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ABSTRACT The Effects of Varying Starch Concentration on a Solution of Amylase: Measurement of Enzymatic Rate Changes Using IKI.

Brooke Good Student, Functional Biology, Section 1003, Southwest Texas State University, San Marcos, TX 78666 The relationship between starch concentration and the enzymatic rate of amylase were investigated. Nine experimental assays were created all of which contained a solution of IKI, starch, saliva, and pH buffer. The nine assays were set up under four separate conditions containing a varying dilution of a 1 % starch concentration. The assays were then timed and their IKI absorbencies were noted. The assays containing the greatest percent starch concentration, 1%, had the highest enzymatic rate. As starch percentage was decreased enzymatic rate decreased. It was concluded that starch concentration has a direct effect on the enzymatic rate of amylase. The findings were consistent with experiments of the past of this nature performed by early scientist. INTRODUCTION Enzymes are perhaps one of the most important proteins of the human body. Enzymes such as amylase, an enzyme that breaks down carbohydrates, work by means of surface catalysis. In other words, the surface of the enzyme enables other molecules to react in a manner they would not be able to without the surface of the enzyme present. Enzymes achieve this by lowering the amount of activation energy needed for anabolic reactions, allowing these reactions to occur as catabolic reactions would. Enzymes are generally large proteins made up of several hundred amino acids, and often contain a non-proteinaceous group called the prosthetic group that is important in the actual catalysis. In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site of the enzyme. The enzyme and substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds (Nichols and Cholewiak, 1991).

Enzymes are not only important because they keep the metabolic pathways free of congestion but they are also key in digestion. Enzymes are needed to perform an infinite number of tasks within the human cellular system. Without enzymes the body could not possibly function properly, let alone sustain life. Enzyme productivity like many other things in our body tend to vary based on outside factors. Environmental parameters such as temperature, pH, and substrate concentration, such as starch, all cause changes in enzyme productivity. How does a varying intake of starch affect the rate of enzymatic catalysis in our body? Or does it? This will be the focus of this particular experiment. Will varying starch concentrations directly or indirectly affect the rate of enzyme catalysis, specifically amylase, an enzyme found in saliva? Starch is a polysaccharide found in many of the foods eaten on a daily basis, thus its importance. It might be assumed that starch concentration does not affect amylases ability to catalyze. However, through the use of IKI, a solution of Iodine and Potassium Iodide (optimal 660nm), which is commonly used as an indicator of starch concentration, we will examine if starch concentration does in fact have an effect on the rate of enzyme catalysis. MATERIALS & METHODS Dilutions of 1 % starch solution, 0.0 % (blank), 0.25 %, 0.50 %, and 0.75 % were prepared for four controls, one for each starch concentration to be used as blanks. We set the blanks up the same as the nine experimental cuvettes; each of which contain 1mL of diluted starch solution and IKI, 1.2 mL saliva, 0.8 mL pH buffer, but without the IKI making up the 4mL solution difference with an increase amount pH buffer. For the experimental cuvettes we first pipet the starch solution and pH buffer into each of the experimental cuvettes. Next we added saliva to all of the cuvettes and began timing. At 30-second intervals we added the IKI solution to each cuvette starting with an immediate zero reading up to 4 minutes and measured each absorbency. At completion, we repeat the previous steps as before only using 0.25 %, 0.50 %, and 0.75 % starch solution respectively, keeping all substance and cuvettes at 37 Degrees Celsius throughout the duration of the experiment.

RESULTS As the starch concentration was increased a higher reading was observed and then a steady decline over the 4 min period (Figure 1) . As seen in Figure 2, 1.00 % was the most optimal of the concentrations. At this point amylase catalysis rate was at its highest.
Absorbance plotted against Time 1.6 1.4 1.2 1 .8 .6 .4 .2 0 -.2 -25 25 75 125 time (sec) 175 225 1.00 % Starch Conc. 0.75 % Starch Conc. 0.50% Starch Conc. 0.25 % Starch Conc.

absorbance

1.00 % Starch Conc. =of 1.15 .01 * time of (sec); R^2 = .9 Figure 1. Absorption Readings IKI-in Solutions Varying Starch. Absorbnce 0.75 % Starch Conce. = .44 2.5E-3 * time (sec); R^2 = .51 (nm) measured over time for varying substrate concentrations (1.0 %, 0.75 %, 0.50 %, Starch Conc. = .37 -of 2.14E-3 * time (sec); R^2 = .43 0.25 %). 0.50% Illustrated with a regression 95% confidence for slope. 0.25 % Starch Conc. = .54 - 3.04E-3 * time (sec); R^2 = .55
.011 .01 .009 .008 .007 .006 .005 .004 .003 .002 .001 .2 .3 .4 .5 .6 .7 .8 % conc. starch .9 1 1.1

Figure 2. Rate of amylase activity (slope) at varying starch concentrations.

Rate of Amylase Activity (abs/sec)

DISCUSSION Outside experimentation, the body perhaps is the greatest test of enzyme adaptability. It uses enzymes in countless ways, and in countless conditions thus the importance of understanding the effects of its environmental parameters. Upon completion of the experiment we found that when temperature and pH are held constant the activity of an enzyme system is determined by the relative concentration of the enzyme and its substrate, starch in this case. Specifically, we found 1% starch solution to result in the highest rate of enzyme activity. If there is an excess of substrate, the rate of catalysis is directly proportional to the enzyme concentration. If enzyme concentration is kept constant, as it was in the following experiment, then the rate of reaction is directly proportional to the amount of substrate present. Thus, an increase in substrate, starch, causes an increase in enzyme catalysis. However, this is only up unto the point when all enzyme molecules are utilized, or saturated. At this point the enzymes have reached their optimal catalyzing rate and will no longer increase in rate of productivity. A much larger experimental group would have been needed in order to find amylases optimal catalysis condition, or saturation point. Results from the experiment supported our hypothesis and support the results from previous work on amylase activity but under different experimental conditions (Jensen et al., 1997; Skrabanja & Tufvesson, 2000). However, it is not clear why there was a precipitous decrease in enzyme activity at 0.075% starch. The inaccurate data could have occurred as a result of imprecise measurements of the parts of the solution. Or perhaps the time delay posed by the fact that time was not properly compensated when all parts of the solution could not be added by one person simultaneously, or even the mixtures of the IKI and starch solutions could have been slightly off. All of these errors could have been reduced however through more careful planning and accuracy during preparation.

REFERENCES

Jensen, M. S. Jensen, S. K., and Jakobsen K. 1997. Development of digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and pancreas. Journal of Animal Science. 75:437-445. Nichols, B.A.D. and Cholewiak L. B. . 1991. A quantitative enzyme study using simple equipment. ABLE. 218:89-99. Skrabanja, V. and Tufvesson, F. 2000. Digestibility of starch systems containing amylose-glycerol monopalmitin complexes. ABLE. 34:131-139.