INTRODUCTIONIntroduction Many enzymes exist in multiple forms which involve proteins that can be separated on the basis of net charge and size (isoenzymes) in the same tissue(Hillis, Moritz, and Mable, 1996). These isoenzymes have the same catalytic activity, but may differ slightly in amino acid sequence or in conformation. The distribution pattern of isoenzymes is often characteristic of the particular tissue, and changes in the pattern have been used to study differentiation during development (Jensen and Fairbrothers,1983). For example, in plants some isoenzymes involved in flower color pigments are present in petal cells but not in root cells (Crawford,1990). Although various parts of a plant -roots, leaves, and flower parts appear to be quite diverse, virtually all their cells have the same organelles, but as the cells develop certain organelles may become modified ( Mauseth, 1995). This research sought to compare the distribution pattern of isoenzymes within the root, stem, and leaf of Zinnia elegans ( common name, Zinnia) by means of gel electrophoresis.
Methods and Materials
The first procedure in conducting this experiment was preparation of the tissue extracts for gel electrophoresis. A sample of root, stem, and leaf from the Zinnia elegans plant was cut up and weighed, the weight for each sample was 0.5 g. Each sample was placed in a mortar with 5ul of extraction buffer and 495ul of H2O and ground well with a pestle. The homogenate was poured into labeled 1.5 ml tubes and centrifuged for 5 min. The supernatants were then transferred into clean labeled 1.5 ml tubes and placed in the refrigerator. Each of the three samples root, stem, and leaf Two of the four primary methods of electrophoresis that were used during the course of this experiment were agarose gel electrophoresis and polyacrylamide gel electrophoresis (Hillis, Moritz, and Mable, 1996). In order to run the agarose gel electrophoresis an agarose gel was prepared, .35 g of agarose and 35 ml of 1X Tris-glycine were mixed in a Erlenmeyer flask which was then heated for 45 sec. in the microwave until the liquid became clear. After a few minutes warm agarose liquid was poured into the mold and the well comb inserted into the center of the gel. Once the gel had set up the comb was removed and sides of the mold were lowered the gel was then placed in the chamber containing 1XTris-glycine buffer. Samples of root, stem, and leaf 10 ul from each was mixed with 5 ul of 5X protein buffer and then loaded. Well (1) 6 ul of HRP 804,well (2) was skipped, well (3) 15 ul of root sample, well (4) 15 ul of stem sample, and well (5) 15 ul of leaf sample. The electrophoresis well top was plugged into the bottom buffer tray and the electrodes were connected to the power supply. Before turning on the power the amperage and running-time were set, amperage at 100 mA and running-time 45 min. During the 45min wait a peroxide stain was prepared in a Erlenmeyer flask by adding the following: 500 ul Choloronapthol, 200 ul Hydrogen peroxide, 600 ul Tris-HCL, Ph 8.3, and 18.7 ml of H20. When the running-time had elapsed the gel was removed placed in plastic dish and covered with peroxide stain. The plastic dish containing the gel was then transferred to a 37C water bath for 30 min.. For this experiment two polyacrylamide gel electrophoresis were run; the gels were made with the following: 11.52 ul of Tris-glycine, 4.4 ul of 30% Acrylamide, 8 ul of Temed, 80 ul of 10% Ammonium persulfate, and 3.52 ml of H20. The polyacrylamide mixture was then poured into the double-sided glass molds and a comb was inserted in the top of each gel. When the gel`s had set-up the combs were removed and the wells were rinsed with water. The double-sided glass molds were transferred to the electrophoresis buffer tray`s which contained Tris-glycine buffer. Polyacrylamide gel ( #1) was loaded in the following order going from left to right : well (1) 6 ul Hemoglobin Albumin, well (2) 6 ul Cytochrome C, well (3) 6 ul HRP Mixture, well (4) 6 ul HRP Basic, well (5) 5X ul protein buffer + 10 ul root supernatant, well (6) 5X ul protein buffer + 10 ul stem supernatant, well (7) 5X ul protein buffer + 10 ul leaf supernatant, well (8) 6 ul Congo Red, and well (9) 6 ul TBO. Polyacrylamide gel (#2) was also loaded from left to right in the following order : well (1) 6 ul Hemoglobin Albumin, well (2) 6 ul Cytochrome C, well (3) 6 ul HRP Mixture, well (4) 6 ul HRP Basic, well (5) 5X ul protein buffer + 10 ul root supernatant, well (6) 5X ul protein buffer + 10 ul stem supernatant, and well (7) 5X ul protein buffer + 10 ul leaf supernatant. The electrophoresis tops was connected to the bottom of the buffer trays in polyacrylamide gels (#1) and (#2). Both (#1) and (#2) had a running time of 20 min., amperage of 20 mA, and a voltage of 126 v. The difference between these two polyacrylamide gels was the way the gels were run. In polyacrylamide gel (#1) the electrodes were plugged in to the power supply backwards and in polyacrylamide gel (#2) the electrodes were plugged in to run a normal gel ( forwards). When electrophoresis was completed the same peroxide stain formula used on the agarose gel electrophoresis was also used on both the polyacrylamide gels. These two gels were then allowed to float in a 37C warm water bath for 30 min.
The results of the agarose gel electrophoresis are clearly visible in (photograph I and photograph I, a) wells 1, 3, 4, and 5 all show migration toward the positive electrode. Well (1) was the standard HRP 804 Mixture, well (3) 5X protein buffer + root supernatant , well (5) 5X protein buffer + leaf supernatant, and well (4) 5X protein buffer + stem supernatant. The results of polyacrylamide gel electrophoresis (#1) can be seen in (photograph III), wells (6) 5X protein buffer + stem supernatant and (7) 5X protein buffer + leaf supernatant show no significant migration however, well (8) standard Congo Red does show migration in the positive direction. The results of polyacrylamide gel electrophoresis (#2) can be seen in (photograph II and II, a), wells 1-7 all show migration toward the positive electrode. Well (1) Hemoglobin Albumin, well (2) Cytochrome C, well (3) HRP Mixture, well (4) HRP Basic, well (5) 5X protein buffer + root supernatant, well (6) 5X protein buffer + stem supernatant, and well (7) 5X protein buffer + leaf supernatant.
In both the agarose gel electrophoresis and the polyacrylamide gel electrophoresis (#2), positive migration could be seen in the root, stem and leaf. However, there was a noticeable increase in migration of the root and leaf that was evident in both the agarose gel and polyacrylamide gel. It is possible that two factors played a role in this outcome: (1). The fact that agarose gel and polyacrylamide gel separate proteins on the basis of size and charge (Chrambach and Rodbard, 1971). (2). The rate of movement will increase with net charge and strength of electric field and decrease with size (Hillis, Moritz, and Malde, 1996). Therefore indicating that there was an increase in the net charge and the size of the root and leaf proteins must be small in comparison to the stem protein. This research sought to compare the distribution patterns of isoenzymes within the root, stem, and leaf of the Zinnia elegans (common name, Zinnia) by means of electrophoresis. Through the findings from this experiment it can be concluded that the distribution patterns of isoenzymes was found to be very similar in the root and leaf during agarose gel and polyacrylamide gel electrophoresis (#2). The results of polyacrylamide gel electrophoresis (#1) proved to be inconclusive.