Methodology

A. Preparation of Starch Standard Curve

Different concentrations of starch solution in different 10 ml test tubes
were prepared by following the table below. The 2ml of each solution were taken
and were diluted to 10.0ml. The different concentrations of starch solution were
given I2 solution just before they were measured for absorbance. The
absorbance of each I2-starch complex were measured at 620 nm. The data was
then plotted as starch concentration vs. absorbance.
Table 1: Different concentrations of starch solution (Manila, 2013)
Test tube #

1
2
3
4
5
6
7

Volume of
0.1% starch
solution
0.00
0.50
1.00
1.50
2.00
2.50
3.00

Volume dH2O
(ml)

Volume I2
solution (L)

3.00
2.50
2.00
1.50
1.00
0.50
0.00

20.0
20.0
20.0
20.0
20.0
20.0
20.0

Starch
Concentration
(%w/v)

B. Reaction of Amylase in Human Saliva

The amylase of 100 ml of 1:10 dilution ratio of human saliva was prepared
in a conical tube. In five separate test tubes, the 1 ml of enzyme solution and the
9 ml of 0.1% starch solution were mixed. The different test tubes were incubated
at room temperatures for 0, 3, 5, 7 and 10 minutes respectively and all were
immersed in boiling water for 3 minutes. The tubes were cooled and were given 2
ml distilled H2O and 1 drop of I2 solution. The 2 ml of each solution were taken
and diluted to 5 ml. The absorbance of the solutions were measured at 620 nm.
The starch concentration vs. the incubation time was graphed by converting the
absorbance measured into starch concentration using the standard curve. The
average reaction velocity was calculated by getting the change in starch
concentration over the change in time per interval. The reciprocal of the starch
concentration vs. the reciprocal of the average reaction velocity was graphed to
determine Km and Vmax.
C. Effect of temperature on Enzyme activity
The following were mixed in each of the four separated test tubes: 1 ml of
0.1% starch solution, 3.5 ml of 0.1 M phosphate buffer of pH 6.7, and 0.50 ml of
0.9 % NaCl solution. Each of the test tubes were pre-incubated separately in
different water baths with temperatures of 10 C , 40 C, 60 C and 80 C for 5
minutes and all were added 5 drops of enzyme solution and all were continued
incubation for 10 minutes more. The four test tubes were immersed in a boiling
water for 3 minutes and were cooled to room temperature. An ml of an aliquot of

each test tubes were taken and were given 2.0 ml of distilled H 2O and a drop of I2
solution before measuring for absorbance at 620. The absorbance data was
converted to starch concentration was graphed vs. temperature.
D. Effect of pH on Enzyme Activity
In each four separated 10ml test tubes, the following were mixed: 1ml of
0.1% starch solution and 0.50 ml of 0.9% NaCl solution. Different pH (4.0, 6.7,
7.4, 9.0) of 3.5 ml 0.1 M phosphate buffers were added respectively on each of
the four separated test tubes. All tubes were added with 5 drops of enzyme
solution and were incubated at room temperature for 10 minutes before
immersing the tubes at boiling water for 3 minutes. The test tubes were cooled
and 1 ml aliquot of each test tubes were mixed with 2 ml of distilled H 2O and a
drop of I2 solution. The absorbance of each mixture were measured at 620 nm
and converted into starch concentration. The data gathered was graph into
starch concentration vs. pH.

Introduction
Enzymes are the catalyses of the reactions undergoing in our body. They
speed up reactions by finding an alternative reaction pathway, lowering the
activation energy and stabilizing the intermediates in the reaction. (Science,
2004).

Figure 1: general equation of enzymes (Massachusetts, 2015)

There are two hypotheses behind how enzymes work. These are the lock
and key hypothesis which states that the substrate simply fits into the active site
to form a reaction intermediate and the induced fit hypothesis which states that
the enzyme molecule changes shape as the substrate molecules gets close. The
change in shape is 'induced' by the approaching substrate molecule. This more
sophisticated model relies on the fact that molecules are flexible because single
covalent bonds are free to rotate. (Massachusetts, 2015)
There are four factors which affects the catalytic activities of enzymes.
They are temperature, concentration of the enzyme and substrate, pH and the
presence of inhibitors. The purpose of the experiment is to find out how these
factors affect the activities of the enzyme amylase which are found in the human
saliva and compute for the affinity constant of the sample saliva and its maximum
reaction velocity at different situations by using the Michaelis-Menten equation
and the Lineweaver-Burke equation. (J.H, 1971)

Figure 2: Michaelis-Menten Equation (Michaelis-Menten Kinetics and BriggsHaldane Kinetics, 1925)

Figure 3: Lineweaver-burke equation (Enzyme Kinetics as an Approach to

Understanding Mechanism, 2011)

References
Enzyme Kinetics as an Approach to Understanding Mechanism. (2011). Retrieved
from Bioinfo.org:
http://www.bioinfo.org.cn/book/biochemistry/chapt08/sim2.htm
J.H, W. (1971). Enzyme kinetics and its relevance to enzyme assay. Journal of
Clinical Pathology, 14-21. Retrieved from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176280/?page=2
Manila, U. o. (2013). Laboratory Manual in Biochemistry. Manila.
Massachusetts, U. o. (2015). Intro Biology. Retrieved from UMassAmherst:
http://bcrc.bio.umass.edu/intro/content/enzyme-kinetics-lab-protocol
Michaelis-Menten Kinetics and Briggs-Haldane Kinetics. (1925). Retrieved from
Michaelis-Menten Kinetics and Briggs-Haldane Kinetics:
http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html
Science, R. A. (2004). Enzymes. Retrieved from RSC:
http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm