Peroxidases are a common enzyme that are found in plant structures and were found to have multiple functions. A lot about this enzyme remains unknown to scientists. They differ in concentrations, location and function depending on the type of plant being used. In this experiment four different procedures; tissue print, dot blot, gel electrophoresis and spectrophotometric assay; were used to determine the concentration, localization and isoelectric points of peroxidase enzymes in carrot and celery extracts. The results of these techniques showed that carrots contain a higher concentration relative to celery. Since both of the vegetables had similar localization, it suggests that there is a correlation between function and location. The enzyme plays a similar role of protection in both vegetables.
Peroxidases have many functions and are composed of a large class of enzymes. They are found distributed throughout plants, rather than in one specific region. They are known to catalyze the oxidation of phenolic compounds while releasing hydrogen peroxide and involvement with the synthesis of the cell wall. The different forms of peroxidases are called isoenzymes. There is over one hundred isoenzymes that differ in protein sequence. Although there were many studies done using peroxidases, there is a lack of understanding of their function. The challenge of uncovering their functions is due to the many forms of peroxidase enzymes.The understanding of this enzyme remains of interest to scientists.
Previous studies identified two roles that peroxidase can play in plants. According to the first hypothesis, peroxidase assists in the synthesis of the plant cell wall. This theory suggests that the enzyme strengthens the cell wall by cross-linking phenolic residues of polysaccharides and glycoproteins in the cell wall, which are both important for cell strength.
The second hypothesis states that peroxidase protects the cell from microorganisms and toxic chemicals. It is involved in cell healing. It can protect the cell from harmful chemicals such as alcohol and hydrogen peroxide. However, the precise mechanism and function of peroxidase in both of these theories is still unknown because the enzyme is still present in the plant system even when the plant is not undergoing stress. Additional research suggests that some peroxidase isoenzymes are involved in normal developmental processes.
The goal of this experiment was to utilize techniques to identify the function of peroxidase in carrots and celery. It is known that peroxidase serves to keep the cell wall strong in these vegetables, therefore it will appear on the edges of the tissues by the cell wall. The epidermis protects the cell, but not the inner softer tissues of the vegetables, and serves for mineral absorption. Peroxidase also serve to protect the cell from harmful chemicals. Harmful chemicals such as microorganisms can enter the cell through vascular systems. Peroxidase is found at vascular bundles.
A sheet of nitrocellulose was dampened by floating it in a petri dish with distilled water. The membrane was placed on a paper towel, and excess moisture was removed by blotting the membrane dry with a paper towel. The membrane was placed horizontally on a paper towel and aligned with a metric ruler. 5 uL of each of the four peroxidase standards (1-4) were pipetted onto the nitrocellulose membrane in specific spots. 5 uL of “10% celery” and “100% carrot were pipetted onto the membrane. This step was repeated with the “10% celery” and “100% carrot”. 5 minutes were given for the solutions to be absorbed on the membrane.
Using a razor blade the celery and carrot were cut to produce a cross section. The surface was carefully blotted onto a dry paper towel to remove excess moisture. The cut vegetable was pressed down onto the nitrocellulose membrane and held in place for 10 seconds. Two prints were made for each vegetable and a fresh cut was done for each print. After all tissue prints were prepared, the nitrocellulose membrane was placed in a petri dish with distilled water. The water was poured out and 15 mL of color development solution was added. The development of a purple color was observed. After 5 minutes the color solution was removed and distilled water was added. Tissues that contain peroxidase were identified. 15 mL of protein Ponceau S, blot stain, was added to the dish. After 5 minutes the staining solution was poured off and the membrane was washed three times with water.
Gel Electrophoresis
Two Eppendorf tubes were labeled, one carrot and one celery. 10 uL of bromophenol blue loading dye was added to each, and then 20 uL of “100% celery” to the celery tube, as well as 20 uL of “100% carrot” to the tube labeled carrot. 6 samples were loaded into the prepared agarose gel, left to right, as follows: lane 1 – 15 uL of Cytochrome C; lane 2 – 15 uL of Hemoglobin-Albumin; lane 3 – 8 uL of HRP-basic; lane 4 – 8 uL of HRP-conjugated; lane 5 – 30 uL of carrot sample; lane 6 – 30 uL of celery sample. The gel was then run at 135 Volts for 30 minutes, until the blue loading dye was close to the positive end of the electrode. The gel was removed from the electrophoresis and transferred to a weighing boat. The positions of the colored standard proteins were recorded. A ruler was used to measure the distance and direction that the standards travelled. 20 mL of prepared peroxidase substrate solution was added to the weighing boat containing the gel, which was then incubated at 37 degrees for 30 minutes. The distance and direction of each peroxidase isoenzyme was noted.
Spectrophotometric Assay
A spectrophotometer was used to determine the concentration of peroxidase enzymes in celery and carrot extracts. Nine test tubes were prepared a blank, 40 uL peroxidase standard, 40 uL peroxidase standard B, 40 uL peroxidase standard C, 40 uL peroxidase standard D, 5 uL 100% carrot extract, 40 uL 100% carrot extract, 5 uL 100% celery extract and lastly, 40 uL 100% celery extract. 5 mL of color development solution was added to each test tube and contents were mixed. After three minutes, absorbance was read and recorded at a wavelength of 575 nm. A standard curve was made based on the known sample concentrations. The concentration of the unknowns were determined using the equation from the linear regression line of the standard curve.
