The Pepsin Pals

 

Abstract:

       Our goal was to compare red cabbage (Brassica oleracea var capita rubra) and
lettuce (Lactuca sativa) through a series of tests to determine similarities and differences in carbohydrates, photosynthetic pigments, and enzymes.  Our hypothesis was that red cabbage and lettuce would have similar carbohydrates and enzymes, but different photosynthetic qualities.
        Lettuce and red cabbage performed differently in the Benedict's and Barfoed's tests with lettuce having free ketones or aldehydes and monosaccharides, and red cabbage with neither.  Bial's test for furanose or pyranose rings showed that lettuce has pentose-furanose rings while red cabbage has hexose-furanose rings.  Selivanoff's test for structure showed that red cabbage has disaccharide ketoses present while lettuce has monosaccharide ketoses.  According to the Iodine test, neither lettuce nor cabbage contains starch.  The paper chromatography test showed similarity between lettuce and red cabbage in that the Rf values infer that both contain carotene, xanthophyll, chlorophyll a, and chlorophyll b.  The differences in absorption spectra are that lettuce peaks at 400nm and shows a small peak at 670nm, whereas red cabbage peaks at 400nm and has a large peak at 610nm.  Lettuce has polyphenoloxidase (PPO) but red cabbage does not.  The effect of pH on lettuce peaked at the pH values 6.5 and 7.5.
        We found that our hypothesis was incorrect.  Red cabbage and lettuce, though they seem similar because of shape and growth, are actually very different.  Our research implies that red cabbage and lettuce differ not only for photosynthetic activity, but also for enzyme activity and carbohydrate structure.


  Table:

Table 1:  Photosynthesis Rf Value Results

    This table shows the results for the paper chromatography test for both red cabbage and lettuce.  This test was done to determine which pigments were present in our samples.  Rf calculations for all of the pigments were found by dividing the pigment distance by the solvent front distance.  They were compared to the controls for each pigment.  The green band is then considered to be chlorophyll b, the bright green band is chlorophyll a, the yellow band is xanthophyll, and the orange/yellow band is carotene.  Both red cabbage and lettuce show bands for all four of these pigments.


      

		Green Band	Green Band	Bright Green Band	Bright Green Band	Yellow Band	Yellow Band	Orang/yellow Band	Oramge/yellow Band	Solvent Front
		Distance (cm)	Rf Value	Distance (cm)	Rf Value	Distance (cm)	Rf Value	Distance (cm)	Rf Value	Distance (cm)
	Trial 1	1.7	0.122	2.8	0.201	4.8	0.345	13.9	1	13.9
Red Cabbage	Trial 2	1.3	0.119	No Band	No Band	3.0	0.275	10.9	1	10.9
	Trial 3	1.1	0.091	1.8	0.149	4.1	0.339	12.1	1	12.1
	Trial 1	2.0	0.180	2.7	0.243	4.1	0.369	11.1	1	11.1
Lettuce	Trial 2	1.5	0.121	2.3	0.185	3.5	0.282	12.4	1	12.4
	Trial 3	2.1	0.186	3.0	0.265	4.6	0.407	11.3	1	11.3

  Discussion:

        In this experiment we examined Brassica Oleracea (cabbage) and Lactuca Sativa (lettuce) in a variety of carbohydrate, enzyme and photosynthesis tests.  Through these tests, we wanted to determine the differences between red cabbage and iceberg lettuce.  For a control, we used the facilitated experiments in the lab manual (Maleszewski, 2003 Pps. 63-84).  Each of the control experiments came out as expected and created a basis to compare each test with and the possible results therein.
        Brassica Oleracea contains many vitamins, anti-cancer and antioxidant compounds.  Aside from enhancing antioxidant effects in the body, red cabbage also aids in detoxification.  Other compounds in cabbage slow tumor growth and act as cancer inhibitors (Wolford et al., 2001).  Phenols present in cabbage can help to prevent the development of carcinogens (Christman, 2002).  Cabbage is often used in coleslaws, various salads, or served by itself either pickled or boiled.
        Lactuca Sativa is a good source of vitamin A, potassium and also contains vitamin C, calcium, iron, and copper.  Just like cabbage, it contains anti-oxidants, which can prevent various forms of cancer (Wolford et al., 2001).  Lettuce is particularly popular in salads and is commonly used as a garnish (Neild, 1990).
        Originally we hypothesized that Brassica Oleracea and Lactuca Sativa would be similarly structured in organic composition and enzymes present but that they would differ in photosynthetic qualities. Our predictions were not completely correct as the results show that red cabbage and lettuce are in fact different in structure, photosynthetic activity and enzyme activity.
         The first set of experiments we did consisted of carbohydrate structure tests.  The results of the carbohydrate tests we conducted imply that red cabbage and iceberg lettuce are not structurally similar. We conducted five different carbohydrate tests: Benedict's, Barfoed's, Selivanoff's, Bial's, and an Iodine test (Table 4). 
        In the Benedict's test it was observed that lettuce contains either a free ketose or free aldose because it formed a red precipitate, telling us that lettuce contains reducing sugars.  In the same test for red cabbage, no precipitate formed, so it does not contain a free ketose or aldose.  “In a solution of pH 8 or higher the [reducing] sugar is capable of reducing certain weak oxidizing agents such as cupric hydroxide along with a resultant oxidation of the carboxyl group of the sugar” (Maleszewski, 2003 pg. 64).  This result could help explain why lettuce is an affective anti-oxidizer. 
        The Selivanoff's test followed the Benedict's test further, not only showing that a free ketose or aldose was present, but also classifying cabbage and lettuce as having either one or the other.  As ketoses are faster to react than aldoses, and because the test for lettuce changed to red in less than a minute a monosaccharide ketose was present.  Red Cabbage took slightly longer, approximately one minute, which implies that disaccharide ketoses are present.
        A green or olive colored product resulting from Bial’s reagent is indicative of pentose-furanose rings.  A muddy brown colored product, however, indicates a hexose-furanose ring. Our results from Bial's test showed that lettuce had a pentose-furanose ring since a color change to olive green was observed.  For red cabbage the test results confirmed that it contains hexose-furanose rings because a muddy brown color resulted.
        “Carbohydrates may contain one sugar molecule (monosaccharides), two sugar molecules (disaccharides) or many sugar units (polysaccharides)” (Maleszewski, 2003 pg. 63). Barfoed's test for lettuce showed rust colored precipitate that implied that monosaccharides were present.  The same test for red cabbage showed no color change and therefore the sugars present were not monosaccharides. 
        The final carbohydrate test we performed was the Iodine experiment, which tests for starch.  When starch is present the sample will turn a blue-black color. The solution of red cabbage did show a color change but it did not turn the blue-black color of starch indication, instead it changed to a gray-brown.  The color change is apparently a mixture of the reddish purple cabbage solution and the yellow-brown of the Iodine.  The lettuce solution did not turn color and therefore starch was not present. 
        Our carbohydrate results imply that the carbohydrate make-up of red cabbage is disaccharide ketoses and pentose-furanose rings.  Lettuce's carbohydrate make-up was found to be free ketoses, monosaccharide ketoses, and pentose-furanose rings. 
        An interesting observation during both the Bial's and Selivanoff's tests was that the red cabbage solutions changed to a pink when the reagents were added prior to the actual tests.  This is due to red cabbage being a pH indicator and both Selivanoff's and Bial's reagents being acidic (Cobb, 1998).
         The results we obtained for photosynthesis disagreed with our hypothesis.  We performed a pigment identification test using chromatogram strips to determine which pigments the cabbage and the lettuce contained.  We were able to determine various pigments by the color and the distance the solvent moved down the strip over a period of time. Both lettuce and cabbage had carotene (orange-yellow), xanthophylls (pale yellow), chlorophyll a (blue-green) and chlorophyll b present (pale green).  The Rf values obtained from the tests represent positive identification of the specified pigments we found (Table 1).  Each Rf value represents the distance each pigment traveled divided by the solvent front distance. 
        The absorption spectrum test was done to show the absorption of wavelengths of light given off by separate pigments present in each species.  Both cabbage and lettuce were different, supporting the original claim. As the wavelength was increased from 400nm to 700nm, the absorption of light by the lettuce solution decreased with one noticeable increase at 670nm.  Most photosynthesis is done at the 400nm to 535nm range with a small area of increased photosynthesis around 670nm.  As the wavelength was increased from 400nm to 700nm, red cabbage overall decreased, but had several points of increase along the way at approximately 400nm and 610nm (Table 3).  Red cabbage photosynthesizes well from 400nm to 640nm with peak photosynthesis occurring at 610nm.  Red cabbage absorbs well in the blue light (~400nm) region as well as the green light (~600) region, but not well in the red light (~700nm) region therefore, red light is reflected and it produces the red color. 
        New solutions of the red cabbage and lettuce were made for the absorption tests because they looked less colorful and had a slight stench (Figure 8).  It was suspected that theses old solutions could affect the results of the photosynthetic tests.  This was found to be partly true as the red cabbage absorbance spectra for the new and old solutions varied drastically (Figures 11 & 12).  Also, the absorption spectra for old solutions were higher in absorbance than those for the new solutions.  It is speculated that this is due to either the deteriorating cells absorbing light or the fact that the new solutions were made with leaves from further within each species.
        The tests for enzymes differed from our original hypothesis that red cabbage and lettuce would contain similar enzymes.  The results we obtained through our experiments, however, showed that the lettuce contained polyphenoloxidase (PPO) while the cabbage did not.  Because PPO catalyzes the oxidation of catechol it was used to test for the presence of PPO.  When catechol was added to the lettuce, it turned a dark brown color from its normal pale green. We tested the effect of pH on the lettuce to see how different levels of pH would react with the lettuce.  The results showed a general increased, in absorbance as pH increased but since the cabbage tested negative for PPO this didn't have any significant bearing on our results.
         During these tests various problems arose that may have affected our data.  These include human error in measurements and perception of color change.  Also mechanical errors may have occurred during the use of the spectrometers.  Contamination of the species involved is also an issue because we did not personally grow them, and therefore could not control both environments in an identical manner.
        After the completion of all of the tests, we found that our hypothesis was mostly incorrect.  The evidence implied that red cabbage and lettuce differ in enzyme activity and carbohydrate structure.  The only similarity between the two was found in the paper chromatography, which showed a similarity in photosynthesis, but according to the absorption spectra, the two are not entirely the same in photosynthesis either.