The difference in the structures of different plant tissues of plants that may be in the same family and genus made it necessary to carry out an experiment to determine the differences that exist between the stem tissues of broccoli and cauliflower. The point of interest was the differences in peroxidase enzyme concentrations and activity in the two plants. The experiment was carried out using dot blot assays, electrophoresis and spectrophotometry to determine various features of the plant tissues. The results show that the peroxidase enzymes in the tow plants are possibly of similar origins and of same molecular weights but are different in concentrations since broccoli recorded higher absorbance activity compared to cauliflower. While the experimental study was effective, it is possible that errors could have occurred during sampling procedures, especially where approximations were used.
Examination of plant tissues for proteins can be carried out through various methods. However, the most common methods in use include the dot blot assay and the spectrophotometry. Each of these methods helps to identify different features of the protein tissues in the sample. For instance, while the dot blot assay can help to determine which proteins are heavier than others based on the distance moved from the origin, spectrophotometry focuses on finding the actual concentrations of various proteins in the sample. Spectrophotometry applies the principle of absorbance, where substances with higher absorbance rates indicate greater concentrations in relation to other proteins (Rendina 48). The objective of this experiment was to compare and contrast the tissue characteristics of broccoli and cauliflower through comparison of the peroxidase enzyme contents in each of them. The comparison was carried out based on molecular weight using dot blot assay as well as through spectrophotometry.
Materials and Methods
Part 1: Dot Blot and tissue imprint
After putting on gloves, stems of broccoli were cut into thin strips using a scalpel and a total of 1g each placed into a mortar containing 1ml of peroxidase extraction buffer. The extraction buffer had been initially prepared using 2mM of MgCl2, 20mM of Nacl; NP40 of concentration 0.01 percent and 10mM Tris. This gave the buffer a pH of 8. The broccoli stem tissue was then ground in the mortar using a pestle until the mixture formed a homogeneous suspension. This suspension was later placed in a centrifuge tube labeled extract 1. The mortar and pestle used in the crushing were washed and dried and the initial process was repeated using cauliflower stems. The resultant extract from cauliflower crushing was labeled extract 2 and also placed in a centrifuge tube.
After preparation of both plant tissue samples, the extracts were centrifuged for 5 minutes each. A clean pipette was then used to draw off samples of extracts from the tubes. The first sample comprising of broccoli stem tissue was placed in a smaller centrifuge tube and labeled 100 percent Extract 1 while the second sample was labeled 100 percent extract 2.
900μl of deionied water was then placed in two small centrifuge tubes to which 100μl of each of the samples were then added separately. The first tube containing the broccoli stem tissue extracts and 900μl of deionized water was labeled 10 percent extract 1 while the one containing cauliflower extracts was labeled 10 percent extract 2.
After this, the first pair of gloves was removed and exchanged with a fresh pair to be used for handling nitro-cellulose, which is harmful for health. A petri dish containing 20ml deionized water was then used to wet a nitrocellulose sheet. Following this, a paper towel was moistened and placed flatly on the laboratory bench. The nitrocellulose membrane was then placed on the moistened paper towel. This was to prevent analytical errors, which would arise in case dry nitrocellulose membranes were used. The deionized water used to wet the membranes would act as carriers for the various constituents of the stem tissue extracts.
A metric ruler was then placed along the shorter edge of the nitrocellulose membrane and used to mark 4 points in a vertical straight line ½ cm from the ruler. The marks were placed 1cm apart. The first mark was placed at ½ cm from the top edge of the membrane. 5μl samples of standard peroxidase solutions were then placed on the marked points with the marks bearing concentrations in order. The concentration of the peroxidase standard at the first point was 0.01μg HRP per ml; this was followed by 0.1μg HRP/ ml, then 1μg HRP/ ml and finally 10 μg HRP/ ml.
5μl of extract 1 and extract 2 were measured in different concentrations and placed alongside the peroxidase standards. The first spot had 10 percent extract 1, followed by 100 percent extract; then 10 percent extract 2 followed by 100 percent extract 2. The extracts were then labeled with the laboratory group’s identity (i.e. group 1) and frozen for later use in electrophoresis. At the same time, the pipette solutions were given 5 minutes to be absorbed by the membrane.
Later, new pairs of gloves were worn and a broccoli stem of approximately 1 inch in diameter were cut using a scalpel. An equal sized cauliflower stem was cut and placed separately. Each of the cut cross-sections was then subdivided into four equal pieces in order to fit the available spaces on the nitrocellulose membrane. A dry paper towel was used to dry the pieces and then the pieces were pressed onto the membranes firmly. The broccoli was pressed on the top row while the cauliflower was pressed onto the bottom row. The membrane was then placed inside a petri-dish containing deionized water.
After formulation of the color development solution, the solution was used to replace the water in the petri dish after draining the deionized water. Only 15 ml of the solution was used to substitute the water over the membrane. The solution was discarded after 5 minutes then the petri-dish was rinsed with Deionized water and finally drained. The color development solution is prepared using 5m; chloronaphthol, 2ml 1M Tris buffer, 0.7ml hydrogen peroxide and 150 ml deionized water. The final step in the protein tissue imprint process involved staining with 15 ml of Ponceau solution in a bath for 5minutes. Pictures of the membrane were taken to indicate the protein imprints.
Part II: Electrophoresis
A solution of 0.6 g agarose was dissolved by heating in a microwave together with 50ml electrophoresis buffer in a 250ml beaker. After this, 35ml of the agarose solution was measured into a casting tray; the comb positioned at the center of the tray then allowed to set for 15minutes. The comb was only removed when the gel was solid enough and the tray gel inserted into the electrophoresis chamber so that the surface of the gel was covered by the buffer. After this, the previously prepared and frozen samples of 100 percent extract 1 and 100 percent extract 2 were thawed and 15μl of each of the samples pippetted into other tubes, which were labeled extract 1and, extract 2 for broccoli and cauliflower samples respectively. Following this, 15μl of the sample buffer for electrophoresis were pipette into new tubes. The sample buffer was prepared using glycerol, bromophenol blue and electrophoresis buffer. After placing the buffer solutions into the fresh tubes, the lanes were then loaded with 15μl of samples corresponding extract numbers i.e. lane 1, extract 1(broccoli); lane 2 loaded with extract 2 (cauliflower); lane 3 loaded with hemoglobin Albumin, lane 5 loaded with Cytochrome c; while lane 7 and 8 were loaded with HRP Basic and HRP mixture respectively. The cover of the electrophoresis chamber was then closed and the anode and cathode attached.
The electrophoresis voltage was adjusted to be 90V and the gel was set to run in the chamber for 35 minutes. Bromophenol blue, red hemoglobin and the blue serum were observed to move towards the anode while the orange cytochrome C moved towards the cathode. The visual differences made it possible to track the progress continuously through the process. After the 35 minutes, the electrophoresis system was disconnected, followed by the removal of the gel casing. The removed gel was stored for spectrophotometric analysis.
Part III: Spectrophotometry
Nine glass tubes were taken and labeled 1 to 9. For each of the tubes, the following were added. In tube 1, nothing was added. In tube 2, 40 μl of the 0.08μg/ml of peroxidase standard solution was added; in tube three, 40μl of 0.4μg/ ml of peroxidase standard solution; tube 4 – 40μl of 2μg/ml of peroxidase standard solution; tube 5 had 40μl of 10μg/ ml standard peroxidase solution; tubes 6 and 7 had 5μl and 40μl of extract 1 respectively while tubes 8 and 9 had 5μl and 40μl of extract 2 respectively. 5ml of color development solution was added to each of the tubes after this. The solution was prepared immediately before use by adding 500ml of water, 8ml of 1M Tris buffer solution, 1.5ml hydrogen peroxide, and 15 ml chloronaphthol. The tubes were then left to settle for about 3 minutes before reading the absorbances of the samples at 575nm using tube 1 as the blank. The absorbance data was recorded.
The objective of the three experimental parts was to compare the concentration of the peroxidase enzyme in the broccoli and the cauliflower stem tissues. From the Dot blot and tissue imprint, the objective was to determine the types of peroxidase enzymes that exist in both of the plant tissues and determine their activities. The following table gives the description of the results obtained from the tissue imprints.
|Source of Peroxidase||Distance from sample Well (mm)||Direction Migrated|
|Vegetable Extract I
Lanes 1 and 5
|Vegetable Extract II
(Lanes 2 and 6 )
|HRP-Basic (lane 7)||18 mm||Positive|
|HRP-Mixture (lane 8)||18 mm||Positive|
Besides the peroxidase enzyme, the dot blot assay also helped to identify the activity of other molecules such as hemoglobin, Albumin and Cytochrome C. The table below shows the results obtained from these particular molecules. The heavier molecules moved for the shortest distances from the point of origin.
|Distance Migrated (mm)||Direction Migrated|
|Hemoglobin (lane 3)||4 mm||positive|
|Albumin(lane 3)||11 mm||Positive|
|Cytochrome C (lane4)||10 mm||Negative|
From the spectrophotometric analysis, the results obtained were expected to give the same implication received from the dot blot assay. Spectrophotometry gives absorbances as functions of the concentrations of the target substances in the sample. Consequently, the higher concentrations are expected to give the higher absorbances. The following table shows the results obtained from spectrophotometry.
|Tube #||Addition||Absorbance at 575nm|
|2||40 μl Peroxidase Standard (0.08 μg/ml)||0.011|
|3||40 μl Peroxidase Standard (0.4 μg/ml)||0.084|
|4||40 μl Peroxidase Standard (2 μg/ml)||0.457|
|5||40 μl peroxidase Standard (10 μg/ml)||1.690|
|6||5 μl Extract 1||0.845|
|7||40 μl Extract 1||2.106|
|8||5 μl Extract 2||0.111|
|9||40 μl Extract 2||1.012|
From the above results, a chart of absorbances at 575nm versus the corresponding peroxidase standard concentrations was constructed as shown below.
This graph was used to calculate the concentrations of the peroxidase enzyme in the different samples based on their absorbances. In the straight-line equation presented above; y= absorbance at 575nm and x= concentration of peroxidase enzyme in μg/ml.
The dot blot assay indicated that the vegetable extracts 1 and 2 moved equal distances from the sample well i.e. 17mm. This was different from the distance moved by the Horse Radish peroxidase (HRP) which was 18mm. This showed that the HRP was lighter than the plant extract peroxidases. However, both cauliflower and broccoli have the same molecular structures for their peroxidase enzymes. The differences between the three sources of peroxidase enzymes show that there may be a possibility that the sources of peroxidase in animals and plants are slightly different. Moreover, the electrophoresis process gave the characterization of the peroxidase enzyme types in the two different plants.
Finally, while working through spectrophotometry, the absorbances of the different samples were compared with standard samples in absorbance. The absorbance of extract 1 was found to be 0.845 and 2.106 for 5μl and 40μl of the sample respectively. From the straight line equation describing the relationship between absorbance and concentration in μg/ ml and substituting for y, x which is equivalent to the concentration can be found as:
Y =0.167x + 0.029 and y = 0.845; x= 4.886μg/ ml of Peroxidase enzyme
For y = 2.106; x = 12.437 μg/ ml of peroxidase enzyme.
For the second extract, the absorbances were found to be 0.111 and 1.012 for 5μl and 40μl respectively. Using the same method used for extract 1 yield concentrations of extract 1 as 0.491μg/ml and 5.886 μg/ml respectively. The differences in concentrations show that broccoli has higher concentrations of peroxidase enzyme per unit mass compared to cauliflower.
Comparing the results obtained from the dot blot essay with those obtained from the spectrophotometry indicates that while the sources of peroxidase enzyme in the two plant stem tissues are the same and have same molecular weights, the activity of the peroxidase in each of the plant tissues is different, with higher activity being in broccoli. The experiment can be said to have been effective in accomplishing its original objectives.
Rendina, George. Experimental Methods in Modern Biochemistry. Saunders: Philadelphia. 1976.