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Juacalla, Katherine Joy C. Kalalo, Donna Marie S. Ledesma and Nenet V. Licuanan Group 6 2F-Pharmacy Biochemistry Laboratory ABSTRACT
Gluten was the protein found in wheat which remained when wheat dough was washed to take away starch granules and water-soluble constituents. The presence of superfluous starch in the dough was tested by addition of iodine solution to the washings, wherein blue-violet coloration indicates such presence. Gluten was then isolated, and situated in an enzymatic substance for hydrolysis, which was then subjected to different qualitative tests. Paper chromatography was performed to analyze the different amino acid components of the protein.

Proteins, also known as polypeptides, are organic compounds made of amino acids arranged in a linear chain and folded into a globular form [5]. They are naturally known for having molecular weights greater than 5000. These macromolecules show great diversity in physical properties, ranging from water-soluble enzymes to the insoluble keratin of hair and horn, and they perform a wide range of biological functions. The amino acids in a polymer are joined together by covalent bonds called peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The physical and chemical properties unique to each amino acid are results of the structure and chemical properties of the R groups. Gluten is a mixture of two proteins, glutenin and gliadin. It is also the composite of a prolamin and glutelin, which exist, conjoined with starch, in the endosperm of various grass-related grains. Of the gluten proteins, gliadrin has a relative molecular mass between 30000 and 80000 Da whereas glutenin is multi-chained with relative molecular mass up to several million Da. It is a long molecule having strong and flexible characteristics. It is described as a yellowishwhite, tough, elastic, and sticky protein. Given these characteristics, gluten became useful in bread-making; for it traps the carbon dioxide (CO2) produced by the reaction of flour and yeast and gives flour its characteristic chewiness, helping it rise and keep its shape. Isolation of proteins is the process for separating a type of protein from a complex mixture. Its importance is to characterize the proteins solubility, acid-base property, function, structure, and interactions. Proteins can be isolated depending on their size, shape, charge, affinity, hydrophobicity, and other physiochemical

characteristics. Some methods used are heat denaturation, chromatography, and ultracentrifugation. Other methods are used in this experiment, which are for casein and myoglobin. As for gluten, it is separated through its difference in solubility. Insoluble proteins are easily isolated, removing other substances that are soluble by washing. Hydrolysis, on the other hand, is a chemical process of decomposition involving the splitting of a bond and the addition of the hydrogen cation and the hydroxide anion of water [4]. In basic terms, it was performed to break the peptide bonds which results to smaller amino acid chains (or peptides), and free amino acids. The solution containing the protein pieced is called a hydrolysate. The protein which is hydrolyzed in the experiment is gluten, obtained from wheat flour. Color reactions are done to determine the specific amino acid components of gluten. The objectives of the experiment involve the isolation of different proteins such as casein and lactalbumin from skimmed milk by isoelectric precipitation, gluten from wheat flour by difference in solubility [1], and myoglobin from beef sample by salt-induced precipitation using ammonium sulfate; analysis of chemical groups responsible for color reactions and explanation of the principles involved for each test; and determination of protein concentration by Bradford Protein Assay method, which is based on the binding of Coomassie dye to proteins in acidic solution leading to an increased absorbance of the sample at 595 nm.

EXPERIMENTAL A. Samples used

Wheat flour, tap and distilled cheesecloth, gluten, iodine (I2) solution. water,

B. Procedure
Water was added to one (1) cup of wheat flour to make thick dough. The dough was then wrapped with cheesecloth, and then placed under running tap water until all starch is removed. The washings were tested with iodine solution until negative results were obtained. Unwanted presence of starch was determined if the washings produce a blue-violet coloration, indicating iodine-starch complex [3]. Then, the sample dough was subjected to refrigeration.

Observations indicated that upon addition of iodine (I2) solution to the washings, blue-violet coloration imply presence of unwanted starch. After opportune washing, no more coloration was observed, indicating that all starch was removed. The insoluble material remaining in the dough was identified as the crude gluten [1]. After refrigeration of the sample, it exhibited a stickier form, with a slight darkening in coloration and emitted a pungent odor. The observations examined were analogous to the given characteristics of gluten.


Figure 1: Thick dough as a result of adding water to wheat flour.

Figure 3: Blue-violet coloration upon adding I2 solution.

Figure 2: Washing of the thick dough wrapped in cheesecloth. Thick dough was formed upon addition of distilled water to one (1) cup of wheat flour, using mortar as container. Portion of the formed dough was wrapped in cheesecloth and placed under running water, with beaker serving as a receiver for the washings. Continuous washing was done in order to remove all the starch. Intermittent testing of washings was done in order to determine overall removal of starch.

Figure 4: Washings remained clear after iodine test, indicating removal of all starch. The principle involved in the isolation of gluten is difference in solubility. The starch is partially soluble in water while gluten is insoluble in water. Thus, gluten can be separated from starch. Iodine solution is used to test the complete removal of starch, which involves the formation of blue-iodostarch complex). Refrigeration keeps the protein from degrading. [2]

[1] Crisostomo, A., et al. (2011). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc. [2] Protein Isolation and Analysis. 0_580/mlab/protein_isolation.htm. 01/16/2012 [3] Dashman, T. Baker N. and Blocker, D.E. (2008) Laboratory Manual for Human Nutrition. 2nd ed. The Netherlands: Harwood Academic Publisher GmBH [4] Retrieved from: sis. 01/15/2012 [5] Demain, A.L. and Pasupuleti, V.K. (2010) Protein Hydrolysates in Biotechnology. New York: Springer Publishing, Inc.