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Localization of Peroxidase Activity, Detection of Multiple Peroxidase Isoforms, and Quantification of Peroxidase Isoenzymes in Carrot and Celery Samples
By: Hannah Mamet

Abstract

 

Peroxidases are a group of enzymes found ubiquitously in plants and are responsible for catalyzing the oxidation of phenolic compounds. Although extensive research has been dedicated to studying peroxidases, much about their exact function remains unknown [1]. In order to characterize peroxidases and further understand their role in plants, peroxidase activity was studied in carrot and celery samples. It was hypothesized that there would be a higher concentration of peroxidase in carrots than celery. Performing orthogonal analysis, the concentration of peroxidase isoenzymes in the vegetable tissues was quantified using both a dot-blot technique and spectrophotometry. The localization of peroxidase was investigated using tissue-printing. Finally, the peroxidase isoforms found in carrot and tissue prints were quantified and characterized using gel electrophoresis. Despite conflicting results regarding the concentration of peroxidase enzymes in carrots and celery, the more conclusive results were in line with the hypothesis that there is a higher concentration of peroxidase enzymes in carrots than celery. Findings from the experiment show that peroxidases have multiple isoforms in both plants and celery, and must play distinct roles within the respective plants. Furthermore, it was found that peroxidase activity is localized to the epidermis, cambium and cortex in the carrot extract, while the celery extract mostly exhibited peroxidase activity in the epidermis and vascular bundles. Findings from the experiment confirm previous research that peroxidase aids in synthesizing and strengthening the cell wall as well as protecting the vegetable from toxins or microorganisms [1]. Findings also suggest that peroxidases play a role in water and food storage and in development.  

 

Introduction

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Peroxidases are a group of isoenzymes that are prevalent in all plants [1]. They are a type of oxidase enzyme, which, among many other functions, use hydrogen peroxide to oxidize phenolic compounds [2,3] Unlike most classes of oxidases, peroxidases do not need a nicotinamide cofactor, making it an inexpensive biocatalyst. This, among many other reasons, has made peroxidases a focus of scientific research and biotechnology applications [3]. 

 

Despite immense research dedicated to peroxidase, its exact function still remains unclear. Currently, peroxidases are believed to aid in synthesizing and strengthening the plant cell wall by cross-linking phenolic residues of cell wall glycoproteins and polysaccharides [1]. It is believed that this is an aspect of a wound-healing response since peroxisome action is induced by stress, an infection, wounding, or elevated salt concentration. Peroxidases can also play a role in the destruction of microorganisms and toxic chemicals. Therefore, it is possible that peroxidases help in protecting the plant. The uncertainties of peroxidase isoenzyme’s functions are complicated by their being  tissue-specific and in the absence of stress stimuli, they can be developmentally regulated. This suggests that some of them might also function in the normal development processes [1].  

 

The current dearth of clarity regarding peroxidase function was the primary motivation of the lab’s research. The goal was to characterize peroxidase on multiple levels including localization of peroxidase in plant tissue, determination of how many peroxidase isoforms are in a plant tissue and estimation of the amount of active peroxidase molecules in plant tissue [1]. Tissue extracts were used from carrots, which is a root vegetable, and celery, which is a stem. Celery is part of a subdivision of flowering plants called monocots in which the embryo and seedling have one cotyledon, or seed leaf. Carrots are dicots, which have two seed leaves [1]. Monocots and dicots both contain plant peroxidases, but differ in their development and structure. Monocot growth is considered primary in origin, meaning that stem and root cells are derived from the apical meristem. In contrast, dicots develop cambium in the root and stem, which undergoes division and produces secondary tissues, thereby increasing the diameter of the plant [1]. Thus, while in dicots most of the tissue is from cambium, in monocots, the tissue is from the apical meristems. Epidermal, vascular and packing elements compose the three major tissue systems in plants. Their arrangement differs in monocots and dicots and varies according to maturity of the tissue [1]. The differences in structure of mature root and stem vegetables can be seen in Figure 1 below. 

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Screenshot 2025-01-30 134715.png

Figure 1. Mature dicot root and monocot stem differ in anatomical structure. Epidermal, vascular and packing elements make up the three major tissue systems in plants. Their arrangement differs in monocots and dicots.

The hypothesis was that peroxidase would be present in both carrot and celery extracts. However, because of dicots’ aforementioned anatomy and need for increased peroxidase activity for its secondary growth, it was expected that the carrot sample would have a higher concentration of peroxidase. Since peroxidase plays a key role in fighting pathogens and in a plant’s wound-healing response, it was also hypothesized that peroxidase enzymes would be localized to the epidermal edges of both the carrot and celery samples. 

 

In order to test the hypothesis and study peroxidase, a dot-blot procedure, spectrophotometric analysis, tissue-print, and gel electrophoresis were performed. Celery and carrot tissue extracts, and protein standards were electrophoresed on agarose gels to determine the number of isoforms of peroxidase isoenzymes found in each vegetable. The standards used were Cytochrome C, Hemoglobin, Serum Albumin, Horseradish Peroxidase (basic isoenzyme) and Horseradish peroxidase (mixture). Cytochrome C is a cell protein pigment found in plant and animal tissues that plays a crucial role in the electron transport system in mitochondria as well as in producing energy for the cell [1]. Hemoglobin transports oxygen in blood and carries a slight negative charge [1]. Serum albumin, dyed blue, is the main protein found in blood plasma [1].

 

Most experiments involved the use of a color development solution containing hydrogen peroxide. Chloronaphthol and hydrogen peroxide (H2O2) were used as substrates, which upon reaction with the enzyme peroxidase, turned from a soluble, colorless substance to a visible, insoluble purple product (Figure 2) [1]. This detectable reaction confirmed the presence of peroxidase. Ponceau S, a protein “blot” stain, was also used in this experiment during the tissue-printing procedure. Ponceau S binds to positively charged amino groups and nonpolar regions of protein and was later used to compare distribution of total protein versus peroxidase in tissue samples. 

Screenshot 2025-01-30 135143.png

Figure 2. Color Development Reaction used in the experiment to detect peroxidase in plant tissue. Upon reaction with substrates chloronaphthol and hydrogen peroxide, peroxidase converts chloronaphthol from a soluble colorless substrate to a detectable, insoluble purple product.

Materials and Methods

 

Dot-blot

Cell-free extracts of carrot and celery were prepared. Using a razor blade, 2 grams of celery and carrot were finely chopped and each placed into small beakers. 2 mL of enzyme extraction buffer, containing 2mMMgCl2, 20 mM NaCl, 0.01% NP40, I0 mM Tris, pH 8.0, was added to each beaker containing a vegetable extract. A tissue homogenizer was used to grind the tissues into a homogeneous suspension. The two suspensions were transferred to eppendorf tubes. After centrifuging the tubes for five minutes, the supernatant from each sample was removed with a pipette and transferred to new eppendorf tubes. 100µL of the carrot and celery extracts were taken and transferred to tubes containing 900µL of distilled water. The tubes were kept on ice [1].

 

One sheet of nitrocellulose was soaked in distilled water and then placed on a paper towel and blotted with another paper towel to remove excess moisture. The nitrocellulose membrane was oriented horizontally. 5µL of each of the four peroxidase standards were pipetted on the nitrocellulose membrane as individual dots in a vertical column approximately 0.5 cm from the short edge of the membrane (Figure 3) [1]. Parallel to the standards and approximately 1 cm from the edge of the membrane, 5µL of the “10% carrot”, “100% carrot”, “10% celery”, and finally, “100% celery” were dotted onto the nitrocellulose membrane. The solutions were left to absorb onto the membrane for five minutes [1]. 

Screenshot 2025-01-30 135350.png

Figure 3. Experimental set- up of Dot-blot and Tissue Prints (A) Approximately 0.5 cm from the short edge of the nitrocellulose membrane, the four peroxidase standards were placed in individual dots in a vertical column. Parallel to the peroxidase standards, approximately 1 cm from the short edge of the nitrocellulose membrane, in a vertical column (from top to bottom): “10% carrot”, “100% carrot”, “10% celery” and “100% celery”. (B) To the right of the dot-blot, are the tissue print samples: two carrot samples (top two) and two celery samples (bottom two).

Tissue printing

Using a razor blade, two samples of carrots and two samples of celery were cut. The surfaces of each vegetable were blotted dry to remove excess liquid. The cut surface of the carrot was placed on the nitrocellulose membrane in an area parallel and to the right of the dot-plot procedure (Figure 3) [1]. Firm pressure was applied for 10 minutes in order to make an imprint [1]. Using a fresh cut of carrot, another imprint was made next to the first. Below the tissue imprints of the carrot, the same procedure was performed with celery (Figure 3). 

 

The nitrocellulose membrane containing the dot-blot and tissue prints was soaked in  15 mL of freshly prepared color development, composed of 5 mL chloronaphthol, 0.7 mL of hydrogen peroxide, and 2 mL of 1M Tris buffer to 150 mL of distilled water [1]. A purple color was observed over a period of 5 minutes, after which, the color development solution was discarded and the membrane was soaked in water [1]. Observations regarding the relative intensities of purple color seen by the four peroxidase standards and vegetable extracts were recorded. 

 

Following peroxidase detection, the membrane was soaked for 5 minutes in 15 mL of protein blot stain (Ponceau S). After 5 minutes, the stain was discarded and the membrane was washed 3 times with water [1].

 

Gel electrophoresis

Two eppendorf tubes — one for carrot extract and one for celery extract — were obtained. 50µL of bromophenol blue loading dye was added to each tube [1]. The 100% celery and carrot extracts were added to the respective tubes. The samples were loaded into the agarose gel sample wells (Lane 1-6) were as follows: 15µL Cytochrome C, 15µL Hemoglobin-Albumin, 8µL Horseradish Peroxidase (HRP)-Basic, 8µL Horseradish Peroxidase (HRP)-Mixture, 30µL carrot, and 30µL celery. The gel was run at 170 V until the bromophenol blue loading dye in the vegetable extract samples migrated to within 1 cm of the positive electrode. The gel was removed from the electrophoresis chamber and using a ruler, the position of colored standard proteins in the gel were recorded. The gel was soaked in 20 mL of peroxidase substrate solution (containing 130 mL of H2O, 2 mL Color Developmental Solution (1M Tris Buffer), 5 mL of chloronaphthol, and 500 µL H2O2) for 30 minutes at 37℃ in the dark [1]. The gel was examined every 5 minutes to check for the appearance of purple bands in the lanes with peroxidase standards (lanes 3 and 4) and the lanes with tissue samples (lanes 5 and 6). Using a ruler, the distance and direction that each peroxidase isoenzyme was recorded. 

 

Spectrophotometry

Nine test tubes were prepared, containing (from Tube #1-9): blank (no addition), 40µL peroxidase standard A (0.08 µg/mL), 40 µL peroxidase standard B (0.4 µg/mL), 40 µL peroxidase standard C (2.0 µg/mL), 40 µL peroxidase standard D (10 µg/mL), 5 µL 100% carrot extract, 40 µL 100% carrot extract, 5 µL 100% celery extract, 40 µL 100% celery extract. 5 µL of color development solution (composed of 500 mL H20, 8 mL 1M Tris buffer, 15 mL chloronaphthol, 1.5 mL H2O2) was added to each tube and the contents were mixed. After 3 minutes, the absorbance at 575 nm was recorded for each of the tubes [1].

 

Results

 

Dot-blot

After treatment with a color development solution, the peroxidase standards and vegetable extracts were compared for relative intensities of purple color (Figure 4). The concentration of the peroxidase standards 1-4  (Figure 4, column A) were (from top to bottom): 0.01 µg/mL, 0.1 µg/mL, 1.0 µg/mL, and 10.0 µg/mL. The 10% celery extract (Figure 4, column B) was lighter in purple color than Standard 1 (0.01 µg/mL). Therefore, the concentration of the 10% celery extract was approximated to 0.001µg/mL. The 100% celery extract looked similar to Standard 2 (0.1 µg/mL), but had a dark purple coloring around the outer rim of the dot-blot, and therefore its concentration was approximated to be 0.3 µg/mL. The 10% carrot extract was much lighter than Standard 3 (1.0µg/mL), and therefore its concentration was determined to be 0.05 µg/mL. The 100% carrot extract was completely colored purple, but still much lighter than Standard 4 (10.0 µg/mL), which was very dark. Therefore, the concentration of the 100% carrot extract was approximated to be 0.5 µg/mL. When comparing the 100% celery extract to the 100% carrot extract, the carrot contained a higher concentration of peroxidase as it contained a deep purple color throughout the whole sample, as opposed to the celery extract, which only had a dark purple in the outer circular area (Figure 4). 

Screenshot 2025-01-30 135650.png

Figure 4. Dot blot of standards, celery and carrot extracts. The standards (A) were overall more concentrated than the vegetable extracts samples (B). 100% carrot extract had a higher concentration of peroxidase than the 100% celery extract.

Tissue-Printing

Following staining with color development solution and Ponceau S, the tissue-prints of carrots and celery were analyzed for localization of peroxidase activity and total protein activity. Figure 5 shows the results of the tissue prints after soaking with color development and Ponceau S. In the carrot tissue-prints, purple coloring was found in the epidermis, cortex, and partially in the vascular cambium. The strong purple coloring throughout the carrot extract indicated that peroxidase activity is found in higher amounts in carrots than in celery, which had less purple color overall. Peroxidase activity in celery was mostly localized to the epidermis and vascular bundles. The Ponceau staining performed after the color development solution resulted in a slight red tinge in areas where the proteins are found in the vegetable tissue prints, such as the epidermis, cortex, and minimally in the vascular cambium in the carrot extract and in the epidermis and vascular bundles of the celery extract print.

Screenshot 2025-01-30 135844.png

Figure 5. Results after Ponceau staining. In the carrot sample, peroxidase activity was localized to the cortex, epidermis, and vascular cambium. In the celery sample, peroxidase was localized mainly to the epidermis and vascular bundles. Because of subsequent soaking with Ponceau S, a faint red tinge can be seen in the same areas where the peroxidase was localized.

Gel electrophoresis

Following electrophoresis of Cytochrome C, Hemoglobin-Albumin, HRP-Basic, HRP-Mixture, carrot and celery extract, bands were found towards the negative and positive ends of the gels (Figure 6). In lane 1, Cytochrome C traveled a distance of 28 mm to the negative electrode, indicating that this protein carries a net positive charge. In lane 2, hemoglobin migrated a distance of 13 mm to the positive electrode and albumin traveled 21 mm to the positive electrode, which indicated that both have a net negative charge. In lane 3, which contained HRP-Basic, band 1 migrated 8 mm to the negative pole, which indicated that HRP-Basic is composed of one isoform of net positive charge. In lane 4, which contained the HRP-Mixture, band 1 migrated 17 mm to the negative electrode, band 2 migrated 8 mm to the negative electrode, and band 3 migrated 24 mm to the positive electrode. This indicated that HRP-Mixture was made of 3 isoforms of peroxidase: two with a net positive charge (B1, B2) and one strongly negative (B3). For the carrot extract (Lane 5), band 1 migrated 11 mm to the negative electrode, band 2 migrated 1 mm to the negative electrode, band 3 migrated 4 mm to the positive electrode, and band 4 migrated 15 mm to the positive electrode. This indicated that the carrot extract was composed of four isoforms: 1 positive (B1), 1 weakly-positive (B2), one negative (B3), and one strongly negative (B4) (Figure 6). For the celery extract, found in lane 6, band 1 migrated 11 mm to the negative electrode, band 2 migrated 7 mm to the positive electrode, and band 3 migrated 16 mm to the positive electrode. This indicated that the celery extract was composed of three isoforms: one positive (B1), one negative (B2), and one very negative (B3). 

Screenshot 2025-01-30 140117.png

Figure 6. Detection of tissue sample isoforms using gel electrophoresis. Purple bands were observed in the peroxidase standards (lane 3 and 4) and in tissue samples (lanes 5 and 6). Carrot extract (lane 5) contains four isoforms: two positive (B1, B2) and two negative (B3, B4). Celery extract contains three isoforms: one positive (B1) and two negative (B2, B3).  

The pIs of standards Cytochrome C, Hemoglobin-Albumin, HRP-Basic and HRP-Mixture are 10.2, 7.2, 4.8, 9, and 9 respectively [1]. Using the known pIs of the standard proteins and calculating the distance traveled of the standards on the gel (Figure 6), a standard curve of migration vs. pI was made (Graph 1). The distance and direction (negative or positive) traveled of the tissue extract isoforms were measured and inserted as y into the equation of the standard curve found in Graph 1.

Screenshot 2025-01-30 140343.png

Graph 1. Standard curve formed from known isoelectric points of protein standards vs. their migration distances (mm) determined from gel electrophoresis. The equation y = -7.9149x + 65.491 was formed. 

The direction (negative or positive) and distance traveled by the carrot and celery isoforms were noted and inputted into the equation formed from the standard curve in order to calculate their unknown isoelectric points. Table 1 is a summary of the results.

Screenshot 2025-01-30 140807.png

Table 1. Summary of  pI, net charge, and distance traveled of electrophoresed standard proteins and tissue samples, including their isoforms.  The direction in which the proteins and tissue sample isoforms traveled indicated their net charges as either positive or negative. This information was inputted into the standard curve equation (see Graph 1) and the unknown pIs were determined.   

Spectrophotometry

The amount of active peroxidase in carrot and celery extracts was determined using spectrophotometric analysis. A standard curve was generated using the peroxidase standards and their absorbance values (Graph 2).

Screenshot 2025-01-30 141031.png

Graph 2. Standard curve formed from known concentrations of peroxidase standards vs. their spectrophotometrically determined absorbances. The equation y = 0.0631x was formed.

Samples of the vegetable tissue samples were also placed into a spectrophotometer and their absorbances were measured at 575 nm. Using the equation provided from the standard curve (y = 0.0631x), the spectrophotometrically determined absorbances of the four vegetable extracts were inserted as y and their concentrations (x) were determined (Table 2).

Screenshot 2025-01-30 141359.png

Table 2. Concentration and absorbance of standards and vegetable tissue extracts determined from spectrophotometric assay. The concentrations of the Standards A-D were known. The absorbances of the standards and the tissue samples were measured by spectrophotometry. Using the equation of the standard curve (Graph 2), the unknown concentrations of the tissue samples were calculated.

The concentrations of peroxidase in the tissue extracts determined from the dot-blot, which is a semi-quantitative method, and the spectrophotometric analysis, which is a quantitative method, were compared. In the dot-blot, 100% carrot extract was approximated to be 0.5 µg/mL . However, both the 100% carrot extracts from the spectrophotometer results were below 0.5 µg/mL — one was 0.158 µg/mL and the other 0.192 µg/mL. In the dot-blot, the 100% celery extract was approximated to be 0.3 µg/mL which is very close to the results of the 100% celery from the spectrophotometric result of  0.349µg/mL but was lower than the other spectrophotometer result of 2.12 µg/mL. Generally, when comparing the results of the dot-blot analysis to the spectrophotometric analysis, the spectrophotometric analysis indicated a higher concentration of peroxidase in celery extract than in carrot extract, while the dot-blot indicated a higher concentration of peroxidase in carrot extract than in celery extract. 

 

Discussion

 

A key aspect in characterizing peroxidase isoenzymes involved finding their concentrations in vegetables, which was done through the semi-qualitative technique of dot-blot as well as the qualitative technique of spectrophotometry. This orthogonal analysis was meant to provide more information in our efforts to assess peroxidase concentration. However, it was surprising that the dot-blot results indicated that carrots had a lower concentration of peroxidase than celery, but the results of the spectrophotometer indicated the opposite. Given that dot-blot is only a semi-qualitative method and spectrophotometry is a qualitative method, the more conclusive evidence would seem to indicate that the celery indeed has a higher concentration of peroxidase activity. These results proved contrary to the hypothesis that carrots would have a higher concentration of peroxidase than celery given dicot anatomy and need for increased peroxidase activity for its secondary growth. When examining the spectrophotometric results closely, the two 100% carrot extract values are close to each other (0.158 µg/mL and 0.192 µg/mL). However, the 100% celery extract results were far apart from one another (0.349 µg/mL and 2.12 µg/mL). Although it is difficult to draw a definitive conclusion, the disparity in results of the 100% celery extract could be a source of question regarding our spectrophotometry results. The spectrophotometry equipment in the lab has previously been unreliable, also leading us to question the accuracy of its results. 

 

Another key element in our objective to characterize peroxidase isoenzymes involved localizing them in carrot and celery samples. Following staining with color development solution and Ponceau S, peroxidase activity was localized to the epidermis, cortex, and somewhat in the vascular cambium of the carrot tissue prints. In the celery extracts, peroxidase activity was localized to the epidermis and vascular bundles. These results aligned with our hypothesis that peroxidase enzymes would be localized to the epidermal edges of both the carrot and celery samples. We conclude from peroxidase’s localization in both plant extracts to the epidermis that peroxidase aids in synthesizing and strengthening the cell wall as well as protecting plants from toxins or microorganisms. We further conclude from peroxidase’s localization to the cortex in both plants that it must play a role in water and food storage since that is the function of the cortex region. Based on the peroxidase’s localization to the vascular cambium in carrots, we concluded that peroxidases play a crucial role in plant development since the cambium is an essential structure in dicot growth. 

 

A difficulty we encountered was the weakness of the Ponceau staining. The red was very light in color and based on previous research, it should have been detectable throughout the entire tissue-print. Instead, the red was mainly localized to the regions of the peroxidase activity — epidermis, cortex, vascular bundles, cambium. Since the red stain was not everywhere, this suggests that the carrot was not pressed enough and the proteins did not transfer to those areas of the tissue. Therefore, it is possible that peroxidase was located in other areas but the proteins did not transfer.

 

The results of our gel electrophoresis suggested that carrot extract contains four isoforms: one positive, one slightly positive, one negative, and one strongly negative. The results also suggest that the celery extract contains three isoforms: one positive, one negative, and one strongly negative. It can be concluded that peroxidases have multiple isoforms in both plants and celery, and therefore each must play distinct roles within the plants, as  confirmed by previous research. The varying amounts of isoforms in each plant suggest that carrots may have more varying types of peroxidases than celery do in order to fulfill a specific function in the various areas it is located in. These results do not necessarily confirm or reject that there are more peroxidases in carrots than celery. 

 

This study investigated peroxidase on multiple levels by localizing it and eliciting concentration in different vegetable samples, as well quantifying and comparing its isoforms in carrots and celery. By examining and engaging in orthogonal analysis, this study contributed to the information available about this critical enzyme and further-confirmed results from previous studies. Future directions for this research include studying different root and different stem vegetables, which may further validate the results and suggest the different roles peroxidase might play in different plant samples. In addition, comparison between young dicots and monocots versus mature dicots and monocots could perhaps determine whether  peroxidase is present in varying concentrations during different stages of development. 

References

1. Characterization of Peroxidase in Plants.  John N. Anderson (2003)

2. Molecular Biology and Application of Plant Peroxidase Genes. K Yoshida, P Kaothien, T Matsui, A 

Kawaoka, A Shinmyo (Epub 2002)

3. Synthetic Methods VI - Enzymatic and semi-Enzymatic. G. Grogan, Comprehensive Chirality (2012)

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