Isolation, Purification, and Characterization of Horseradish Peroxidase (HRP) J. Kane, T. Schweickart

Isolation, Purification, and Characterization of Horseradish Peroxidase (HRP) J. Kane, T. Schweickart From the Department of Chemistry, Elon University, Elon, North Carolina 27244 Running title: Analysis of horseradish peroxidase To whom correspondence should be addressed: Jennifer A. Kane, Department of Chemistry, Elon University, 9653 Campus Box, Elon, North Carolina 27244, Telephone: (772) 766-9687; E-mail: jkane3@elon.edu or Tucker H. Schweickart, Department of Chemistry, Elon University 9234 Campus Box, Elon, North Carolina, 27244, Telephone: (606) 923-1533; E-mail: tschweickart@elon.edu Keywords: Horseradish Peroxidase ------------------------------------------------------------------------------------------------------------------------------- ABSTRACT Peroxidases are a class of oxidoreductase enzymes. Horseradish peroxidase (HRP) was the peroxidase of study. HRP was isolated from horseradish root by homogenization, vacuum filtration, sonication, ammonium sulfate fractionation, and centrifugation. Isolated HRP was purified through column chromatography. HRP was characterized by ligand-binding and Michaelis-Menten kinetics analytics. Resorcinol and orcinol, both phenols, were used as ligands to determine HRP s binding affinity. HRP had a similar high affinity for both ligands as indicated by their KD values: 0.001529 ± 0.000662 M for resorcinol and 0.002369 ± 0.002663 M for orcinol. The kinetic efficiency of HRP was determined using sodium azide as a competitive inhibitor and ABTS as substrate. The K M and V max for uninhibited HRP were 0.00171 µm and 18550 µmol/min respectively. The K M and V max for the inhibited HRP were 0.00718 µm and 19230 µmol/min confirming that sodium azide acted competitively with ABTS substrate while bound to HRP protein. INTRODUCTION Peroxidases are a class of enzymes predominantly involved in oxidation-reduction reactions with important environmental and industrial applications (1). Over the past decade, it has been found that peroxidases play key roles in the detoxification of water sources, defense mechanisms against disease in plants and mammals, and western blots and ELISA assays as a source of detection (1-2). It has also been experimentally observed that the heme group, consisting of an Iron in its reduced state (Fe2+), is the reducing agent to hydrogen peroxide to reduce it to water (2-3). Such heme groups favor the oxidation of specific groups of substrates typically aromatic phenols, halides, or amines in order for the oxidized Iron (III) to return to its stable, reduced Iron (II) form (2, 3). Horseradish peroxidase (HRP) is an enzyme that falls into the peroxidase class of proteins. HRP is most commonly observed as a catalyst of an oxidation reaction involving hydrogen peroxide and a substrate. HRP requires the presence of a heme group for proper function. In the reaction, the enzyme reduces hydrogen peroxide, which in turn, produces a kinetically favorable environment for the oxidation of the desired substrate. The general redox reaction can be seen below (1): H 2 O 2 + substrate red. 2H 2 O + substrate oxid. (1) This particular study focuses specifically on HRP, which has many of the same industrial and environmental applications as most peroxidases (2). As more research of horseradish peroxidase evolves from the demands of industry and society, it has become increasingly important to discover new methods for isolating and purifying the enzyme so that it can be further characterized. Key studies by Lavery, Matera, Shannon, and Critchlow et al are used as the experimental basis of this multi-week project to

isolate, purify, and characterize HRP from horseradish root. Economic feasibility of the protocols played a major role in the development of this study. Each of these studies provides the most cost-effective plans for analyzing HRP, a relevant and adaptable protein that is ubiquitous to biochemistry. All experiments involving HRP are conducted under physiological conditions in vitro using phosphate buffer. The overarching goal of the study is to observe the kinetic enzymatic ability of HRP as an oxidizing enzyme of the substrate, 2,2 -azino-bis (3- ethylbenzthiazoline-6-sulfonic acid) (ABTS), as well as analyze its ligand-binding capabilities to phenols--resorcinol and orcinol. In the kinetic characterization of HRP, comparisons can be made when the heme group is normally functioning and when it is inhibited using a kinetic assay (5). Ligand-binding of HRP to resorcinol and orcinol is analyzed by concentration of protein and protein-ligand complex (6). In completing this experiment, the protein will be isolated, purified, and characterized with sufficient supporting data to conclude its functionality and the structural characters that predict its behavior. METHODS HRP was isolated using a referenced protocol from Lavery et al (1). The procedure was modified to include a vacuum filtration step of the homogenized horseradish sample before lysing it through sonication. Furthermore, the phosphate buffer was made at a ph of approximately 7.0. Finally, all centrifugations, both of crude and 30/65% fractionated salt extracts, were conducted for a period of 30 minutes. The isolated HRP sample was purified through vertical column chromatography by size using a Sephadex G-50 matrix and phosphate buffer to pack the column. Blue Dextran was used as a control to record the void volume. The fractionated ammonium sulfate HRP sample was run through the column and collected in 1 ml fractions approximately 3 ml before and 3 ml after the void volume. The concentration of each fraction was analyzed using a spectrophotometer at a wavelength of 402 nm. Fractions three and four were collected into a 15 ml conical tube and centrifuged down. The presence of pure HRP enzyme was qualitatively confirmed by running a vertical SDS-PAGE electrophoresis gel using 10% acrylamide gel for 30 minutes at 150 V. SeeBlue Marker Dye was used as a molecular weight ladder. Coomassie Blue dye was used to stain the gel. The gel was destained over two 18-hour periods. A Bradford Assay was used for quantitative analysis of HRP concentration within the sample. The characterization of the purified HRP sample was first accomplished through analysis of its ligand-binding capabilities. 0.1 M resorcinol and 0.1 M orcinol were phenols uses as ligands to HRP protein. The HRP sample was diluted from 1 mg/ml to 0.1 mg/ml for a 100:1 dilution using phosphate buffer. One cuvette was used to add 1000 µl of the diluted HRP and increasing concentrations of resorcinol in 5 µl increments. The absorbance of pure HRP was taken, and with each consecutive addition, the absorbance was recorded. The first four additions involved 0.1 M resorcinol, the fifth and sixth addition involved 0.5 M resorcinol, and the final addition (the saturated addition) was 1.0 M. The procedure was repeated for orcinol. GraphPad (Prism) was utilized to develop plots for both resorcinol and orcinol that displayed the fraction of ligand bound to HRP as compared to the concentration of ligand added. The fraction of ligand bound to HRP was calculated from the absorbance recorded using the equation below: Y! = A!"#!! A!"# A! A!"# Michaelis-Menten kinetics was used to analyze HRP by determining the rate of enzymatic activity through absorbance per minute. 0.0087 M ABTS was used to represent the uninhibited HRP-substrate complex while 0.5 M NaN 3 was used to represent inhibited enzymatic activity with ABTS. 0.3% H 2 O 2 solution was used to initiate the oxidation-reduction reaction. For both the uninhibited and inhibited complexes, a constant amount of H2O2 and HRP was added to a 1 ml cuvette. For the inhibited trials, 100 µl of NaN 3 was added before adding increasing

amounts of ABTS (50 µl, 100 µl, 200 µl, 400 µl, 600 µl, 800 µl) and phosphate buffer to the reaction mixture until it reached a total volume of 1000 µl. With each addition of ABTS, the absorbance was recorded. The H 2 O 2 was added after the HRP, ABTS, NaN 3, and buffer and an initial absorbance was immediately recorded. The final absorbance was recorded after 1.5 minutes for each addition of ABTS. A similar procedure was used for the uninhibited trials, without the addition of NaN 3. GraphPad (Prism) was utilized to determine the V max and K M values for each of these variables via a Michaelis-Menten hyperbolic curve comparison and a Lineweaver Burke Plot. Absorbance was converted to change in concentration (µmol/min) using Beer s Law, which can be seen below: A = ε L c t From analysis of the BSA Standard Curve seen in Figure 2, the concentrations of pure HRP and 10% diluted HRP were determined to be 0.584 mg/ml and 0.218 mg/ml respectively. From the two hyperbolas plotted on the ligand-binding curve, seen in Figure 3, the K D values for the binding of HRP to resorcinol and orcinol were determined as 0.001529 ± 0.000662 M and 0.002369 ± 0.002663 M respectively. From the Lineweaver-Burk plot shown above in Figure 4, the K M and V max values for uninhibited ABTS and HRP were found to be 0.001708 µm and 18551 µmol/min respectively. This was compared to the K M and V max values for sodium azide inhibition of ABTS and HRP, which were found to be 0.00718 µm and 19230 µmol/min respectively. The extinction coefficient for ABTS was 36.8 mm. The change in concentration was plotted over the total concentration of ABTS added. RESULTS The SDS-PAGE electrophoresis gel can be seen in Figure 1. Coomassie Blue dye was used to track each protein band as it moved down the vertical acrylamide gel. As seen in Figure 1, from left to right the wells contained: the SeeBlue protein standard (MW), crude (A), 30/65% ammonium sulfate (B), pure HRP (C), commercial HRP (D). The crude sample contained bands at 62 kda, 44 kda, 39 kda 35 kda, 32 kda, 28 kda, and 26 kda. The 30/65% ammonium sulfate sample contained bands at 62 kda, 28 kda, and 26 kda. The pure HRP contained bands at 62 kda, 44 kda, 39 kda, 28 kda, and 26 kda. The commercial product did not produce any relevant bands. A Bradford Assay Standard Curve with concentrations ranging from 0 mg/ml to 1 mg/ml of BSA standards was made and can be seen in Figure 2. At a wavelength of 595 nm, the data points in Figure 2 represent each of the six standards as well as the two values for absorbance. The data set displays a linear trend line based on the concentration and absorbance of each data point. Figure 1. SDS-PAGE of HRP at Three Stages of Isolation and Purification Compared to Commercial HRP. The technique was conducted with vertical mini-acrylamide gel (10% acrylamide), bromophenol blue tracking dye, and a 150 V current for approximately 30 minutes. The gel was stained with Coomassie dye and destained over two 18-hour intervals. The MW well contained SeeBlue pre-stained protein standard for a molecular weight marker (kda). A, crude isolated HRP. B, isolated HRP after 30/65% ammonium sulfate fractionation. C, column chromatography purified HRP sample. D, commercially isolated and purified HRP.

0.8 Absorbance (595nm) 0.6 0.4 0.2 y = 1.259x + 0.0434 R² = 0.97256 10% Diluted Pure HRP: 0.218 mg/ml Pure HRP: 0.584 mg/ml Standard BSA HRP 0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration (mg/ml) Figure 2. Bradford Assay with Bovine Serum Albumin (BSA) Standard Curve Quantifying HRP Concentrations. A serial dilution was made and an assay was run at a wavelength of 595 nm. The 1.0 mg/ml coordinate was taken out of the data set to improve linear fit. The concentration of the pure HRP sample was determined via the trend line equation provided by the BSA Standard Curve. HRP was analyzed as pure sample and as a 10% diluted sample. HRP concentrations were found to be 0.584 mg/ml and 0.218 mg/ml respectively to pure HRP and 10% diluted HRP. A 1.0 B 1.0 Y L 0.5 Y L 0.5 0.0 0.000 0.005 0.010 0.015 Concentration (M) 0.0 0.000 0.005 0.010 0.015 Concentration (M) Figure 3. Ligand-Binding Curves for HRP to Resorcinol (A) and Orcinol (B). Each graph shows the fraction of ligand bound to HRP versus the concentration of ligand added for one observation of HRP sample. The fraction of ligand bound (Y L ) was calculated using absorptions. Varying concentrated additions were made through dilution. K D values were graphically calculated for both resorcinol and orcinol to be 0.002271 M and 0.007867 M respectively.

Table 1. Average and Standard Deviation for Dissociation Constants (K D ) Average K D (M) Standard Deviation (M) Resorcinol 0.001529 0.000662 Orcinol 0.002369 0.002663 Using fifteen distinct observations for both resorcinol and orcinol, an average K D and standard deviation was calculated for each after using a Grubb s Test to remove any outliers. 0.00025 0.0002 y = 3E-07x + 9E-05 R² = 0.95668 1/V0 (min/µmol) 0.00015 0.0001 y = 8E-08x + 6E-05 R² = 0.9847 0.00005 0-600 -100 400 900 1400 1900 2400 Concentration of ABTS (µm) Figure 4. Lineweaver-Burk Plot for Inhibited (A) and Uninhibited (B) HRP Protein with ABTS as Substrate. The plot displays the V max and K M values for HRP bound to ABTS substrate uninhibited and HRP bound to ABTS while competitively inhibited by sodium azide (NaN 3 ). K M and V max values for uninhibited ABTS and HRP were found to be 0.001708 µm and 18551 µmol/min respectively. K M and V max values for inhibited ABTS and HRP were found to be 0.00718 µm and 19230 µmol/min respectively. DISCUSSION HRP was isolated, purified, and characterized using the methodology described. Past studies have proven that micro volumes of HRP could be used to exemplify the specific kinetic and binding activity of the enzyme (1). Therefore, for the purpose of this study, all procedures were run on a micro-scale. To ensure a high yield of protein recovery in relation to the scale, it was crucial that all of the protein was recovered from the column and that the SDS- PAGE gel electrophoresis was run to confirm successful separation at various points in the procedure. The SDS-PAGE Gel, seen in Figure 1, displayed crude product from isolation (A), 30/65% ammonium sulfate fractionation (B), pure HRP (C), and commercial HRP (D). From the gel it was determined that the crude extraction of HRP was unsuccessful as seen by low protein content. This was unexpected, and thus limited the amount of HRP that was recovered from the column purification. Approximately 1.0 mg/ml of HRP was expected as a yield, but only 0.584 mg/ml was actually

recovered. The purified HRP sample in well C and the 30/65% fractionated sample in well B further show that there was little to no separation of the protein from other biomolecules in the horseradish root. The error was most likely in the lysing step of sonication where the vacuum filtrated homogenous horseradish root was supposed to be broken down into its various parts. Furthermore, the commercial HRP protein that was going to be used to compare the purity of the experimentally purified sample did not run through the gel properly, as seen by the blank well, D. Therefore, it could not be determined that the protein that ran in the sample well, C, was 100% pure HRP protein, and thus the chance was taken to continue characterizing the sample regardless of its purity. Some relevant bands could be seen around 28-30 kda as well as at approximately 62 kda in comparison to the samples that included various stages of isolated HRP protein, but there was no indication whether these bands represent pure horseradish peroxidase protein or not because the remaining wells, A and B, involved impure HRP protein. Nonetheless, the concentration of protein was quantified using a Bradford Assay. The Bradford Assay, seen in Figure 2, was conducted to quantify the concentration of the HRP sample acquired from purification. The bovine serum albumin standard was plotted on a linear curve and used as a standard for pure HRP protein and 10% diluted HRP protein. The R 2 value from this line of best fit, 0.973, indicated that the data from the standard showed almost 100% correlation to the surrounding points. The plot of the HRP data (in orange on the graph in Figure 2) showed similar correlation to the standard. This data was crucial to ensuring that a sufficient amount of enzyme was available for characterization. Although the amount of HRP recovered was about half of what was expected (0.584 mg/ml), there was still a sufficient amount of the sample to continue with characterization. HRP was first characterized through ligand-binding analysis. Graphpad (Prism) was used to plot a one-site binding hyperbola with nonlinear fit regression, which allowed for the retrieval of the given dissociation constant values (K D) for each ligand to HRP. The respective average values for fifteen observations of both resorcinol and orcinol were found to be 0.001529 ± 0.000662 M and 0.002369 ± 0.002663 M respectively. The proximity between these two values is indicative that HRP has a similar affinity for both ligands and overall a high affinity for both. Both ligands are phenols, and are only different by one methyl substituent on the ring of resorcinol. These structural similarities play a major role in HRP s similar affinity for both ligands. Finally, Michaelis-Menten analytics were used to further characterize the kinetic efficiency of HRP as an enzyme. This was done by comparing a control sample of HRP and increasing substrate concentrations to a sample of HRP and increasing substrate concentrations in the presence of a competitive inhibitor (NaN 3 ). The calculated efficiency constant (K M ) and maximum rate (V max ) values for the uninhibited sample were 0.00171 µm and 18550 µmol/min respectively. The K M and V max values for the inhibited HRP were 0.00718 µm and 19230 µmol/min confirming that NaN 3 acted competitively with ABTS while bound to HRP protein as indicated by an increased Km and constant V max comparatively. Both hyperbolic and Lineweaver-Burk plots were developed to analyze the data. Lineweaver-Burk, the reciprocal of the hyperbolic plot, was determined to be the most accurate method for visually representing the data in this study. This style of plot allows for less error in visually interpreting the data because the changes in rate and efficiency can be seen more clearly with a line of best fit as opposed to a hyperbolic curve. This study ultimately set out to analyze the effectiveness of horseradish peroxidase via the protocol set forth in by Lavery et al. Through various techniques it can be understood that horseradish peroxidase is an efficient and effective enzyme with ubiquitous applications in biochemistry. REFERENCES (1) Lavery, C.B., MacInnis, M.C., MacDonald, M.J., Williams, J.B., Spencer, C.A., Burke, A.A., Irwin, D.J,G., D Cunha, G.B. (2010).

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