, the spectrophotometric method is inhibited by traces of metal ions [ 9,101. Similar interference is known for the fluorimetric method with, for example, homovanillic acid as donor [ 111. The potentiometric method [ 121 with an iodide-selective elec-
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers B.V.
174
trode as the sensor and iodide as donor is not specific for HRP because many other substances can oxidize iodide to iodine [ 9,101, and the method is unsuitable for protein-containing samples because HRP also catalyzes the iodination of proteins [ 121. In this report, the development and optimization of experimental parameters in a continuous-flow system with a fluoride-selective electrode as detector and p-fluoroaniline as hydrogen donor are described for the determination of HRP at concentrations as low as 0.001 U ml-‘. HRP activities were determined in aqueous solutions, in turnip extract and in milk, based on the continuous-flow detection of the liberated fluoride after the enzymatic cleavage of the C-F bond inp-fluoroaniline. The method is selective because HRP has the ability to cleave a C-F bond under mild reaction conditions [ 10,131 and because the fluoride electrode is very selective for fluoride [ 141. However, interfering effects are shown to be caused by many inhibition factors which alter the rate of the HRP-catalysed reaction. EXPERIMENTAL
Instrumentation, reagents and solutions Potentiometric measurements were made with an Orion pH/mV meter Model 701A coupled to a Mace FBQ-100 strip-chart recorder. The electrodes were an Orion fluoride-selective electrode (Model 94-09) and an Orion double-junction reference electrode (Model 90-02) with 10% (w/v) potassium nitrate in the outer compartment. Solution pH was measured with an Activon digital pH/mV meter fitted with a combination pH glass electrode. The constanttemperature bath was of the circulating type (B.T.L. Long Reach Circon, England; Model 69/3926/0182). Spectrophotometric studies were done with a Zeiss PMQ-II spectrophotometer. Grade II horseradish peroxidase (donor: hydrogen peroxide oxidoreductase ) of specific activity 100 U mg-l, lot No. 1259124, was obtained from Boehringer-Mannheim Corp. Stock solutions of 27.7 U ml-’ were prepared in glassdistilled water and stored at 4’ C. Solutions remain stable for over a year [ 71 if stored as such. The p-fluoroaniline was obtained from Sigma Chemical Co. For routine analysis, a 0.104 M p-fluoroaniline solution was prepared in a 0.16 M sodium acetate/acetic acid buffer pH 4.6. Hydrogen peroxide (Pacific Manufacturing, Sydney, Australia; 30% (w/v) hydrogen peroxide, 1.1 g ml-’ specific gravity) was used at a concentration of 4.0 mM in distilled water. Other concentrations of hydrogen peroxide in the range 0.1-4.0 mM were used for the optimization studies. Diluted enzyme solutions, p-fluoroaniline in the acetate buffer and other solutions required were prepared freshly on the day of measurement. The milk
175
sample was a 10% (w/v) solution of skimmed milk powder. Fresh turnips were purchased from a vegetable market. Procedures The flow system used for the studies with HRP (Fig. 1) was as described earlier [ 11. A Watson-Marlow type proportioning pump fitted with a rapid speed motor (100 rpm) was used to pump solutions through the individual pump channels at the flow rates shown. The electrode flow-through cell was as previously described [ 151. The baseline potential was set by pumping the bufferedp-fluoroaniline solution (0.104 M), hydrogen peroxide (4.5 mM) and the water wash into the flow system with air-segmentation of the stream at a constant flow rate. The stream was debubbled just prior to the flow cell. A calibration plot for standard fluoride solutions was first measured to test the response of the fluoride electrode in the continuous-flow system in the presence of the acetate buffer pH 5.6. Peak heights corresponding to various concentrations of fluoride (0.01-10 mM) were plotted against log molarity. The reaction parameters for the assay of HRP were studied by passing water, hydrogen peroxide and buffered p-fluoroaniline through the system to establish the baseline and then passing HRP sample solutions (1.4 x 10m3-1.7 x 10-l U ml-‘) through the sample line for 30 s, followed by a wash for 4 min or as required for the peak to return to baseline. The HRP sample solutions were prepared by diluting 5-600 ~1 of stock (27.7 U ml-l) HRP solution to 100.0 ml. Optimization studies. To examine the effect of HRP reaction time, the length
_
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waste
’ ” , 3.316.8 _
i r\ I air l.Sl3.4
/ “, I r\ ’!~ ;” !~
I sample or wash ; 2.Ll6.0 1substrate ; 2415.1 ! buffer and donor / I420
La -I 2.L’6.0
Fig. 1. Schematic diagram of the flow system used: (a) indicator electrode; (b) reference electrode; (c) flow-through cell; (d) proportioningpump; (e) mV/pH meter; (f) recorder; (g) mixing coils; (h) debubbler; (i) buffer solution. Flow rates are given in ml min- ‘: the larger flow rates are used for HRP determination, and the lower flow rates for calibration of the electrode.
176
of the mixing coils (g, and g,, Fig. 1) were varied. For the study of different substrate concentrations, calibration plots of HRP concentration against potential were constructed for various concentrations (1.8-44.0 mM) of hydrogen peroxide. Calibration plots for HRP at various fixed p-fluoroaniline concentrations (0.21, 0.042, 0.104 M) were also established. To evaluate the effects of pH and buffer concentration on the HRP calibration plot, buffers of various pH and concentrations were pumped through the buffer channel (Fig. 1). For interference studies, Co2+, Fe3+, Cu2+, S2- and CN- were individually mixed with the HRP sample before its introduction into the system. The effect of temperature was examined by varying the temperature (16-50’ C ) of the water circulating through the jacketted mixing coil (82) in Fig. 1. Analysis offood samples. Samples of turnip extract and milk containing HRP were analysed in the above flow system. The turnip samples were prepared as described by Mann and Saunders [ 161: fresh turnips were washed, weighed, chopped and blended in a Waring blender; the mass was filtered through cheesecloth, and then through a sintered-glass Buchner funnel and the filtrate was diluted to 11. A lO.O-ml aliquot of the extract was diluted to 1 1 and analyzed by standard addition in the flow system; Gran plots were used for data evaluation. The samples were also analysed by using the spectrophotometric method of Luck [ 171. The sample solutions (2.0 ml of turnip extract), 1 ml of phosphate buffer (0.067 M Na2HP0,/KH2POd, pH 7.0), 0.1 ml of hydrogen peroxide solution (3.0 mM) and 0.1 ml ofp-phenylenediamine solution (l%, w/v) were mixed thoroughly in a cuvette and the absorbance was read at 485 nm every 30 s for 3 min. The control cuvette was prepared similarly, except that distilled water was used instead of hydrogen peroxide. Initial rates were plotted as a function of concentration to obtain calibration or standard addition plots. In this technique, standard addition was the only viable method because erratic results were obtained when the data for the turnip sample were compared directly with a calibration plot. For the electrode method, however, both calibration and standard addition plots were satisfactory. Milk was tested to investigate the effect of protein on the percentage recovery of HRP. Various volumes (50-400 ~1) of stock HRP were added to 100.0 ml of milk (lo%, w/v); the peak heights corresponding to these solutions were evaluated from the usual calibration plot. RESULTS AND DISCUSSION
The dynamic response of the fluoride electrode to fluoride in a continuousflow system has been reported by many authors (see, e.g. [X3-21] ) . Here, the sensitivity of the electrode response for determination of HRP was optimized by studying the effect of temperature, buffer composition, pH, substrate and hydrogen donor concentrations. The effect of several substances known to in-
117
terfere with HRP detection was studied and finally the flow method was applied for the analysis of food samples. Optimizatioti of the HRP reaction Initially, the electrode response in the flow system was calibrated against aqueous fluoride standards in the range 0.01-100.0 mM. Slow response was observed but a near-Nernstian slope of - 57 mV pF-’ was obtained after plotting the relevant peak heights. Measurements were done at 26’ C. The response of the electrode in the flow system for the HRP-catalyzed reaction is shown in Fig. 2. The response was again slow and showed peak widths at baseline of ca. 4 min. The response is attributed [ 131 to the reaction: H-donor + Hz O2-Products+H++F-+H,O
(I)
where the hydrogen donor is p-fluoroaniline and the oxidation products are 2amino-p-fluoroanilinobenzoquinone and tetra-p-fluoroazophene. The production of fluoride is relatively fast [ 221 and the rate of appearance of fluoride ion is proportional to the HRP activity. Calibration plots are shown in Fig. 3, corresponding to the initial experimental conditions (A) used in the flow system, and after modification (B) where it can be seen that as little as 0.001 U ml-’ HRP can be detected. Conditions used to obtain the data for curve C gave optimum linearity and were used in subsequent studies. These results were obtained after the optimization study outlined below. Effect of incubation time, temperature, buffer and PH. The optimum incu-
Fig. 2. Continuous-flow output for the determination of HRP with the fluoride electrode at 24 samples h-l. (A) Peaks for HRP samples: (a) 0.139, (b) 0.278, (c) 0.554, (d) 1.39, (e) 2.78, (f) 5.54, (g) 11.1, (h) 16.6~ lo-’ U ml-’ HRP. (B) Steady-state signal for 0.11 U ml-’ HRP. (C) Replicates for 0.11 U ml-’ HRP. Conditions: 8.9 mM hydrogen peroxide, 0.104 Mp-fluoroaniline in 0.5 M acetate buffer, pH 4.7,16”C. Fig. 3. Calibration plots for HRP corresponding to the following conditions: (A) 0.104 Mp-fluoroaniline in 0.5 M acetate buffer, pH 4.7, 8.9 mM hydrogen peroxide, 16°C; (B) 0.104 M pfluoroaniline in 0.16 M acetate buffer, pH 4.6,4.45 mM hydrogen peroxide, 37°C; (C) as for (B) but at 45°C.
178
bation time for the reaction was obtained by varying the length of the delay coils in the flow system. An incubation time of 60 s was chosen in order to allow sufficient time for the reaction to reach completion. Incubation at temperatures between 19 and 36°C gave useful reaction rates. A marked decrease in response was obtained at 5O”C, probably because of deactivation of the enzyme. Figure 4 shows the effect of pH on the rate of catalytic release of fluoride. The use of a buffer is necessary to maintain enzyme activity and to give reliable electrode response. Below pH 4.5, fluoride exists as HF or HF,- and above pH 8.0 hydroxide interferes with the response [ 141. Here, the choice of pH is limited by the optimum pH for maximum activity of the HRP which is generally considered to be pH 7.0 [23] but others maintain to be pH 4.8 [24]. The optimum pH depends on the nature of the hydrogen donor. In the case of pfluoroaniline, a slightly acidic pH is required for its dissolution in aqueous solutions. The results in Fig. 4 show that the sensitivity was greatest at pH 4.6. It was also found that an increase in the concentration of the buffer decreased the sensitivity. Effect of substrate and donor concentration. Figure 5 shows an increase in the sensitivity for the determination of HRP at low hydrogen peroxide concentration, in agreement with previous studies [ 25,26 J. Hydrogen peroxide concentrations < 10.0 mM are essential because an excess of hydrogen peroxide is known to inactivate HRP [lo]. An optimum concentration of 4.5 mM was used in subsequent determinations. Figure 6 shows that an increase in the hydrogen
Fig. 4. Plots of peak height against pH for various HRP activities ( X lo-’ U ml-‘): (A) 0.278, (B) 0.554, (C) 1.39, (D) 2.78, (E) 5.54, (F) 11.1. The various pH values were provided by the following buffers: (1) 0.05 M acetate, pH 4.73; (2) 0.16 M acetate, pH 4.55; (3) 0.25 M acetate, pH 4.49; (4) 0.4 M acetate with pH values of 4.42,4.99,6.17; (5) 0.1 M KH2P04/NaOH, pH 6.29. Conditions: 0.106 M p-fluoroaniline in the buffer, 4.45 mM HzOz, 15’ C. Fig. 5. Plots of peak height against log[ H,O,] for different HRP activities ( X lo-* U ml): (A) 0.554, (B) 1.39, (C) 2.77, (D) 5.54, (E) 11.1. Conditions: 0.104 M p-fluoroaniline in 0.16 M acetate buffer, pH 4.6,15”C.
179
Fig. 6. Effect of p-fluoroaniline concentration on the electrode response for different HRP activities ( x lo-‘U ml-‘): (A) 1.38, (B) 2.77, (C) 4.20. Conditions: 0.5 M acetate buffer, pH 4.7,8.9 mM Hz02, 15’ C. Fig. 7. Effect of inhibitor concentration on the electrode response for 0.014 U ml-’ HRP for different inhibitors in the concentration range 0.001-1.0 mM: (A) Cu*+, (B) CN-, (C) S*-, (D) Fe3+, (E) Co*+. Conditions as for curve A, Fig. 3.
donor concentration in the range 0.016-0.10 M results in increased sensitivity for the HRP determination. An optimum concentration of 0.104 M p-fluoroaniline in 0.16 M acetate buffer was shown to give maximum sensitivity. Effect of inhibitors The effect of inhibitors on the rate of the HRP-catalysed reaction was tested. Inhibition by various ionic species including Cu2+, Co2+, Fe3+, cyanide and sulphide ions has been reported previously [ 10,111. In this study, little effect from Cu2+ , Fe3+ and Co2+ on the HRP calibration plot was observed but considerable inhibition was found for cyanide and sulphide (Fig. 7). Interferences of Cu2+, Fe3+ and Co2+ ions have been reported [ 111 for a peroxidase system. However, Cu2+, Fe3+ and Co2+ inhibit peroxidase activity only in the presence of certain hydrogen donors [lo], e.g., Cu2+ with guaiacol, and many other metals are known to interfere with the p-phenylenediamine reaction with hydrogen peroxide [25,26]. The key to the selectivity of this system, therefore, is the ability of HRP to cleave the C-F bond in the p-fluoroaniline donor. Cyanide and sulphide cause strong inhibition whatever the method of assay, because they bind with HRP at the active site, causing inactivation [lo]. Analysis of food samples The continuous-flow system was applied in the analysis of some food samples for HRP. The samples chosen were turnip extracts and milk, treated ini-
180 TABLE 1 Recoveries of HRP added to milk samples diluted 1: 50 HRP added ’ (10-2U ml-‘) Recovered (10-2U ml-‘) Recovery ( % )
1.39
2.77
5.54
11.1
1.54 111
2.84 102
5.53 100
11.9 107
tially to produce appropriate dilutions and then introduced into the flow system shown in Fig. 1 at a sampling rate of 12 h-l. The turnip extract was also analysed by the conventional spectrophotometric method [ 171. The HRP content found in the turnip extracts, diluted 1: 100, was 1.8 2 0.3 x lo-’ U ml-’ (n= 3) by the proposed method and 1.8~ 10m2 U ml-’ by the spectrophotometric method. The recovery study after standard addition of HRP to milk, however, gave some high results (Table 1); the high protein content of milk may have caused the high recoveries but the reasons remain unclear. Interferences in HRP-catalysed reactions may occur in several ways. The detection system and/or the reaction scheme may suffer from interference effects. Unlike spectrophotometry, the electrode method is oblivious to sample colour and turbidity. With the fluoride electrode, interference with the detector response is minimal. HRP is not donor-specific, and so interferences with the reaction scheme depend on the donor used. Normally, a donor is chosen on the basis of the uniqueness of the product of the HRP-catalyzed reaction with hydrogen peroxide such as the reaction [ 10,131 of p-fluoroaniline with hydrogen peroxide to produce fluoride ion. However, HRP is substrate-specific to hydrogen peroxide, so that any substance able to react with hydrogen peroxide may interfere with the present technique, or with any other HRP assay for that matter.
REFERENCES P. W. Alexander and C. Maitra, Anal. Chem., 54 (1982) 68. I. W. Siddiqi, Clin. Chem., 28 (1982) 1962. M. H. Ho and T. G. Wu, ISA Trans., 24 (1985) 61. M. H. Ho and T. G. Wu, Ann. N. Y. Acad. Sci., 434 (1984) 523. R. Lauterburg, Eur. Pat. Appl. EP 85,276, (Cl. GOlN33/58), Aug. 1983; CH Appl. 81/7, Dec. 1981. I. Siddiqi and J. Brochot, Eur. Pat. Appl. EP 125, 368 (Cl. GOlN33/54), Nov. 1984; Appl. 83/810,563, Dec. 1983. K. J. Grime and K. R. Lockhart, Anal. Chim. Acta, 106 (1979) 251. L. Ambler and G. E. Peters, Anal. Biochem., 137 (1984) 66. A. C. Maehly and B. Chance, in D. Glick (Ed.), Methods of Biochemical Analysis, Vol. I, Interscience, New York, 1954, p. 357.
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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 26
B. C. Saunders, A. G. Holmes-Sidle and B. P. Stark, Peroxidase, Butterworth, London, 1964. G. G. Guilbault, P. J. Brignac, Jr. and M. Zimmer, Anal. Chem., 40 (1968) 190. M. Morrison, Gunma Symp. Endocrinol., 5 (1968) 239. G. M. K. Hughes and B. C. Saunders, J. Chem. Sot., (1954) 4630. A. K. Covington, Crit. Rev. Chem., 3 (1974) 355. P. W. Alexander and P. Seegopaul, Anal. Chem., 52 (1980) 2408. F. G. Mann and B. C. Saunders, Practical Organic Chemistry, Longmans Green, London, 1952, pp. 420-421. H. Luck, in H. U. Bergmeyer, (Ed.), Methods of Enzymatic Analysis, Academic, London, 1963, p. 895. J. Mertens, J. Van den Winkel and D. L. Massart, Anal. Chem., 48 (1976) 272. D. C. Cowell, Ann. Clin. Biochem., 14 (1977) 269,275. D. E. Davey, D. E. Mulcahy and G. R. O’Connel, Anal. Lett., 19 (1986) 1387. W. Frenzel and P. Brat&r, Anal. Chim. Acta, 185 (1986) 127; 187 (1986) 1. B. C. Saunders, in G. L. Eichhorn (Ed.), Inorganic Biochemistry, Elsevier, Amsterdam, 1973, Chap. 28. G. Guilbault, Handbook of Enzymatic Methods of Analysis, Dekker, New York, 1976, pp. 107,147-149,311. L. H. Piette, I. Yamazaki and H. S. Mason, in M. S. Blois, Jr., H. W. Brown, R. M. Lemmon, R. 0. Lindblom and M. Weissbluth (Eds.), Free Radicals in Biological Systems, Academic, New York, 1961. B. Chance, in D. Glick (Ed.), Methods of Biochemical Analysis, Vol. 1, Interscience, New York, 1954, p. 411. J. Putter, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2, Academic, New York, 1974, p. 685.
173 Printed in The Netherlands
CONTINUOUS-FLOW POTENTIOMETRIC DETERMINATION OF HORSERADISH PEROXIDASE WITH A FLUORIDE-SELECTIVE ELECTRODE
PETER W. ALEXANDER*
and CARMELITA MAITRA
School of Chemistry, University of New South Wales, P.O. Box 1, Kensington, New South Wales (Australia) (Received 9th September 1987)
SUMMARY A continuous-flow system is reported for the determination of horseradish peroxidase (HRP) with a fluoride-selective electrode. Reaction conditions are optimized for the HRP-catalysed oxidation ofp-fluoroaniline (0.104 M) with hydrogen peroxide (4.0 mM) as the oxidant in 0.16 M acetate buffer at pH 4.6. HRP is determined in the concentration range 0.016-0.12 U ml-’ in the flow system at a sampling rate of 24 h-i. Interfering effects caused by known HRP inhibitors, including metal ions, cyanide and sulphide, are reported for the range 1 PM-1 mM. Applications of the system for determination of the enzyme in turnip extract and milk are described.
The use of the fluoride-selective electrode for the detection of horseradish peroxidase (HRP) with p-fluoroaniline as the hydrogen donor for enzyme immunoassay was reported by Alexander and Maitra [ 11. Since then, several publications and patents have appeared in which the fluoride electrode was used in conjunction with various hydrogen donors [2], including p-fluorophenol, for the determination of glucose, cholesterol and glucose oxidase [ 2-41, and in enzyme immunoassays [ 5,6]. However, the experimental conditions affecting the rate of the HRP-catalysed reaction, and inhibition factors, have not been studied in detail. Determinations of HRP are usually based on the spectrophotometric or fluorimetric measurement of the initial increase in absorbance or fluorescence of the oxidized donor. Many donors have been used. Other techniques include potentiometric, enthalpimetric and potentiostatic methods [ 71. An immunospecific enzyme assay involving the use of an anti-HRP antibody has also been reported [ 81. Interference effects, sensitivity, precision and accuracy with these methods depend mainly on the choice of hydrogen donor. With some hydrogen donors, such as pyrogallol, guiaicol and p-phenylenediamine, the spectrophotometric method is inhibited by traces of metal ions [ 9,101. Similar interference is known for the fluorimetric method with, for example, homovanillic acid as donor [ 111. The potentiometric method [ 121 with an iodide-selective elec-
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers B.V.
174
trode as the sensor and iodide as donor is not specific for HRP because many other substances can oxidize iodide to iodine [ 9,101, and the method is unsuitable for protein-containing samples because HRP also catalyzes the iodination of proteins [ 121. In this report, the development and optimization of experimental parameters in a continuous-flow system with a fluoride-selective electrode as detector and p-fluoroaniline as hydrogen donor are described for the determination of HRP at concentrations as low as 0.001 U ml-‘. HRP activities were determined in aqueous solutions, in turnip extract and in milk, based on the continuous-flow detection of the liberated fluoride after the enzymatic cleavage of the C-F bond inp-fluoroaniline. The method is selective because HRP has the ability to cleave a C-F bond under mild reaction conditions [ 10,131 and because the fluoride electrode is very selective for fluoride [ 141. However, interfering effects are shown to be caused by many inhibition factors which alter the rate of the HRP-catalysed reaction. EXPERIMENTAL
Instrumentation, reagents and solutions Potentiometric measurements were made with an Orion pH/mV meter Model 701A coupled to a Mace FBQ-100 strip-chart recorder. The electrodes were an Orion fluoride-selective electrode (Model 94-09) and an Orion double-junction reference electrode (Model 90-02) with 10% (w/v) potassium nitrate in the outer compartment. Solution pH was measured with an Activon digital pH/mV meter fitted with a combination pH glass electrode. The constanttemperature bath was of the circulating type (B.T.L. Long Reach Circon, England; Model 69/3926/0182). Spectrophotometric studies were done with a Zeiss PMQ-II spectrophotometer. Grade II horseradish peroxidase (donor: hydrogen peroxide oxidoreductase ) of specific activity 100 U mg-l, lot No. 1259124, was obtained from Boehringer-Mannheim Corp. Stock solutions of 27.7 U ml-’ were prepared in glassdistilled water and stored at 4’ C. Solutions remain stable for over a year [ 71 if stored as such. The p-fluoroaniline was obtained from Sigma Chemical Co. For routine analysis, a 0.104 M p-fluoroaniline solution was prepared in a 0.16 M sodium acetate/acetic acid buffer pH 4.6. Hydrogen peroxide (Pacific Manufacturing, Sydney, Australia; 30% (w/v) hydrogen peroxide, 1.1 g ml-’ specific gravity) was used at a concentration of 4.0 mM in distilled water. Other concentrations of hydrogen peroxide in the range 0.1-4.0 mM were used for the optimization studies. Diluted enzyme solutions, p-fluoroaniline in the acetate buffer and other solutions required were prepared freshly on the day of measurement. The milk
175
sample was a 10% (w/v) solution of skimmed milk powder. Fresh turnips were purchased from a vegetable market. Procedures The flow system used for the studies with HRP (Fig. 1) was as described earlier [ 11. A Watson-Marlow type proportioning pump fitted with a rapid speed motor (100 rpm) was used to pump solutions through the individual pump channels at the flow rates shown. The electrode flow-through cell was as previously described [ 151. The baseline potential was set by pumping the bufferedp-fluoroaniline solution (0.104 M), hydrogen peroxide (4.5 mM) and the water wash into the flow system with air-segmentation of the stream at a constant flow rate. The stream was debubbled just prior to the flow cell. A calibration plot for standard fluoride solutions was first measured to test the response of the fluoride electrode in the continuous-flow system in the presence of the acetate buffer pH 5.6. Peak heights corresponding to various concentrations of fluoride (0.01-10 mM) were plotted against log molarity. The reaction parameters for the assay of HRP were studied by passing water, hydrogen peroxide and buffered p-fluoroaniline through the system to establish the baseline and then passing HRP sample solutions (1.4 x 10m3-1.7 x 10-l U ml-‘) through the sample line for 30 s, followed by a wash for 4 min or as required for the peak to return to baseline. The HRP sample solutions were prepared by diluting 5-600 ~1 of stock (27.7 U ml-l) HRP solution to 100.0 ml. Optimization studies. To examine the effect of HRP reaction time, the length
_
r,y
waste
’ ” , 3.316.8 _
i r\ I air l.Sl3.4
/ “, I r\ ’!~ ;” !~
I sample or wash ; 2.Ll6.0 1substrate ; 2415.1 ! buffer and donor / I420
La -I 2.L’6.0
Fig. 1. Schematic diagram of the flow system used: (a) indicator electrode; (b) reference electrode; (c) flow-through cell; (d) proportioningpump; (e) mV/pH meter; (f) recorder; (g) mixing coils; (h) debubbler; (i) buffer solution. Flow rates are given in ml min- ‘: the larger flow rates are used for HRP determination, and the lower flow rates for calibration of the electrode.
176
of the mixing coils (g, and g,, Fig. 1) were varied. For the study of different substrate concentrations, calibration plots of HRP concentration against potential were constructed for various concentrations (1.8-44.0 mM) of hydrogen peroxide. Calibration plots for HRP at various fixed p-fluoroaniline concentrations (0.21, 0.042, 0.104 M) were also established. To evaluate the effects of pH and buffer concentration on the HRP calibration plot, buffers of various pH and concentrations were pumped through the buffer channel (Fig. 1). For interference studies, Co2+, Fe3+, Cu2+, S2- and CN- were individually mixed with the HRP sample before its introduction into the system. The effect of temperature was examined by varying the temperature (16-50’ C ) of the water circulating through the jacketted mixing coil (82) in Fig. 1. Analysis offood samples. Samples of turnip extract and milk containing HRP were analysed in the above flow system. The turnip samples were prepared as described by Mann and Saunders [ 161: fresh turnips were washed, weighed, chopped and blended in a Waring blender; the mass was filtered through cheesecloth, and then through a sintered-glass Buchner funnel and the filtrate was diluted to 11. A lO.O-ml aliquot of the extract was diluted to 1 1 and analyzed by standard addition in the flow system; Gran plots were used for data evaluation. The samples were also analysed by using the spectrophotometric method of Luck [ 171. The sample solutions (2.0 ml of turnip extract), 1 ml of phosphate buffer (0.067 M Na2HP0,/KH2POd, pH 7.0), 0.1 ml of hydrogen peroxide solution (3.0 mM) and 0.1 ml ofp-phenylenediamine solution (l%, w/v) were mixed thoroughly in a cuvette and the absorbance was read at 485 nm every 30 s for 3 min. The control cuvette was prepared similarly, except that distilled water was used instead of hydrogen peroxide. Initial rates were plotted as a function of concentration to obtain calibration or standard addition plots. In this technique, standard addition was the only viable method because erratic results were obtained when the data for the turnip sample were compared directly with a calibration plot. For the electrode method, however, both calibration and standard addition plots were satisfactory. Milk was tested to investigate the effect of protein on the percentage recovery of HRP. Various volumes (50-400 ~1) of stock HRP were added to 100.0 ml of milk (lo%, w/v); the peak heights corresponding to these solutions were evaluated from the usual calibration plot. RESULTS AND DISCUSSION
The dynamic response of the fluoride electrode to fluoride in a continuousflow system has been reported by many authors (see, e.g. [X3-21] ) . Here, the sensitivity of the electrode response for determination of HRP was optimized by studying the effect of temperature, buffer composition, pH, substrate and hydrogen donor concentrations. The effect of several substances known to in-
117
terfere with HRP detection was studied and finally the flow method was applied for the analysis of food samples. Optimizatioti of the HRP reaction Initially, the electrode response in the flow system was calibrated against aqueous fluoride standards in the range 0.01-100.0 mM. Slow response was observed but a near-Nernstian slope of - 57 mV pF-’ was obtained after plotting the relevant peak heights. Measurements were done at 26’ C. The response of the electrode in the flow system for the HRP-catalyzed reaction is shown in Fig. 2. The response was again slow and showed peak widths at baseline of ca. 4 min. The response is attributed [ 131 to the reaction: H-donor + Hz O2-Products+H++F-+H,O
(I)
where the hydrogen donor is p-fluoroaniline and the oxidation products are 2amino-p-fluoroanilinobenzoquinone and tetra-p-fluoroazophene. The production of fluoride is relatively fast [ 221 and the rate of appearance of fluoride ion is proportional to the HRP activity. Calibration plots are shown in Fig. 3, corresponding to the initial experimental conditions (A) used in the flow system, and after modification (B) where it can be seen that as little as 0.001 U ml-’ HRP can be detected. Conditions used to obtain the data for curve C gave optimum linearity and were used in subsequent studies. These results were obtained after the optimization study outlined below. Effect of incubation time, temperature, buffer and PH. The optimum incu-
Fig. 2. Continuous-flow output for the determination of HRP with the fluoride electrode at 24 samples h-l. (A) Peaks for HRP samples: (a) 0.139, (b) 0.278, (c) 0.554, (d) 1.39, (e) 2.78, (f) 5.54, (g) 11.1, (h) 16.6~ lo-’ U ml-’ HRP. (B) Steady-state signal for 0.11 U ml-’ HRP. (C) Replicates for 0.11 U ml-’ HRP. Conditions: 8.9 mM hydrogen peroxide, 0.104 Mp-fluoroaniline in 0.5 M acetate buffer, pH 4.7,16”C. Fig. 3. Calibration plots for HRP corresponding to the following conditions: (A) 0.104 Mp-fluoroaniline in 0.5 M acetate buffer, pH 4.7, 8.9 mM hydrogen peroxide, 16°C; (B) 0.104 M pfluoroaniline in 0.16 M acetate buffer, pH 4.6,4.45 mM hydrogen peroxide, 37°C; (C) as for (B) but at 45°C.
178
bation time for the reaction was obtained by varying the length of the delay coils in the flow system. An incubation time of 60 s was chosen in order to allow sufficient time for the reaction to reach completion. Incubation at temperatures between 19 and 36°C gave useful reaction rates. A marked decrease in response was obtained at 5O”C, probably because of deactivation of the enzyme. Figure 4 shows the effect of pH on the rate of catalytic release of fluoride. The use of a buffer is necessary to maintain enzyme activity and to give reliable electrode response. Below pH 4.5, fluoride exists as HF or HF,- and above pH 8.0 hydroxide interferes with the response [ 141. Here, the choice of pH is limited by the optimum pH for maximum activity of the HRP which is generally considered to be pH 7.0 [23] but others maintain to be pH 4.8 [24]. The optimum pH depends on the nature of the hydrogen donor. In the case of pfluoroaniline, a slightly acidic pH is required for its dissolution in aqueous solutions. The results in Fig. 4 show that the sensitivity was greatest at pH 4.6. It was also found that an increase in the concentration of the buffer decreased the sensitivity. Effect of substrate and donor concentration. Figure 5 shows an increase in the sensitivity for the determination of HRP at low hydrogen peroxide concentration, in agreement with previous studies [ 25,26 J. Hydrogen peroxide concentrations < 10.0 mM are essential because an excess of hydrogen peroxide is known to inactivate HRP [lo]. An optimum concentration of 4.5 mM was used in subsequent determinations. Figure 6 shows that an increase in the hydrogen
Fig. 4. Plots of peak height against pH for various HRP activities ( X lo-’ U ml-‘): (A) 0.278, (B) 0.554, (C) 1.39, (D) 2.78, (E) 5.54, (F) 11.1. The various pH values were provided by the following buffers: (1) 0.05 M acetate, pH 4.73; (2) 0.16 M acetate, pH 4.55; (3) 0.25 M acetate, pH 4.49; (4) 0.4 M acetate with pH values of 4.42,4.99,6.17; (5) 0.1 M KH2P04/NaOH, pH 6.29. Conditions: 0.106 M p-fluoroaniline in the buffer, 4.45 mM HzOz, 15’ C. Fig. 5. Plots of peak height against log[ H,O,] for different HRP activities ( X lo-* U ml): (A) 0.554, (B) 1.39, (C) 2.77, (D) 5.54, (E) 11.1. Conditions: 0.104 M p-fluoroaniline in 0.16 M acetate buffer, pH 4.6,15”C.
179
Fig. 6. Effect of p-fluoroaniline concentration on the electrode response for different HRP activities ( x lo-‘U ml-‘): (A) 1.38, (B) 2.77, (C) 4.20. Conditions: 0.5 M acetate buffer, pH 4.7,8.9 mM Hz02, 15’ C. Fig. 7. Effect of inhibitor concentration on the electrode response for 0.014 U ml-’ HRP for different inhibitors in the concentration range 0.001-1.0 mM: (A) Cu*+, (B) CN-, (C) S*-, (D) Fe3+, (E) Co*+. Conditions as for curve A, Fig. 3.
donor concentration in the range 0.016-0.10 M results in increased sensitivity for the HRP determination. An optimum concentration of 0.104 M p-fluoroaniline in 0.16 M acetate buffer was shown to give maximum sensitivity. Effect of inhibitors The effect of inhibitors on the rate of the HRP-catalysed reaction was tested. Inhibition by various ionic species including Cu2+, Co2+, Fe3+, cyanide and sulphide ions has been reported previously [ 10,111. In this study, little effect from Cu2+ , Fe3+ and Co2+ on the HRP calibration plot was observed but considerable inhibition was found for cyanide and sulphide (Fig. 7). Interferences of Cu2+, Fe3+ and Co2+ ions have been reported [ 111 for a peroxidase system. However, Cu2+, Fe3+ and Co2+ inhibit peroxidase activity only in the presence of certain hydrogen donors [lo], e.g., Cu2+ with guaiacol, and many other metals are known to interfere with the p-phenylenediamine reaction with hydrogen peroxide [25,26]. The key to the selectivity of this system, therefore, is the ability of HRP to cleave the C-F bond in the p-fluoroaniline donor. Cyanide and sulphide cause strong inhibition whatever the method of assay, because they bind with HRP at the active site, causing inactivation [lo]. Analysis of food samples The continuous-flow system was applied in the analysis of some food samples for HRP. The samples chosen were turnip extracts and milk, treated ini-
180 TABLE 1 Recoveries of HRP added to milk samples diluted 1: 50 HRP added ’ (10-2U ml-‘) Recovered (10-2U ml-‘) Recovery ( % )
1.39
2.77
5.54
11.1
1.54 111
2.84 102
5.53 100
11.9 107
tially to produce appropriate dilutions and then introduced into the flow system shown in Fig. 1 at a sampling rate of 12 h-l. The turnip extract was also analysed by the conventional spectrophotometric method [ 171. The HRP content found in the turnip extracts, diluted 1: 100, was 1.8 2 0.3 x lo-’ U ml-’ (n= 3) by the proposed method and 1.8~ 10m2 U ml-’ by the spectrophotometric method. The recovery study after standard addition of HRP to milk, however, gave some high results (Table 1); the high protein content of milk may have caused the high recoveries but the reasons remain unclear. Interferences in HRP-catalysed reactions may occur in several ways. The detection system and/or the reaction scheme may suffer from interference effects. Unlike spectrophotometry, the electrode method is oblivious to sample colour and turbidity. With the fluoride electrode, interference with the detector response is minimal. HRP is not donor-specific, and so interferences with the reaction scheme depend on the donor used. Normally, a donor is chosen on the basis of the uniqueness of the product of the HRP-catalyzed reaction with hydrogen peroxide such as the reaction [ 10,131 of p-fluoroaniline with hydrogen peroxide to produce fluoride ion. However, HRP is substrate-specific to hydrogen peroxide, so that any substance able to react with hydrogen peroxide may interfere with the present technique, or with any other HRP assay for that matter.
REFERENCES P. W. Alexander and C. Maitra, Anal. Chem., 54 (1982) 68. I. W. Siddiqi, Clin. Chem., 28 (1982) 1962. M. H. Ho and T. G. Wu, ISA Trans., 24 (1985) 61. M. H. Ho and T. G. Wu, Ann. N. Y. Acad. Sci., 434 (1984) 523. R. Lauterburg, Eur. Pat. Appl. EP 85,276, (Cl. GOlN33/58), Aug. 1983; CH Appl. 81/7, Dec. 1981. I. Siddiqi and J. Brochot, Eur. Pat. Appl. EP 125, 368 (Cl. GOlN33/54), Nov. 1984; Appl. 83/810,563, Dec. 1983. K. J. Grime and K. R. Lockhart, Anal. Chim. Acta, 106 (1979) 251. L. Ambler and G. E. Peters, Anal. Biochem., 137 (1984) 66. A. C. Maehly and B. Chance, in D. Glick (Ed.), Methods of Biochemical Analysis, Vol. I, Interscience, New York, 1954, p. 357.
181
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 26
B. C. Saunders, A. G. Holmes-Sidle and B. P. Stark, Peroxidase, Butterworth, London, 1964. G. G. Guilbault, P. J. Brignac, Jr. and M. Zimmer, Anal. Chem., 40 (1968) 190. M. Morrison, Gunma Symp. Endocrinol., 5 (1968) 239. G. M. K. Hughes and B. C. Saunders, J. Chem. Sot., (1954) 4630. A. K. Covington, Crit. Rev. Chem., 3 (1974) 355. P. W. Alexander and P. Seegopaul, Anal. Chem., 52 (1980) 2408. F. G. Mann and B. C. Saunders, Practical Organic Chemistry, Longmans Green, London, 1952, pp. 420-421. H. Luck, in H. U. Bergmeyer, (Ed.), Methods of Enzymatic Analysis, Academic, London, 1963, p. 895. J. Mertens, J. Van den Winkel and D. L. Massart, Anal. Chem., 48 (1976) 272. D. C. Cowell, Ann. Clin. Biochem., 14 (1977) 269,275. D. E. Davey, D. E. Mulcahy and G. R. O’Connel, Anal. Lett., 19 (1986) 1387. W. Frenzel and P. Brat&r, Anal. Chim. Acta, 185 (1986) 127; 187 (1986) 1. B. C. Saunders, in G. L. Eichhorn (Ed.), Inorganic Biochemistry, Elsevier, Amsterdam, 1973, Chap. 28. G. Guilbault, Handbook of Enzymatic Methods of Analysis, Dekker, New York, 1976, pp. 107,147-149,311. L. H. Piette, I. Yamazaki and H. S. Mason, in M. S. Blois, Jr., H. W. Brown, R. M. Lemmon, R. 0. Lindblom and M. Weissbluth (Eds.), Free Radicals in Biological Systems, Academic, New York, 1961. B. Chance, in D. Glick (Ed.), Methods of Biochemical Analysis, Vol. 1, Interscience, New York, 1954, p. 411. J. Putter, in H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2, Academic, New York, 1974, p. 685.