Macromolecule Analysis and HPLC Protein Identification
to Analyze Differences in the Nutritional Content of Red Delicious Apples
Caused by Pesticide Treatment
By: G. Boyd, R. Howard, K. Jackson, K. Salvati, and R. Smith
Team
http://www.msu.edu/user/smith698/index1.htmÊ
LBS Biology II: Cell and Molecular Biology
Wednesday Section 1
Susannah Cooper
February 28, 2006
Abstract
Written by: Kyle Salvati
Revised by: Greg Boyd
Final Draft by: Kimberly Jackson
Over recent years,
there has been much debate about the differences between organically grown and
conventionally grown crops.Ê An
investigation using Red Delicious apples subjected to both organic and
conventional (i.e. treated with pesticides) growing methods was carried out to
determine any differences in types of carbohydrates present, protein content
(both overall protein content and specific proteins present), amount of
photosynthetic pigments, and overall appeal to consumers which are caused by
pesticide treatment.Ê Barfoedâs
Test, Selivanoffâs Test, and the Iodine Test for
Coiled Polysaccharides allowed for the determination of qualitative difference
in the structures of carbohydrates and sugars present in the apples.Ê A
Discussion
By: Richard Smith
Final Draft by: Kyle Salvati
The purpose of this experiment was to determine whether or not treatment of Red Delicious apples with pesticides affected the macromolecule content (specifically, the carbohydrate, photosynthetic pigment, and protein content) and overall taste of the apples.Ê Based on previous studies, we hypothesized that pesticide treatment would result in a few small differences between the macromolecule content (with the main difference being in photosynthetic pigment content) of the two treatments of apple, which would result in the organic apple being preferred.Ê Prior studies on the effects of pesticide treatment on carbohydrate content in various organisms showed that effects varied depending on both the species being treated and the pesticide being used in the treatment; the results of prior studies on the effects of pesticide treatment on protein content were even more conflicting, with no distinct relationship between pesticide use and protein content established.Ê However, studies conducted on the effects of pesticides on photosynthetic pigment content and overall test were quite decisive; in almost all studies investigated, pesticide use decreased the amount of photosynthetic pigments or activity in autotrophic organisms and one study showed that organically produced apples tasted better than pesticide treated apples.Ê Based on the results of tests conducted to analyze these four aspects (carbohydrate content, photosynthetic pigment content, protein content, and taste preference) of each treatment of apple, our hypothesis was partially, but not completely, supported.Ê As we predicted in our hypothesis, pesticide treatment did in fact alter the macromolecule content in the conventionally produced apples, which was shown by the results of experiments conducted to analyze photosynthetic pigment and protein content in the apples.Ê However, as the results show, these minute changes actually led to the apples treated with pesticides being preferred by consumers.
When analyzing the results of the many tests performed to investigate the effects of pesticides on apples, it was very clear which experiments supported our hypothesis and which refuted it.Ê Barfoedâs Test, Selivanoffâs Test, and the Iodine Test for Coiled Polysaccharides were used to examine the carbohydrate content of the two treatments of apples; none of these three tests showed any difference between organic and pesticide-treated apples.Ê After performing thin-layer chromatography and photospectrometry on the apple samples, it was determined that the pesticide-treated apples contained pheophytin B and xanthophyll while the organic apples did not (all other discovered pigments were shared by both apples).Ê The Bradford assay showed that the pesticide-treated apples had a slightly higher protein amount than the organic apples; HPLC analysis yielded a possible reason for this disparity in protein content, as the chromatogram produced by pesticide-treated apples showed the presence of a specific protein not found in organic apples.Ê Ultimately, these small differences amounted to a consumer preference of the apples treated with pesticides, which was shown by the taste test and accompanying survey.Ê Though these results seem fairly straightforward, in-depth analysis of the results leads to some interesting conclusions, especially for the carbohydrate tests.
Though none of the three carbohydrate tests showed a difference in carbohydrate content between organic and pesticide-treated apples, this evidence is nowhere close to conclusive due to the fact that all three tests are qualitative.Ê Since each of these tests is designed to indicate the presence or absence of particular types of carbohydrates, using these tests to create a comparison of carbohydrate content between two different samples cannot offer conclusive results. This is because a positive result, which marks the presence of one class of carbohydrate, will mask any potential differences in the class of carbohydrate represented by a negative result.Ê For example, Barfoedâs test indicates the presence of monosaccharides through the precipitation of a rust-colored solid; however, once this precipitant forms, no other conclusions about the carbohydrate content of the sample can be formed except that it contains monosaccharides.Ê Treatment with pesticides may eliminate all polysaccharides in Red Delicious apples, but as long as one monosaccharide remains, Barfoedâs test cannot verify this assumption.Ê
The same problem can be found in the use of Selivanoffâs test because it uses the speed of a color change to denote ketose or aldose presence; if the color changes quickly, ketoses are definitely present in the sample, but the presence or lack thereof of aldoses cannot be determined.Ê In the case of the Iodine test, which uses color change to denote starch presence or absence, a negative result occurred in both apple solutions, which does indicate that neither solution contained starch.Ê However, since both solutions had positive results for Barfoedâs and Selivanoffâs tests, their alleged similarity in carbohydrate makeup cannot be strongly supported.Ê In fact, based on previous experiments, this lack of difference in carbohydrate content after pesticide treatment seems very unlikely, seeing as three studies observed either a negative effect on overall carbohydrate concentration caused by pesticide use (Reganold et al., 2001), a positive effect (Saladin et al., 2003), or a varying degree of effect between the two treatments (Mostafa and Helling, 2002). ÊMuch like the carbohydrate tests, experiments conducted on photosynthetic pigment content of the two treatments of apples produced evidence which seemingly refutes prior research.
In our hypothesis, we stated that the main difference in the chemical composition of the apples would occur in the photosynthetic pigments they contained; after performing thin layer chromatography and photospectrometry on the two apple samples, this was shown to be the case, albeit with some confusing results that are not supported by prior research.Ê Based on the results of the TLC and photospectrometry tests, organically grown apples contain more total chlorophyll than pesticide-treated apples, which should indicate that organically grown apples can carry out more photosynthetic reactions than conventionally grown apples and thus benefit from having more ATP and NADPH.Ê However, the fact that pheophytin B was not present on any of the TLC strips for the organically grown apples contradicts this theory; this result is extremely puzzling because it does not make biological sense, nor is it born out by previous studies.Ê In the photosynthetic process, pheophytin is a key component because it acts as an electron acceptor and commences the whole electron transport chain which produces ATP and NADPH, molecules necessary for life.Ê For the organic apple to be completely devoid of pheophytin is biologically impossible and is shown as such by the very fact that the organic apple exists; therefore, the only possible conclusion is that pheophytin exists in small enough quantities in organic apples that it was not detected by thin-layer chromatography.Ê However, this conclusion is not supported by previous research; in fact, one study on a cyanobacterium species showed that certain insecticides inhibit the activity of photosystem II (Prasad 2004), which is the photosystem of which pheophytin is a part.Ê If pesticides have the same effect on apples, then the organic apples should contain more pheophytin than the apples treated with pesticides; however, according to our results, the opposite is true.Ê Thus, it appears as though our results are in direct contrast with previous research, meaning that either our results legitimately refute prior evidence or that we experienced a major error in the experimental process.Ê Experimental support of prior research or lack thereof is nowhere near as well-defined when analyzing the results of the protein tests, however.
Testing the
overall protein content of both treatments of apples by using a
Based on the chromatograms produced by the two different apple samples subjected to High Performance Liquid Chromatography, pesticide-treated apples contain an additional protein abundant enough to be detected by a photospectrometer which organic apples lack; however, this conclusion may not be too concrete due to the general low abundance of proteins in apples.Ê In HPLC, all the different proteins in a sample begin in the same stationary phase and are subjected to a constantly altering mobile phase (in this case, a mixture of acetonitrile and water with continuously changing concentrations).Ê When the correct (ãcorrectnessä is dependent on the specific protein) concentration of acetonitrile and water in the mobile phase passes through the stationary phase, a given protein will travel with the mobile phase to a photospectrometer.Ê The photospectrometer is a key component of HPLC because of its ability to constantly monitor the absorbance values at two different wavelengths: 214nm, which is the wavelength absorbed by peptide bonds between amino acid residues in a protein, and 280nm, which is the wavelength absorbed by phenyl groups present in the sides chains of three amino acids (tryptophan, tyrosine, and phenylalanine).Ê Because these two wavelengths are measured, the peaks produced on the chromatogram can be assumed to be proteins because the only molecules which would exist in a high enough concentration in the sample to create the peak and contain both peptide bonds and phenyl groups are proteins.Ê However, the relative height of the peaks presents a problem with the results.
ÊBy analyzing the chromatograms and determining
where peaks occur at both measured wavelengths, it is clear that
pesticide-treated apples contain three abundant proteins whereas organic apples
contain only two.Ê When comparing the
height of these peaks to the height of the peaks produced by the ribonuclease/BSA standard, however, it is equally clear
that the absorbance values of the proteins in the apples are minute.Ê This result is to be expected though, because
the amount of protein per gram of apple in the samples is miniscule (0.417 µg/g
in the pesticide-treated sample and 0.371 µg/g in the organic sample); it is
obvious that such a small protein content would lead
to small peaks.Ê Unfortunately, it also
complicates the results because the lone peak which conventional apples do not
share with organic apples is so close to a peak that is shared and has such a
low absorbance that this novel peak may be due to a small portion of the shared
peak which took longer to adhere to and travel with the mobile phase.Ê If this truly is a unique peak, however, then
the HPLC analysis corroborates the results from the
According to the results of the taste test, overall taste, appearance, and sweetness of apples can also be affected by pesticide treatment.Ê After surveying sixty students as to whether organic or conventional apples had the better appearance, sweeter taste, and overall better taste, it was determined that conventional apples were overwhelmingly preferred in each of the categories.Ê These results are surprising because they are completely contradictory to results obtained by previous studies.Ê One such study specifically states that growth via organic standards produce apples that are sweeter, less tart, and, since that was the defining characteristic for overall taste, better tasting overall (Reganold et al., 2001).Ê Thus, it appears that our survey data directly contrasts previous research, although this contradiction (and contradictions generated by other tests) may be attributable to flaws in the experimental process.
Over the course of the experiment, several flaws and unexpected problems arose which may have affected the results obtained in three of the tests.Ê These flaws predominantly occurred in the photosynthetic pigment test, due to both human and mechanical error.Ê The photospectrometers used to determine the absorption spectra of the various pigments were extremely finicky and did not consistently produce reliable results, which accounts for the erratic nature of the absorption spectra for almost every single pigment (Figure 6).Ê Also, untimely spilling of cuvettes containing the only sample of specific pigments led to rushed recording of measurements, which could have caused improper use of the photospectrometers and easily affected the data.Ê An unforeseen difficulty arose when conducting the HPLC analysis: due to the fact that the peaks in absorbance created by proteins were very small, it was impossible to distinguish between these rises in the absorbance value and the gradual rise in absorbance over the course of the entire experiment.Ê As a result of this, the initial experimental plan of specific protein identification was not carried out because extracts of specific proteins were unable to be collected and the identity of each specific protein was unable to be determined.Ê Finally, due to schedule constraints of the experimenters, the apples provided to the sixty students who participated in the taste test were prepared the night before and then were not refrigerated.Ê This could have affected the taste of the apples (especially if commercially produced apples contain some form of preservative) and led to aberrant data.Ê These errors more than likely created inaccurate data and led to unjustified conclusions, but these problems could be remedied with continued experimentation.
After analyzing the results of this experiment, it becomes apparent that several experiments (or alterations of the existing experimental process) could be carried out to increase the knowledge on the effect of pesticide treatment on apples.Ê Testing the carbohydrate content of the apples in a quantitative manner would produce very interesting (and much more useful) results concerning any potential differences in carbohydrate content caused by pesticides.Ê Also, a test could be conducted on the apple skins to quantitatively determine the amount of pheophytin in both organic and pesticide-treated apples; this would both help immensely to clear up the confusing data produced during this experiment by completely eliminating a large source of error (photospectrometer use) and either directly support or refute prior research.Ê Running HPLC with the two samples again would also be beneficial because, now that an approximate time for the appearance of the peaks has been established, the samples could be easily collected and then subjected to a tryptic digest and MALDI mess spectrometry to determine the identity of the specific, abundant proteins.Ê On a much larger scale, these same tests could be performed on a different cultivar of apple (for example, Granny Smith) to determine whether the effects of pesticides on the nutritional content of apples is contained to a few specific species of apple or if pesticide use has a universal effect on all kinds of apples.
Table
Authored by: Kimberly Jackson
Final Draft by: Greg Boyd
Table 1:Ê Rf valuesa of photosynthetic pigments present in organic and conventional (pesticide-treated) apple solutions and a spinach control tested using thin-layer chromotography.Ê
|
|
Spinach |
Organic |
Conventional |
|
|
Solvent |
1 |
1 |
1 |
|
|
Carotene |
1 |
0.96 and 0.75 |
0.77 |
|
|
Pheophytin B |
0.76 |
0 |
0.51 |
|
|
Chlorophyll A |
0.62 |
0.61 |
0.44 |
|
|
Chlorophyll B |
0.57 |
0.55 |
0.42 |
|
|
Xanthophyll |
0.52 |
0 |
0.28 |
|
a The Rf value was found by dividing the distance the pigment travelled up the TLC strip by the distance travelled by the solvent.Ê