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).
2
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 amylase’s 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.
3
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.
Figure 1. Absorption Readings of IKI in Solutions of Varying Starch. Absorbnce
(nm) measured over time for varying substrate concentrations (1.0 %, 0.75 %, 0.50 %,
0.25 %). Illustrated with a regression of 95% confidence for slope.
Figure 2. Rate of amylase activity (slope) at varying starch concentrations.
.001
.002
.003
.004
.005
.006
.007
.008
.009
.01
.011
Rate of Amylase Activity (abs/sec)
.2 .3 .4 .5 .6 .7 .8 .9 1 1.1
% conc. starch
-.2
0
.2
.4
.6
.8
1
1.2
1.4
1.6
absorbance
-25 25 75 125 175 225
time (sec)
0.25 % Starch Conc.
0.50% Starch Conc.
0.75 % Starch Conc.
1.00 % Starch Conc.
0.25 % Starch Conc. = .54 - 3.04E-3 * time (sec); R^2 = .55
0.50% Starch Conc. = .37 - 2.14E-3 * time (sec); R^2 = .43
0.75 % Starch Conce. = .44 - 2.5E-3 * time (sec); R^2 = .51
1.00 % Starch Conc. = 1.15 - .01 * time (sec); R^2 = .9
Absorbance plotted against Time
4
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 amylase’s 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.
