Michelle Campeau-Analytical Problem-Proteins and Fruit Allergies
Food allergies are a serious medical concern for some people and a mere annoyance to others. Unfortunately, food allergies, particularly those to fruits and vegetables, can be a side effect of another common allergy: seasonal pollen allergies, or hay fever. This condition is called oral allergy syndrome, in which the body reacts to proteins in fruits and vegetables in the same way it can react to pollen, causing an allergic reaction (1). Fortunately, the type of pollen allergy a person has can determine what kind of fruits and vegetables they're allergic to. As someone who suffers from this, this is an important issue to me. The allergic reactions I've experienced to fruits have gotten steadily worse over time, starting as just watery eyes while eating an apple about 7 years ago to having my lips swell up for over a day after eating a peach last week. I personally do not want to accidentally ingest a fruit that I am allergic to and have my throat close on me. Consequently, one thing that concerns me is how genetics can affect the fruit's proteins. Genetically altered foods are showing up on the market now (2), but how do these foods differ from "natural" or organic foods? Are there any different proteins? Are the allergen proteins even there?
My hypothesis is that any minor genetic altering of a plant will not affect the proteins that cause the allergic reactions. A conclusion can be reached by analyzing fruit samples of genetically altered fruits, "organic" fruits, and fruits that are neither genetically altered or "organic." The analyte could be a variety of things. For example, one could test the skin of the fruit for protein content as well as juices from the meat of the fruit. The matrix of the juice would also contain carbohydrates and other organic molecules.
1. Mayo Clinic. Food Allergy: Symptoms. http://www.mayoclinic.com/health/food-allergy/DS00082/DSECTION=symptoms
2. Harry A. Kuiper, Gijs A. Kleter, Hub P. J. M. Noteborn, Esther J. Kok. Assessment of the food safety issues related to genetically modified foods. The Plant Journal 2001, vol. 27, issue 6, p. 503-528.
UV-Vis Absorption Spectrometry
With fruit allergies stemming from oral allergy syndrome, different proteins in different fruits can trigger reactions. Also, the proteins allergic reactions will result from are due to an individual's allergy to a specific kind of pollen. Consequently, the wavelengths and extinction coefficient depend on which allergy and protein one is analyzing. For example, the proteins Art v 1 and Act c 1 are the proteins in kiwi that cause allergic reactions due to complications from mugwort pollen allergies. In order to determine the concentration of these proteins, HPLC-UV is used, analyzing the separated proteins at 280 nm. The solvent used in analysis for Art v 1 and Act c 1 were acetate and NaCl, and Tris and citrate, respectively.
In the analysis of allergen content of the kiwi and mugwort samples, the extinction coefficients used were found to me 0.59 +/- 0.06 for Art v 1 and 1.72 +/- 0.02 for Act c 1.
As stated earlier, the values for both the absorption and the extinction coefficient should differ, depending on which allergy and protein are being examined.
2. Milan Blanusa, Iva Perovic, Milica Popovic, Natalija Polovic, Lidija Burazer, Mina Milovanovic, Marija Gavrovic-Jankulovic, Ratko Jankov, Tanja Cirkovic Velickovic. Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method. Journal of Chromatography B 2007, vol. 857, issue 2, p. 188-194.
Blog 6: Chemical Structure and Standards
2. For the mal d 1 protein, the amino acid sequence is (1):
3. I was unable to find a company that sells the mal d 1 protein, or the mal d 2,3, and 4 proteins as well. However, the proteins can be extracted from an apple and purified (2). The concentration could be calculated by using Beer's law and a spectrometer (as mentioned in a previous entry) if the extinction coefficient is known.
1. All four electrophoresis methods seem like they would work just fine.
2. CGE seems like it would be the best choice. Although I could not find an example where the mal d proteins were analyzed via a CE method, CGE was used as a means to analyze the concentrations of other proteins in apples.
3. Since I was not able to find information directly on the mal d proteins, the following information is based off other proteins found in apples (proteins analyzed not known):
A coated fused-silica capillary is used with an internal diameter of 100um and a total length of 32.5 cm. TRIS, aspartic acid, SDS, and acrylamide were used as the buffer (pH 8). The voltage was 7kV, and the separation took less than 15 minutes. (1)
4. In the above example, they used a UV-vis spectrometer with two wavelengths analyzed (215 and 280). They had very low concentrations of analyte in the end (20 ug/ml). Although this would probably work for my problem, I think using a fluorimeter might be a better choice since fluorimeters are more sensitive, especially since the analyte concentration is so low. Also, the fluorescence excitation and emission wavelengths are known for my analyte.
1. Blanco Gomiz, Domingo et all., Size-based separations of proteins by capillary
electrophoresis using linear polyacrylamide as a sieving medium: Model studies and analysis of cider proteins. Electrophoresis, vol 24, issue 9, pg. 1391-1396.