Enzyme Kinetics
...me. Molecules that have been altered can no longer have an effect on the conversion of the substrate into a product and the initial velocity for the reaction is reduced. At pHs lower or higher than the optimum, alteration of the hydrogen bonds responsible for the tertiary structure of the enzyme occurs. When this shift occurs, the enzyme molecule can not affect the conversion of substrate into product and the initial velocity of the reaction is reduced. My hypothesis was that the optimum temperature and pH will be at the temperature and pH inside the barley embryo during the germination process. Since the barley seed releases the alpha-amylase during the germination process, I believe that the optimum temperature and pH will be the temperature and pH of the actual seed which should be approximately 55°C and 5.0 for the pH (Ohdan, 56). Materials and Methods There are two separate, but similar procedures which were done for the experiment. One was to witness the reaction of the enzyme with the starch solution under the effect of temperature and the other was to view it under the effect of pH. The spectrophotometer was turned on fifteen minutes prior to use and the wavelength was set at 560 nm. Six baths were prepared at temperatures: 15, 35, 45, 55, 65 and 70°C where flasks filled with 35 ml of starch solution and 35 ml of distilled water were placed in each water bath for the temperature effect. Six different starch solutions were prepared from 35 ml of stock starch solution mixed with 35 ml of water buffered at the pHs: 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 for the pH effect. 5 ml of distilled water and 0.1 ml of I2KI (iodine) indicator was added to a cuvette and was used to set the spectrophotometer to zero absorbance for the temperature effect. For the pH effect, 5 ml of the water buffered at each pH and 0.1 ml of I2KI indicator was added to a cuvette to zero absorbance. At each pH a new blank was prepared to zero absorbance. For each temperature and pH, 11 cuvettes were filled with 0.1 ml of I2KI indicator. Before timing took place, the 0 reading was taken when 5 ml of the solution from the reaction flask was added to the cuvette containing I2KI indicator and was mixed. The cuvette was then placed into the spectrophotometer and the absorbance reading was recorded. One ml of the alpha-amylase solution was added to the reaction flask and mixed thoroughly, timing began immediately after. Absorbances were read on the spectrophotometer, at 1 minute intervals throughout the first 8 seconds, 2 minute intervals from 8 to 10 seconds and then 10 minute intervals from 10 to 20 seconds for the temperature effect and at 2 minute intervals throughout the entire 20 minutes of the procedure for the pH effect, and all were recorded. For each following reading after the 0 reading, 5 ml of the solution from the reaction flask was added to a cuvette containing I2KI indicator and mixed. Then the absorbance reading was taken on the spectrophotometer. This procedure took place for each temperature and pH and was recorded. Once all data was recorded, graphs of absorbance vs. time were p...