Chromatography and oxidase assays show genetically altered peppers express low levels of PHS pigments & PPO enzyme

By Katherine Ruby, Jaime Murphy, Jon Selbig

Abstract completed by Katherine Ruby

This experiment explores three types of bell peppers: green, red and yellow. Two questions were analyzed through this experiment. Since green and red peppers come from the same plant, the first question sought an explanation for the differences between them. The second question explored the difference between the fruit of a genetically altered plant (yellow peppers) and that of a “natural plant.”

Several methods were used to test for sugars, the presence of photosynthetic pigments and enzyme characteristics. The first series of tests explored the presence of different types of sugars in the peppers. All peppers tested negative for starch. Both the yellow and red peppers were found to contain monosaccharides, while the green pepper did not. All three peppers appeared to be ketoses.

The pigments in each pepper were tested using paper chromatography. The red pepper contained six pigments, while the green and yellow contained three. Pigment abundance was also analyzed by the intensity of the color bands on the chromatogram strip.

The enzyme investigation confirmed the presence of PPO in all three peppers. It appeared that red and green peppers contain a greater amount of PPO than the yellow, however, our results were inconclusive.

The results of this experiment both supported and contradicted our hypothesis that yellow peppers would be completely different from the red and green. While some evidence did support this, our results yielded more similarities than expected.

Table 4: R_f values for pigments found in red, yellow and green peppers.

R_f = pigment distances/solvent front (measured in cm)

PEPPER	Pigment 1	Pigment 2	Pigment 3	Pigment 4	Pigment 5	Pigment 6
Red	.247	.461	.753	.899	.955	1
Yellow	.517	.719	1	N/A	N/A	N/A
Green	.124	.247	1	N/A	N/A	N/A

Discussion completed by Jaime Murphy and Katherine Ruby

This experiment analyzed two questions. Taking into consideration that green and red peppers come from the same plant, the first question sought an explanation for their differences. The third pepper studied, yellow, comes from a genetically altered plant. Therefore, the second question explored the difference between a fruit of an altered plant, compared to that of a “natural plant” (the green and red pepper plant).

We hypothesized that the green and red peppers would be quite similar in composition (although we expected slight differences). Since the yellow pepper was the product of genetic altering, we originally predicted vast differences from the red and green. First, a series of sugar tests were done to test our hypotheses. All except one test produced unanimous results for all three peppers.

A green solution in Bial’s test indicated pentose furanoses, while an olive/brown solution indicated hexose furanose. Each solution turned an aqua green shade. Therefore, it is probable that all three peppers tested are hexose furanoses. It can be inferred from this that the sugars found in each pepper have similar structures. We originally predicted that perhaps this structure would change through maturation.

The next test also produced similar results for all three peppers. When positive, the Iodine test for coiled polysaccharides produces a bluish black solution. This indicates the presence of starch. The solutions of all three peppers showed no color change when heated.

Starch is stored in underground organs, which includes storage roots, rhizomes, corms, tubers and bulbs (Armstrong, 2001.) Therefore it makes sense that peppers would not contain starch since they are grown externally on a plant. This contradicted our previous hypothesis. We predicted that at least one pepper would contain some amount of starch.

Used to differentiate between ketoses and aldoses, the solutions of Selivanoff’s test will react positively to form a red color. The primary difference between ketoses and aldoses is the position of the carbonyl on the carbon chain. The key in distinguishing between ketoses and aldoses in this experiment is the time frame it takes for the solution to react. Ketoses will react within one minute of heating, while aldoses may require several minutes. All three peppers reacted in under one minute, thus it can be inferred that the dominant sugars found in the bell peppers are ketoses (Luckie, et al., 2002). Barfoed’s was the only test where different results arose among the three peppers. This test is used to distinguish monosaccharides from di- and polysaccharides. The formation of a rusty color within two minutes of heating indicates monosaccharides. Both the yellow and the red peppers turned slightly rusty in color. The green, however, showed no reaction. Therefore, green bell peppers indicate di- or polysaccharides. Since the red pepper is simply a matured green pepper, it is likely that sugar composition changes during maturation.

Paper Chromatography was used to identify certain pigments in the peppers. Each pepper was expected at least to have some green pigment in the skin, in order to perform photosynthesis. However, because yellow peppers come from a genetically altered pepper plant, we anticipated that the yellow pepper would yield different results.

Using paper chromatography on a sample of skin from each of the peppers, we sought out to identify the quantity of pigments found in each pepper. By looking at the R_f values calculated for the three pepper samples, it can be seen that each pepper has its own unique set of pigments.

Before we analyze the pigments found in each pepper, it is relevant to note the importance of flavor. Flavor and pigmentation have a direct relationship: The stronger the color the pepper the stronger its flavor. We found red to be the most flavorful pepper, with green a close second. The yellow pepper was by far the most mild. The difference in taste of the peppers results from several aromatic substances affecting flavor, that occur in capsicums (Andrews, 57).

Red peppers are made up of the cartenoid capsanthin (35%) and 5 other pigments (Andrews 58). Our paper chromatography test supports this. We also found evidence that red peppers contain 6 pigments (Table 4). Therefore, it makes sense that the most pigment-abundant pepper has the strongest flavor and the richest color.

The specific pigments found in the green pepper have not been specifically identified, but studies have shown they do not contain capsanthin (Andrews 58). Since a red pepper is simply a matured green pepper, we can infer that capsanthin pigments form throughout maturation

Yellow peppers are the most mildly flavored of the three. They receive their yellow hue from the beta-carotene cucubitene, which is also a cartenoid (Andrews 58). Our paper chromatography test indicated that yellow peppers have three pigments. The pigments were extremely faint on the paper strip.

Another important aspect to consider is pigment abundance. The color bands on the chromatogram strips for both the yellow and green peppers were extremely light. From this we can infer that the pigments found in these peppers are not very numerous. On the other hand, the color bands on the red pepper’s chromatography strip were quite vivid. Therefore, it is probable that red peppers are very pigment abundant (Andrews 58).

As Table 2 and 3, as well as Figures 6 and 7 show, the light absorbency of a substance varies with differing wavelengths. Each pepper showed a distinct absorption pattern that was unique from each of the other two peppers. The green pepper showed a very low level of absorption for a prolonged series of wavelengths. This can be easily explained by simply looking at the pepper. Because the pepper appears green in color, it is apparent that green light is being reflected and therefore not absorbed. The level of absorbency in the green pepper began to decrease as the wavelengths corresponding to green light were approached. The level then began to slowly increase as the wavelength decreased and moved towards blue light.

In a similar manner, the red pepper showed a high level of absorbency at wavelengths corresponding to violet/indigo light and then continuously decreased as the wavelengths continued to increase and approach values corresponding with red light. Because the lowest absorbance was as the red wavelengths, these results are analogous to our original predictions that a pepper showing a red color would absorb red wavelengths the least.

The yellow pepper gave results that were surprising and unexpected. The peak of absorbance was at violet/indigo wavelengths similar to red, although the level continuously decreased throughout the remaining wavelengths. We did expect a decrease as the yellow wavelengths were tested, however we also expected to see an increase as we began testing in the orange and red wavelengths. The only explanation for this abnormal behavior of the yellow pepper is that the range of wavelengths that can be absorbed by the pepper may have been altered as a result of the genetic changes. The higher wavelengths may simply have been too high in energy for the pepper to be able to absorb.

Finally, we tested for the presence of PPO. The catechol placed directly on the peppers yielded no result. However, due to previous research, we knew that bell peppers did in fact contain PPO (Andrews 47). Therefore we found it necessary to test using an alternate method. Using the spectrophotometer, we measured the absorption of the stock solution versus the stock solution with catechol. The results of this test confirmed our hypothesis that PPO is indeed present in all three bell peppers (Table 5).

To verify our results, we ran the test again. Much to our surprise, we gathered drastically different data. This caused us to closely examine our procedure and to seek any errors that may have occurred. At this point, we came to the realization that we neglected to refrigerate our pepper solutions. Ideally, the solutions should have immediately been placed in ice.

Prior to performing the experiment, our peppers were stored in a refrigerator. Since we made our solutions directly after removing the peppers from 4 degrees Celsius, we believe that our first trial was more accurate. In this trial, red and green had similar absorbency differences (between the pure solution and solution with catechol). We analyzed that this may be due to the fact that they are grown from the same plant. Yellow on the other hand, had a very low absorbency difference. We assumed that this was a result of genetic altering.

Enzymes require very specific temperatures in order to function optimally. If the optimum temperature is exceeded, the shape of the enzyme (PPO) can be altered. As a result, this may alter the active site and inhibit the conjunction of the substrate (catechol) (Campbell 99-100). We believe this is what happened in our experiment because PPO must require very low temperatures and we failed to provide these conditions.

However, we also realize that there is a logical objection which someone might make against this explanation for the varied results. In a great deal of places where peppers are sold, they are not refrigerated. Therefore, one would assume that those conditions would be sufficient to cause the altering of the enzyme mentioned above. This objection is entirely legitimate and true in it’s basis. However, the temperatures endured by the enzyme both in the store and in the laboratory when we failed to refrigerate them were most likely not temperatures high enough to permanently denature the protein. Enzymes can be temporarily altered when subjected to temperatures higher than their optimum temperature, but may then return to the active state when given the proper temperature at which it may function. With this realization in mind, we realize that our experiment may have been able to produce more accurate results if we had returned the pepper solutions to the refrigerator for a period of time to allow the enzymes to become active again.

Our results can be supported by the data from our peers. Team Red did a similar experiment. However, they analyzed red, orange and yellow peppers. The results of the peppers common between our group and theirs (yellow and red) are equivalent.

As with any experiment, there are factors that can cause error in the results that are obtained. The first error that may have been encountered originated with the purchase of the pepper. As we looked over the peppers, we searched for a green pepper that showed only green, with no signs of red yet. Likewise, we did the same for the other two peppers, in order to find the purest peppers possible for our experiment. If, for example, the green pepper had begun the process of maturing into a red pepper, the results of the tests for the green pepper would have been much closer to the results of the red pepper tests. Therefore, we tried to find the greenest pepper possible to ensure that the pepper was in the earliest stage of maturation possible. However, because we were unable to grow the peppers ourselves and know when they were planted and how well the conditions were for their growth, it is impossible for us to know exactly what phase of growth the peppers were in.

In order to get the best and most accurate results from this experiment, it would be ideal to grow the peppers ourselves to ensure that each pepper was grown under identical conditions. If, for example, the purchased peppers had been sprayed with a type of pesticide that somehow altered the growth of the pepper, that particular pepper may then have different characteristics than the other two peppers that were purchased. By knowing when the peppers were planted, how they were raised, and when each pepper ripened and was picked, a great number of potential errors could be removed. If more time were available to perform the experiment, this would be the ideal way to start, ensuring that no sources of error were introduced before the actual testing began.

In the future, if we were able to perform this experiment again with an unlimited amount of time and resources, there are many things we would like to do differently. First of all, it would be beneficial if the experiments could be carried out in a more compact space of days. Because the experiments were spread out to once or twice a week for three weeks, we were forced to use three different batches of peppers in order to ensure that old or degraded peppers would not alter our results. This in itself causes a small source of error because the peppers that are being compared are not the same peppers in each investigation. Under ideal circumstances, we would like to perform this series of experiments numerous times. We would prefer to be able to run the full series of experiments on the same red, green, and yellow peppers to get a base idea of what we can expect the results to look like. However, because not all peppers are the same, we would also like to be able to run this experiment again on a large sample of peppers to gather more accurate results for the respective colors of pepper. We feel that this would be a very good basis on which a greater understanding of the similarities and differences between these three peppers may be achieved.