Determination of horseradish peroxidase based on the oxidation reaction of phenol with H by using resonance light scattering technique

Li Zhengping, Gao Ruiguang, Lv Xuechong, Liu Xiuxian
(College of Chemistry and Environment Science, Hebei University, Baoding 071002)

Abstract A new assay of horseradish peroxidase (HRP) is proposed by using of resonance light scattering (RLS). The oxidation reaction of phenol with hydrogen peroxide (H) catalyzed by HRP results in a great enhancement of RLS intensity in the wavelength range from 200 nm to 700 nm characterized by the RLS peak around 313 nm. The enhanced intensity of RLS at 313 nm is proportional to the concentration of HRP in the range of 0.05~5.0×10-7 g/mL and the detection limit (3, n=11) is 1.1×10-10 g/mL. Therefore, a simple and sensitive method for HRP determination is established.

The enzymatic assays have been widely used in analytic biochemistry because of their rapidity and high selectivity[1,2]. Horseradish peroxidase (HRP) catalyzed reaction is one of the most widely used enzymatic reactions in bioanalytical chemistry, such as enzyme-linked immunoassy. The characteristic of HRP was systematically studied using hydrogen peroxide (H) as an oxidizing agent and various substances as substrates. A variety of techniques, such as spectrophotometry, chemiluminescence, fluorescence and electrochemistry, have been used for HRP detection[3-5]. However, to the best of our knowledge, resonance light scattering (RLS) technique has not been proposed for HRP detection.
Recently, RLS has become a new interesting technique for determination of micro amounts of biomolecules[6], such as nucleic acids[7], proteins[8], and drug[9]. The RLS methods are very attractive because high sensitivity can be obtained with a common spectrofluorimeter by using inexpensive reagents.
In this paper, it is found that the oxidation reaction of phenol with H catalyzed by HRP results in a great enhancement of RLS signals. The enhanced RLS intensities at 313 nm are proportional to the concentration of HRP in the range of 0.05~5.0×10-7 g/mL and the limit of detection (LOD) of 1.1×10-10 g/mL can be achieved without preconcentration.

2.1. Apparatus
The RLS spectra and intensity were measured with a Hitachi F-4500 Fluorescence Spectrophotometer (Tokyo, Japan) equipped with a quartz cell (1cm×1cm). A QL-901 Vortex mixer (Jiangsu Clinical Instruments Plant, Haimen, China) was employed to blend the solutions.
2.2 Reagents
A 1.0 mg/mL stock solution of HRP (300 units/mg, Xin Jing Ke Biotechnology Co. Ltd. Beijing, China) was prepared by dissolving 1.0 mg HRP in 1 mL sterilized water and stored in a refrigerator at 4ºC. Phosphate buffered saline (PBS, pH 6.4) was containing 0.3 mol/L NaCl and 10 mmol/mL sodium phosphate buffer. 0.625% (m/v) phenol was prepared by dissolving 0.625 g phenol in 100 mL water. HRP working solution was prepared by diluting the stock solution immediately prior to use. 0.6% (v/v) H was prepared by dissolving 30% H
All reagents were of analytical grade without further purification. Doubly distilled water was used throughout expect preparation of HRP using sterilized water.
2.3 Procedures
In a 10 mL colorimetric tube, 1.5 mL of phenol (0.625%, m/v), 1.5 mL of PBS buffer (pH 6.4), a certain volume of HRP working solution, 50L of H (0.6%, v/v) were added in turn. The mixture was diluted to the mark with water, and stirred thoroughly. After incubation at room temperature for 30 min, 200L citric acid (0.3 mol/mL) was added in to stop the reaction. Then the RLS spectra were measured against the blank solution treated in the same way without HRP.
The RLS spectrum was obtained by scanning simultaneously with the same excitation and emission mono- chromators of F-4500 spectrofluoro- meter from 200 to 700 nm. The intensity of RLS was measured at 313 nm. The silt width and PMT voltage of the measurements were 5 nm and 400 v, respectively.

3.1. Spectral Features of HRP

Fig. 1 shows the RLS spectra of the mixture of phenol and H in the absence and in the presence of HRP. In the absence of HRP, the mixture has a weak RLS peaks at 280 nm, which should be produced by phenol molecules. Up adding HRP in the mixture solution, HRP catalyzes the reaction of phenol and H to produce the free radicals of phenol, which cause the polymerization and produce polyphenol products. According to the RLS theory[10,11] the increase molecular size and the conjugated polymer structure of polyphenol products would result in greatly enhanced RLS. From Fig. 1, one can see that the RLS peak located at 313 nm and the RLS intensity can be greatly increased with increasing the HRP concentration, which implies that the RLS technique can be applied to sensitively determine HRP.
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Fig.1 Resonance light scattering spectra of HRP-phenol system. HRP (g/mL), from bottom to top 0, 5×10-9, 1×10-8, 1×10-7, 5×10-7, 1×10-6, respectively. 0.09% phenol, 0.003% H

3.2 Optimization of the general procedure
The pH value of the reaction solution plays an important role for the HRP-catalyzed polymerization because the enzyme activity can be greatly affected by the pH value. Fig. 2 shows the influence of the pH value on the RLS intensity produced by 1×10-8 g/mL HRP. With increasing the pH value in the pH range of 5.8-8.0 adjusted by PBS buffer, the RLS intensity firstly increases and then decrease. The maximum RLS intensity can be obtained at pH 6.4. Therefore, pH 6.4 was chosen for use to determine HRP.
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Influence of the pH value on RLS intensity. HRP (g/mL): 1, 1×10-8, 2, 0. 0.2% phenol, 0.006% H

The effect of ionic strength was also examined by the addition of NaCl in the PBS buffer solution. A little effect of RLS intensity with the NaCl concentration in the range of 0.015-0.075 mol/L when1.5 mL PBS buffer was used in the 10 mL reaction solution. So the PBS buffer solution containing 0.045 mol/L NaCl was used in the subsequent work.
The concentration of phenol has great effect on the formation of the polyphenol. The influence of phenol concentration on the RLS intensity was investigated in the range of 0.125%-1.25%. As can be seen from Fig. 3 that the RLS intensity are stable when the concentration of phenol was in the range of 0.02%-0.2% and the enhanced RLS intensity reaches its maximum at 0.09%. So 0.09% phenol was selected for subsequent work.
Fig.3 Effect of concentration of phenol on RLS intensity. HRP (g/mL): 1, 1×10-8, 2, 0. 0.006% H
Fig.4 Effect of concentration of H on RLS intensity. HRP (g/mL): 1, 1×10-8, 2, 0. 0.09% phenol.

The effect of H concentration on the RLS intensity was also studied. As shown in Fig.4, with increasing the H concentration, the enhanced RLS intensity was increased when H concentration was less than 0.003%, and decreased when the concentration was greater than 0.003%. So 0.003% H was selected as the optimum in this assay.
The influence of the incubation time of the HRP-phenol solution on the intensity was also investigated. The RLS intensity reached its maximum after the solutions were mixed 30 min, and remained a constant at least for 30 min. The results indicated that the stability of the RLS signal is practical for the determination of HRP.
3.3 Calibration and detection limits
According to above standard procedure, the calibration curve for HRP determination is constructed under the optimal conditions. There are good linear relationships between the enhanced RLS intensity (IRLS) and the HRP concentrations in the range of 0.05~5.0×10-7 g/mL (Fig. 5). The correlation equation was IRLS=184.6 + 1.14 CHRP, the correlation coefficient r=0.9996, and the detection limit (3, n=11) is 1.1×10-10 g/mL.
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Fig.5 Relationship between enhanced RLS intensity and concentration of HRP.

In this contribution, we demonstrate that HRP catalyze the oxidation reaction of phenol with H can form a stable associated complex polyphenol, which produces the strong RLS signal. The enhanced RLS intensities are proportional to the concentration of HRP in a wide range. Based on this, a highly sensitive and very convenient RLS method for HRP determination can be developed. Therefore, the proposed RLS method may open up a new possibility for HRP determination and has great potential applications for bioanalytical chemistry.