Unequal amounts of sugars, photosynthetic action and enzymes found using comparative analysis in apple extract
Abstract:
Do apples at
different stages of development contain the same sugar structures, photosynthetic
action, and enzyme rates? Apples were obtained from an orchard at immature
and ripe stages. We then created extract by blending the solid apples up
and using cheesecloth to extract liquid solution. To determine what types
of organic macromolecules were present Benedict’s, Barfoed’s and Selivanoff’s
tests were run. Photosynthesis pigment tests were performed using paper
chromatography to determine how active the apples were in the photosynthesis
process at each stage of their development. Absorbance tests on a spectrometer
were run to determine if there was a difference in absorbing light energy
between the two apple stages. Thirdly, the apples were tested to determine
if enzymes action of polyphenoloxidase was present by observing a color change
in the catechol substrate. Through our tests and observations we found
that immature and ripe apples have the same photosynthetic action. However,
during early stages of development Chlorophyll a and b were present, while
in mature apples additional xanthophyll and carotene was present. We
also found that the monosacchardies and ketoses were present in both extracts.
Immature apples seem to have more free aldehyde and ketone groups than ripe
ones according to the Benedict’s test. Finally, both stages have the enzyme
PPO present. The pH test confirmed the mature apples were more acidic
than the immature ones. Finally, the Km value for immature apples was
5.3, while in mature apples it was 5.7.
Figure 1.
Benedict’s test results. This figure shows the immature apples having a brighter red color precipitate after the benedicts test was performed. Mature apples test are labeled with an M and immature with an I. Glucose was our positive control.
Discussion:
Fall is time of year that brings about thoughts of picking apples, cider mills and those wonderful donuts. This is the time when people can actually hand pick the best apples, which are fully-grown and ripe. This was the idea that drove our research, what makes those apples taste so good, and why do we only pick the apples when they are fully-grown? It was our original prediction that mature apples would contain more organic macromolecules, photosynthetic activity and enzymes than that of immature and unripe apples. This prediction was purely based on our limited knowledge of mature fruits versus immature fruits, and also by the information presented to us in the Course Packet (Maleszewski et al, 2002). We found that some of these predictions were true, while the rest of the testing proved us wrong.We ran several tests on the specimens, beginning with carbohydrate testing. While testing for different carbohydrates in our mature and immature apples, most of our research yielded the same results. In Benedict’s test, both the extracts of our ripe apples and immature apples turned red. We used glucose for a positive control for this experiment which turns a brownish red when reacted with Benedict's reagent because it is a reducing sugar. However, the mature apples turned a brownish red color, while our immature apples turned to more of an orange red color. The difference in color could be becuase there were different amounts contained in the samples. These results tell us that there were free or potentially free aldehyde and/or ketone groups present in both of the two groups of apples. When using Barfoed’s test, the immature apples yielded a dark brown chunky precipitate, while the mature apples formed a reddish-brown smooth precipitate. To compare these results to a positive control, we used glucose, which forms a precipitate. This insinuates to us that both the immature apples and the mature apples contained monosaccharides. This information also told us that both types of apples must contain polysaccharides since we know that sugars are present and monosaccharides were ruled out. The next test we performed in the carbohydrate lab was Selivanoff’s test, which would tell us if either ketoses or aldoses were present in our apples. We used a positive control of fructose for this test. Our findings were that both the mature and immature apples reacted within one minute by turning red. This revealed to us that not only were both our apples ketose, but that they were also both monosaccharide ketoses, which corresponds with Barfoed’s test.
Our prediction was that the mature apples would contain more carbohydrates than immature apples, but the findings were inconclusive because all the tests were positive and nearly the same for both samples. This would probably not explain why the apples taste different when they are mature and immature.
Next we took a look at the different photosynthetic properties in the two samples of apples. We were concerned with the photosynthetic activity of our mature apples versus our immature apples. By performing the pigment identification on the leaves of the samples, we found that carotene, xanthophyll, chlorophyll A, and chlorophyll B were all present in our mature apples, where as only chlorophyll A and chlorophyll B were present in the immature apples. We also found that according to the absorbance test, there was very slight difference in the wavelengths of the ripe apples and the unripe apples. Although the wavelength tests didn't show much evidence of difference in the absorbance of the pigments, the mature still contained more of them. There could have been human error, mechanical error, and just natural error that could have influenced our findings.
Our hypothesis stated that the mature apples would contain more photosynthetic activity than the immature, and this proved to be true from the thin layer chromatography. The mature contained all four pigments being tested for, and the immature only had two of the four pigments being tested for.
The final tests we ran on our apples were the test for enzymes. We tested for presence of the PPO enzyme in our apples, in which both the immature and the mature apples turned black when catechol was added. This indicates that there was indeed PPO enzyme activity present in both the mature and immature apple. We also did a pH test with pH paper and the mature apples proved to be more acidic than the immature apples. We had a little bit of trouble with finding the maximum rate and the Km, which is the affinity between an enzyme and its substrate (Maleszewski et al, 2002), but we found with the experiment of varying substrate concentrations that the mature apples had a slightly higher Km than the immature apples. These results were not certain which could be due to human error, machine error, and natural error.
Although the Vmax of mature apples was less than that of the immature, the Km of the substrate was greater. This means that there could be more enzymatic activity in the mature apples than in the immature apples. There could have been many sources of error in this lab including the spectrometers used and time intervals when the absorbances were taken may not been in absolute uniform intervals.
This project was accomplished over several weeks, containing many different tests to show the difference in the components of mature and immature apples. Although we had predicted that the mature apples would contain more carbohydrates, more photosynthetic activity, and more enzymatic activity, we were only correct on the latter two. The carbohydrate tests were inconclusive as to which specimen contained more but did tell us a great deal about the types of organic macromolecules present. However, the photosynthetic and the enzymatic activity were greater in the mature apples.
A test that could be done to find more conclusive evidence about carbohydrate concentrations could be the use of glucose testing strips, not on blood plasma, but on the apple extracts. For the test strips, we would place dry reagents on the strips and watch for a color change that could be read on a meter. We could use different enzymes to measure the amounts of glucose present in each sample. These enzymes could be glucose oxidase, hexokinase, and glucose dehydrogenase as reagents (Brown). It would be the same idea as testing blood glucose levels, but would be used on testing apple glucose levels. This would be a way to test for a difference in concentration in at least one sugar of the apples.