Experiment 2: Starch Hydrolysis by Amylase

Theoretical Background Polymers of carbohydrates are called polysaccharides, and make up some of the most important naturally occurring compounds [1]. They have thousands of monosaccharide units linked to each other by oxygen bridges. They include starch, glycogen, and cellulose, all three of which yield only glucose when completely hydrolyzed [2].

1, 4 glycosidic bond

Figure 1. Starch (amylose) (A) and cellulose (B)

Starch occurs naturally in plants, which use it to storage glucose units for energy. It is often found in seeds and tubers (e.g., potatoes). It consists of two kinds of polymers of glucose. The simpler kind is called amylose, and it makes up about 20% of starch. It is basically a chain of glucose units linked by 1,4 glycosidic bonds. During digestion, the oxygen bridges are hydrolyzed and the glucose units are broken up. 80% of starch is a water insoluble fraction called amylopectin [2], which is a branched chain polysaccharide with again 1,4 glycosidic bonds. At approximately every 25 glucose units, a branching of glucose units, exists. Upon treatment with acid or under the influence of enzymes, the components of starch are hydrolyzed progressively to dextrins (mixture of low melting polysaccharides, made up of 3 8 glucose units), maltose and finally D-glucose [3].
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Starch obtained by animals from plants is stored in the animal body in the form of glycogen. Digestive processes in both plants and animals convert starch to glucose, a source of energy. Starch is one of the major nutrients in the human died. Its presence in foods and other substances can be detected by the blue-black color produced when iodine solution is added to a sample of the material to be tested [5]. Starch + I2 blue-black color Amylase enzyme Proteins which catalyze the chemical reactions are called enzymes. Non-biological catalysts work at wide ranges of temperature and pH, whereas enzymes must work in mild conditions in cells, between 32 0C and 40 0C and at a pH between 6.5 and 7.5 [6]. Enzymes are highly specific both in the reaction catalyzed and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. To catalyze a reaction, a substrate must have a matching shape into the active site of the enzyme [2]. The products of the reaction then leave the active site, freeing it up for more similar reactions to take place. Amylase, an enzyme secreted by the salivary glands and the pancreas, rapidly hydrolyzes amylopectin and amylose [2]. Salivary amylase, which is also known as ptyalin, begins the polysaccharide digestion in mouth. The digestion process is completed in the small intestine by pancreatic amylase [5]. Spectrophotometry A solution appears colored because it absorbs certain wavelengths of light in the visible spectrum and reflects the others [7]. A spectrophotometer measures the transmission or absorption of liquids or solids as a function of wavelength [8]. Biologists use the spectrophotometer for two different purposes: 1. to determine the absorption spectrum of a pure substance in solution 2. to determine the concentration of a solution While the determination of the concentration of a solution, the spectrophotometer shines light at a set of color (at different wavelengths) through the sample and measures the percent of light that is absorbed by the sample. By comparing this amount with the graph constructed with known concentrations (calibration curve), the concentration of the unknown solution can be determined [9]. A spectrophotometer consists of a white light source (light of all visible wavelengths), a prism or diffraction grating that separates the light into different wavelengths, a slit through which a
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narrow beam of the desired wavelength passes (the incident light- I 0), a sample solution holder, a photosensitive tube which measures the energy of light transmitted through the solution (I), and a recording device that displays the amount of transmitted light energy digitally or on a dial.

Figure 2: Schematic diagram of the components of a spectrophotometer. The arrows indicate the pathway of light [7]

Transmittance is the ratio of the transmitted light energy (I) to the incident light energy (I 0); percent transmittance is 100X ratio (I/I0). Transmittance, however, is not proportional to solute concentration, so it is usually converted into absorbance which is proportional to solute concentration. Digital spectrophotometers have readouts for both percent transmittance and absorbance, but the absorbance is measured always [7]. % T = (I / I0) x 100 ABS. = log 10 (100 / % T)

APPARATUS Equipment 1. Test tubes 2. Pipettes 3. Pasteur pipettes 4. Beaker, 250 mL 5. Test tube rack 6. Plastic cuvettes

Buffers and Chemicals

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1. Human salivary enzyme 2. Starch solution, 20 g/L 3. HCl stopping solution, 0.1N HCl 4. Iodine reagent stoch solution (in aqueous solution) Iodine: 5 g/L ; KI: 50 g/L; Dilute to 1:100 5. Potassium phosphate buffers KH2PO4 (monobasic phosphate) (MW=136.1 g/mol) K2HPO4.3H2O (dibasic phosphate) (MW=228.23 g/mol) PROCEDURE Preparation of 20 g/L starch solution 1. Mix 20 g of soluble potato starch in approx. 50 mL of cold water. 2. While stirring, add the slurry to approx. 900 mL of gently boiling water in a large beaker. 3. Mix well and cool the gelatinized starch solution to room temperature. 4. Add more water to bring the total volume to 1 liter. 5. Put a few drops of the starch solution on a glass plate. Add 1 drop of the iodine reagent and see that a deep blue color is developed. The blue color indicates the presence of starch in the solution. Preparation of Enzyme Solution Dilute 1 mL of your saliva with 9 mL water. Then, add 60 mL of 0.5% NaCl solution. Effect of pH 1. Prepare 0.1 M pH buffer solutions ranging from pH = 4.5 to pH = 9.0 in increments of one pH unit. 2. Add an equal volume of one of the above buffer solutions to 5.0 mL of the 20 g/L starch solution prepared in Step 1. The resulting solution should contain 10 g/L of starch in a buffered environment. 3. Start the enzymatic digestion process by adding 1 mL of human salivary enzyme solution; shake and mix. 4. Let the hydrolysis reaction proceed for exactly 10 minutes at 25 0C. 5. Add 0.5 mL of the reacted starch solution to 5 mL of the HCl stopping solution (0.1 N). 6. Add 0.5 mL of the above mixture to 5 mL iodine solution to develop color. Shake and mix. The solution should turn deep blue if there is any residual, unconverted starch present in the solution. The solution is brown-red colored for partially degraded starch, while it is clear for totally degraded starch. 7. Meaure the absorbance with a spectrophotometer at 620 nm. Effect of Temperature
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1. Prepare the temperatures of the temporary water baths in 250 mL beakers, and adjust the temperatures ranging from 30 0C to 90 0C in increments of 20 0C. 2. Prepare the starch substrate by diluting the 20 g/L starch solution prepared in Step 1 with an equal volume of pH = 7.0 phosphate buffer solution. This results in a working starch concentration of 10 g/L. Add 5 mL of the starch solution to each of the test tubes. 3. Allow the temperature of each of the starch solutions to come to equilibrium with that of the water bath. 4. Add 1 mL of human salivary enzyme solution to each of the thermostated test tubes to start the reaction. 5. Stop the reaction after exactly 10 minutes and analyze the starch content by following the procedures outlined in Step 3. DATA TABLE pH ABS

T ABS REPORT OBJECTIVES 1. Plot the enzyme activity (absorbance) versus pH. From this curve, what is the optimal pH? Explain why enzyme activities depend on the pH. Similarly plot the enzyme activity versus temperature. Report the optimal temperature. Discuss why do you choose this optimal temperature and pH. 2. How could you make an acetate pH buffer solution? List the required chemicals and the composition needed to make one liter of acetate buffer as a function of the pH (5 7 9 11 13). (Phosphate, acetate, and citrate buffers are the most commonly used buffer.)

REFERENCES
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1. Morrison, R.T., and Boyd, R.N., Organic Chemisty, 6th Ed., Prentice Hall, New Jersey, 1992 2. CHEMystery, Organic Chemistry, Carbohydrates, 2003 http://library.thinkquest.org/ 3. Stryer, L., Biochemistry, 4th Ed., W.H. Freeman Company, New York, 1999 4. Perona, M., Analysis of Foods for Starch and Vitamin C, 2000 http://science.csustan.edu/perona/2000/Exp8/bkg.htm 5. Starch, The Columbia Electronic Encyclopedia, Columbia University Press, 2003 http://reference.allrefer.com/encyclopedia/S/starch.html 6. Amylase, GreenWood Health Inc., 2003 http://greenwoodhealth.net/np/amylase.htm Spectrophotometer Resources 7. Campbell, M., Introduction to Spectrophotometry, Davidson College, 2000 http://www.bio.davidson.edu/Courses/Bio111/Bio111LabMan/Lab%201.html 8. Dzierba, A., Spectrophotometer, Indiana University, 2003 http://dustbunny.physics.indiana.edu/~dzierba/P360n/KPAD/Exps/Spectro/spectro.html 9. Heitholt, J., Spectrophotometry, Texas A&M University, 2000 http://www7.tamu-commerce.edu/agscience/clasnote/pls497/SpecLecture/