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Biology Internal Assessment

Effect of chloride ions on rate of ∝- amylase hydrolysis

By: Christina Wong
1

Exploration

Introduction
Amylase is any member of a class of enzymes that catalyse the hydrolysis of starch into smaller carbohydrate
molecules such as maltose.∝-amylase is widespread among living organisms. In the digestive systems of
humans and many other mammals e.g. ptyalin is produced by the salivary glands, whereas the pancreas
secretes pancreatic amylase into the duodenum. (The Editors of Encyclopædia Britannica, 1998)

Allosteric activators, such as chloride ions, can increase reaction rates for a certain enzyme. They bind to an
allosteric site and induce a conformational change that increases the affinity of the enzyme’s active site for its
substrate, which increases the reaction rate. (Boundless, 2017)

Activators can play an important role in maintaining the conformation of
the protein, forming an essential component of the active site, or they may
be part of the substrate of the enzyme. The amount of enzyme activator
formed is equal to the amount of activator present in the mixture.

An example of an activator of an enzyme is the chloride ion with ∝-
amylase. In this case, ∝-amylase has some activity in the absence of
chloride. With saturating levels of chloride, the ∝ -amylase activity
increases about fourfold. Other anions, including F-, Br- and I- (halogen
ions) also activate ∝-amylase.

The idea for this investigation came about when I was studying Molecular
Biology, unit 7.5 Proteins and 7.6 Enzymes. Enzyme inhibition was part of
the syllables, yet it excludes activation. Thus, I was intrigued to investigate
such mechanism. ∝- Amylase was specially selected, as it is such an
important enzyme in the body, and sodium chloride (salt) is ingested
frequently; hence it sparks my interest to see if chloride ions would
actually affect the rate of hydrolysis of amylase, which could in term aid
Figure 1 illustrating the with digestion if the rate of catabolism increases. (Aghilan, Chaeroseshinie,
mechanism of allosteric activation Aishah Dewi)Concentration of sodium chloride ions (0.0, 0.3, 0.6, 0.9, 1.2,
1.5, 1.8 )/ %

Research Question
How does the concentration of sodium chloride ions (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )% affect the rate in which
enzyme ∝ - amylase hydrolyses starch, measured as the rate of change of absorbance values/ Au s-1(±0.001) of
starch in iodine solution using a spectrophotometer (light wavelength set at 430nm) in 300s?
Independent variable
Concentration of sodium chloride ions (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )/ % ( ±0.2)
Justification of range selected: extreme high concentration of sodium chloride ions is unnecessary as the
substrate is a limiting factor. Moreover, highly concentrated ions may alter the electrostatic interactions
between charged amino acids, causing conformational changes which in turn destroys the active sites, which
may decrease rate of reaction. Thus, such low concentration is selected as the most suitable, to investigate the
activating property of Cl- ions. An interval of 0.3 % in terms of sodium chloride concentration is selected as it
gives considerable change in rate of absorbance value of starch in iodine solution, yet not a significant jump.
This is the appropriate increment for measurements as it is able to show a clear trend, correlation between Cl-
concentration and rate of change in absorbance values of starch in iodine solution. Moreover, this is also the
range and increment of measurement selected by other Scientist conducting similar experiments. Reference to
other scientists’ research: (Aghilan, Chaeroseshinie, Aishah Dewi)

2

Dependent variable

Rate of change of absorbance values of starch in iodine solution with a
spectrophotometer (light wavelength set at 430nm) in 300s /Au s-1
(±0.00100) in different concentration of sodium chloride ions (0.0,
0.3, 0.6, 0.9, 1.2, 1.5,1.8)/ %

Explanation of dependent variable
Equation showing hydrolysis of starch using amylase:
∝ 𝑎𝑚𝑦𝑙𝑎𝑠𝑒
Starch+ water ---------------> maltose
(appear blue- black in iodine) (appear orange in iodine)

Figure2

Diagram showing hydrolysis of starch into
maltose at molecular level

When starch is mixed with iodine, the coils of beta amylose molecules found in starch trap iodine, causing the
mixture to turn into a shade of blue black. (Senese, 2015) When starch is broken down into maltose/ other
sugar, the disaccharide/ monosaccharide does not react with iodine. Therefore, iodine does not change colour
(appear orange). Correspondingly, when drops of amylase are inputted into a blue-black mixture of starch and
iodine, the starch molecules will be broken down causing the mixture to turn colourless appear orange). Thus,
the rate of reaction of amylase correlates to the absolute value of the rate of change in absorbance of the
solution. A rapid decrease in the absorbance value of the blue-black colour equates to a high rate of reaction of
amylase. In the experiment, an external variable-activator of sodium chloride will be manipulated into the
amylase enzyme to determine the effect of Cl- on the rate of reaction of amylase. (Lee, 2011)

∆ !"#$%"&'() !"#$%
Rate of Reaction = ∆ !"#$

Unit= Arbitrary unit per second (Au s-1)
Method of measuring:
Using a spectrophotometer set at 430nm(wavelength of dark blue colour) and Logger Pro, linked to Mac
computer. Rate of reaction calculated using above equation, absorbance v.s. time graph can be used for
reference and confirm results.

Controlled Variables
Controlled variable Reasons for controlling Methods to control
Concentration of enzyme ∝ - Increased concentration of ∝ -amylase Use 2.0 % bacterial amylase,
amylase used will increase rate of reaction, thus measured using measuring cylinder
affecting the results. (± 0.2)
Volume of enzyme ∝ - amylase Increased volume of ∝ -amylase will Use 1.0 cm3 of bacterial amylase,
used increase rate of reaction, thus affecting measured using measuring cylinder
the results. (± 0.2)
Time limit for reaction Rate of reaction would slow down as Time limit set at 300s, set time limit
time progresses as the substrate iodine is in computer/ data logger
used up, thus longer time span would
yield unfair results.
Temperature is a factor which will affect
Temperature of surrounding rate of enzyme activity. As kinetic energy Turn on air conditioner in lab, set at
of enzymes increases, rate of reaction 24 ℃
would increase, but extreme high
temperatures could cause enzyme
denaturation. So need to be controlled to
keep experiments fair (altering only one
variable)
3

Temperature of amylase and Amylase like all enzymes work at an Place starch and amylase in 37 ℃
starch optimum temperature, too warm or cold water bath for 10 minutes before
will lead to difference in rate of reaction. use.
Thus need to be constant to keep
experiment fair.
Same source of ∝-amylase from different sources may Use amylase from bacteria Bacillus
∝-amylase have different rate of reaction. spp. B. amyloliquefaciens
Concentration of substrate Increased concentration of substrate will Use 1.0% starch solution, measured
increase product (oxygen) produced. using measuring cylinder (± 0.2)
Volume of substrate Increased volume of substrate will Use 1.0 cm3 starch solution,
increase product (oxygen) produced. measured using measuring cylinder
(± 0.2)
pH level pH level is also a factor which affects the Use water as buffers to maintain the
rate of enzyme activity. Thus need to be neutral condition
constant to keep experiment fair.
Amylase works at a pH optimum of
around 7.
Volume of iodine More drops of iodine will darken the Use 3 drops via a plastic dropper
colour of the mixture, leading to
inaccurate measure of absorbance value
Volume of sodium chloride used Concentration of sodium chloride is the Use 1.0 cm3 measured using
independent variable, increased volume measuring cylinder (± 0.2)
would increase activation of amylase,
leading to unfair experiment.
Wavelength of light of Light of a particular wavelength will pass Set at 430 nm
spectrophotometer through the cuvette in the
spectrophotometer, then the absorbance
value recorded. If the wavelength is
different, the absorbance value may be
altered, leading to an unfair result.

Hypothesis
As concentration of sodium chloride increases, the rate of reaction/Aus-1 (hydrolysis of starch by ∝- amylase)
should increase, absorbance value will decrease at faster rate due to the increase in substrate activator, which
could catalyse the reaction better.

Materials/ Apparatus
Materials/ Apparatus Justification for choice of use
100 cm3 of 2.0% ∝-amylase 2% amylase is a suitable concentration as if too concentrated will lead to a rate of
from bacteria Bacillus spp. reaction which may be too fast for record of results. It may also be dangerous/
B. amyloliquefaciens hazardous to use highly concentrated enzyme.
The amylase sources from Bacillus spp. B. amyloliquefaciens is used as it is the most
common commercialised microbial amylase produced, thus can be obtained easily
and at low price.
100 cm of 1.0% starch
3 Substrate for hydrolysis by amylase1% starch is a suitable concentration as if too
solution concentrated will lead to very slow rate of reaction, inefficient.
A bottle of iodine Iodine test is used to determine concentration of starch in solution, important for
measuring dependent variable.
100 cm of 2.0% Sodium
3 Activator for ∝-amylase
chloride solution
Excess Distilled water Used as buffers to dilute sodium chloride solution to alter concentration for
independent variables.
LoggerPro + computer A digital way of recording absorbance value more accurately, avoids human errors.

4

Spectrophotometer/Au s-1 To measure change in absorbance value of blue-black colour in mixture, important as
(±0.00100) it is the equipment used to measure the dependent variable. Chosen over colorimeter
as it has a wider range of wavelength, more suited for dark blue colour of starch
solution in presence of iodine.
3 Cuvette To contain the mixture and place inside spectrophotometer. Its transparent property
allows light to pass through in order to accurately measure the absorbance value.
Dropper To obtain the solutions in small volume easily
10cm3 measuring cylinder/ To measure 1 cm3 of starch solution, amylase, sodium chloride, better for small
cm3( ±0.2) volume as the uncertainty is lower.
50cm3 measuring To measure volume of water and sodium chloride for serial dilution.
cylinder/cm3 (±0.4)
250 cm3 Conical flask / cm3 To contain/ store the sodium chloride solution of various concentration. Use bung to
(±0.2) , with bung prevent evaporation or contamination from air.
Water bath at 37℃ To warm starch solution and amylase before experiment to enhance optimum
temperature of ∝-amylase.
1 glass rod To stir the starch and amylase solution so molecules are more evenly spread in the
solution. Ensures the absorbance values recorded are accurate.

Safety, ethical, environmental precautions
- ∝-amylase from microbial sources is chosen over human/animals sources for ethical and economical
issues.
- Be careful to dispose left over chemicals such as enzyme ∝-amylase, pour into sink and flush with
excess water to prevent environmental hazards/ pollution.
- Do not ingest any chemicals from the lab, including sodium chloride as there may be toxic substances
which may cause contamination.
- Be careful when warming solutions in water bath, do not touch the electricity plug with wet hands if
setting up water bath.
- Wear goggles at all times to ensure safety, avoid contact of eyes with any chemicals, if occurred, flush
with clean water immediately and seek medical attention.

Preliminary trial
Conducted 2 times to ensure Logger Pro and Mac computer are working. Experiment was then conducted 3
times with 0.0 % NaCl, then 0.3% and 0.6% NaCl respectively to ensure the increment for measurement
selected is suitable. The concentration range selected for independent variables corresponds to published
research and the results show a trend during preliminary trial, thus do not require any alterations.

Methodology
*Make sure experiment is carried in a lab with uniform temperature, air conditioner turned on to 24 ℃.
I. Prepare sodium chloride solution with varying concentration
Conduct serial dilution using table below:

Concentration/% (±𝟎. 𝟐)of Volume of 2% NaCl/ Volume of H2O/
sodium chloride solution cm3(±𝟎. 𝟒) cm3(±𝟎. 𝟒)
0.0 0 20
0.3 3 17
0.6 6 14
0.9 9 11
1.2 12 8
1.5 15 5
1.8 18 2
Volume ratio calculated using formula: M1V1=M2V2
e.g. to make 0.3% sodium chloride solution
1. Measure 3.0 cm3 of 2% NaCl, using 50.0 cm3 measuring cylinder.
2. Measure 17.0 cm3 of distilled water using 50.0 cm3 measuring cylinder.
3. Pour the liquid into a conical flask and swirl.
4. Repeat for all concentration.
5

II. Conduct experiment
1. Set up spectrophotometer, set light to 430 nm wavelength.
2. Calibrate spectrophotometer using distilled water in cuvette and wait for absorbance to drop to 0.
3. Connect loggerPro to computer and spectrophotometer, set the time limit at 300s and record an
absorbance value at 5sec interval.
4. Measure 1.0cm3 of starch solution, amylase, 0.3% sodium chloride using different 10.0 cm3 measuring
cylinder. Make sure to read the meniscus at eyelevel to avoid error of parallax.
5. Pour the starch solution and sodium chloride into a cuvette and add 3 drops of iodine solution using a
plastic dropper.
6. Place the cuvette into the spectrophotometer.
7. Pour in the ∝-amylase and immediately close the lid of spectrophotometer and start the loggerPro on
computer. Data recording commences.
8. Wait for 300s and loggerPro will automatically stop and record the final value. Observe the curve
drawn.
9. Repeat step 1-7, 15 times. Observe if the curves overlap, suggests accurate results. *Important to ensure
results are reliable.
10. Then repeat step 1-8 using sodium chloride of different concentration.
III. Analyse results
- Compare final absorbance value and initial absorbance value/ Au (±𝟎. 𝟎𝟎𝟏𝟎0) and calculate the rate of
change of absorbance value/ Au s-1
- Calculate the average rate of change of absorbance value/ Au s-1 and observe the correlation between
concentration of NaCl/% and average rate of change of absorbance value. Note: average refers to mean.
- Statistical test(Spearman’s rank coefficient) is to be carried out (more detail below)
Analysis

Table #1: Raw data table showing initial and final absorbance value/ Au(±𝟎. 𝟎𝟎𝟏𝟎𝟎) within 300sec
for mixture in various concentration of NaCl (0.0,0.3, 0.6, 0.9, 1.2, 1.5, 1.8)/%(±𝟎. 𝟐)
Initial Initial

Concentration absorbance Final absorbance Concentration absorbance Final absorbance
of NaCl/% Trial value/Au value/Au of NaCl/% Trial value/Au value/Au
(±𝟎. 𝟐) number (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟐) number (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟎𝟎𝟏𝟎𝟎)

1 1.26200 0.67000 1 1.04800 0.76000
2 1.02100 0.78000 2 0.83500 0.59000
3
0.98700 0.56000 3 0.98600 0.66000
4 1.25900 0.83000 4 1.07600 0.47000
5 1.10700 0.75000 5 1.05400 0.91000
6 1.34000 0.98000 6 0.85000 0.43000
7 1.23000 0.88000 7 0.98000 0.44000
0 8 1.45000 0.96000 0.3 8 0.87000 0.37000
9 1.26000 0.89000 9 1.09000 0.34000
10 0.98000 0.65000 10 1.23000 0.58000
11 1.13000 0.77000 11 1.12000 0.54000
12 0.87000 0.64000 12 1.32000 0.78000
13 0.96000 0.74000 13 1.24000 0.88000
14 1.03000 0.59000 14 0.89000 0.43000
15 1.23000 0.96000 15 0.97000 0.47000
1 0.91900 0.34000 1 0.32755 0.67000
2 0.95400 0.25000 2 0.26224 0.78000
3 1.11800 0.37000 3 0.32765 0.56000
4 1.21200 0.39000 4 0.30405 0.83000
5 1.12600 0.24000 5 0.30495 0.75000
6 1.14000 0.45000 6 0.28273 0.98000
7 1.25000 0.56000 7 0.27669 0.88000
0.6 8 0.98000 0.32000 0.9 8 0.25987 0.96000
9 0.86000 0.29000 9 0.39894 0.89000
10 1.05000 0.43000 10 0.30090 0.65000
11 0.94000 0.34000 11 0.32882 0.77000
12 1.02000 0.47000 12 0.32882 0.64000
13 1.09000 0.32000 13 0.45000 0.74000
14 0.78000 0.45000 14 0.34000 0.59000
15 1.13000 0.49000 15 0.29000 0.96000
6


Concentration Initial Concentration Initial
of absorbance Final absorbance of absorbance Final absorbance
Trial Trial
NaCl/% value/Au value/Au NaCl/% value/Au value/Au
(±𝟎. 𝟐) number (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟐) number (±𝟎. 𝟎𝟎𝟏𝟎𝟎) (±𝟎. 𝟎𝟎𝟏𝟎𝟎)
1 1.39394 0.76000 1 1.13473 0.34000
2 1.59600 0.59000 2 1.57239 0.25000
3 1.61159 0.66000 3 1.58933 0.37000
4 1.05574 0.47000 4 1.55099 0.39000
5 1.71171 0.91000 5 1.61509 0.24000
6 0.97343 0.43000 6 1.66376 0.45000

7 1.42454 0.44000 7 1.49397 0.56000
1.2 8 1.22990 0.37000
1.5 8 1.64247 0.32000
9 1.35602 0.34000 9 1.63464 0.29000
10 1.27616 0.58000 10 1.37761 0.43000
11 1.48003 0.54000 11 1.43370 0.34000
12 1.58096 0.78000 12 1.54134 0.47000
13 1.58933 0.88000 13 1.80365 0.32000
14 1.45557 0.43000 14 1.42505 0.45000
15 1.49338 0.47000 15 1.48636 0.49000

1 1.65128
0.38685
2 1.35925 0.16000
3 1.46624 0.23000
4 1.41410 0.17000
5 1.50564 0.18000
6 1.66245 0.37157
7 1.31956 0.32174
1.8 8 1.65555 0.20000
9 1.04711 0.21000

10 1.51329 0.14000
11 1.38352 0.24000
12 1.72157 0.29000
13 1.51267 0.19000
14 1.40032 0.23000 Figure 3 showing the change in colour of starch
15 1.47917 0.12000 solution, 300sec after amylase is added

Qualitative observation:
- The colour of mixture in cuvette was initially black- dark blue coloured.
- After mixing amylase and waited 300 seconds, the colour of mixture is light yellow, and it has become a
transparent solution.
- There is some blackish sediments at the bottom of the cuvette after 300 seconds.

Table #2 : Processed Data showing rate of change of absorbance value/Aus-1 and its average
within 300sec for various concentration of NaCl (0.0,0.3, 0.6, 0.9, 1.2, 1.5, 1.8)/% (±𝟎. 𝟐)

Trial Average Rate Trial Average Rate
Rate of change of Rate of change
Concentration number of change of Concentration number of change of
absorbance value of absorbance
of -1 absorbance of -1 absorbance
/ Au s -1 value / Au s
NaCl/%(±𝟎. 𝟐) value / Au s NaCl/%(±𝟎. 𝟐)
-1
value / Au s
1 0.00197 1 0.00096
2 0.00080 2 0.00082
3 0.00142 3 0.00109
4 0.00143 4 0.00202
5 0.00119 5 0.00048
6 0.00120 6 0.00140
7 0.00117 7 0.00180
0.0 8 0.00163 0.00121 0.3 8 0.00167 0.00154
9 0.00123 9 0.00250
10 0.00110 10 0.00217
11 0.00120 11 0.00193
12 0.00077 12 0.00180
13 0.00073 13 0.00120
14 0.00147 14 0.00153 7
15 0.00090 15 0.00167

Trial Average Rate Trial Average Rate of
Rate of change Rate of change
number
of absorbance
of change of number change of
Concentration of -1 absorbance of absorbance
value / Au s -1 Concentration of -1 absorbance
value / Au s
NaCl/%(±𝟎. 𝟐) value / Au s NaCl/%(±𝟎. 𝟐)
-1
value / Au s
1 0.00193
1 0.00022
2 0.00235
2 0.00016
3 0.00249 3
0.00029
4 0.00274 4
0.00015
5 0.00295 5
0.00030
6 0.00230 6
0.00020
7 0.00230 7
0.00022
0.6 8 0.00220 0.00219 8
0.9 0.00024 *0.00026
9 0.00190 9
0.00036
10 0.00207 10
0.00024
11 0.00200 11
0.00030
12 0.00183 12
0.00033
13 0.00257 13
0.00037
14 0.00110 14
0.00023
15 0.00213 15
0.00033
1 0.00390 1
0.00246
2 0.00449 2
0.00387
3 0.00416 3
0.00397
4 0.00276 4
0.00373
5 0.00458 5
0.00404
6 0.00185 6
0.00440
7 0.00369 7
0.00377
1.2 8 0.00292 0.00362 1.5 8 0.00384
0.00428
9 0.00329 9
0.00432
10 0.00319 10
0.00340
11 0.00354 11
0.00349
12 0.00410 12
0.00380
13 0.00423 13
0.00485
14 0.00371 14
0.00351
15 0.00395 15 0.00372
1 0.00421
2 0.00400
3 0.00412
4 0.00415
5 0.00442
6 0.00430
7 0.00333
8 0.00485
1.8 0.00414
9 0.00279
10 0.00458
11 0.00381
12 0.00477
13 0.00441
14 0.00390
15 0.00453

*data in red indicates anomalous result
Note: Data are recorded to 5 decimal places because the rate of change of absorbance value/ Au s-1 is too
accurate, the values are very small, if rounded up to 3 decimal places, all the data would just be 0.001 or 0.002
which is not useful for graphing (only a horizontal line would be seen). Thus, Raw data are also recorded to 5
decimal places to keep the decimal places in tables consistent.

8

Table #3 :Processed Data Table showing concentration of NaCl/% (±𝟎. 𝟐) and the average rate of
-1
change of absorbance value / Au s

Concentration of NaCl/%(±𝟎. 𝟐) Average rate of change of absorbance value/ Au s-1
0 0.00121
0.3 0.00154
0.6 0.00219
0.9 0.00026
1.2 0.00362
1.5 0.00384
1.8 0.00414
*red numbers indicate anomalous data which would be eliminated when drawing linear trend line.

Calculations for processed data:
Calculation Formula Example
Rate of change of absorbance
!"!#!$%!!"#$% !"#$%"!&'( !"#$%
value Aus-1 Trial number 1 for 0.3% NaCl:
!""
(1.04800-0.76000)÷300
=0.00096
Standard deviation
Calculated using excel
𝑒. 𝑔 for 0.0% NaCl,
SD≈ 0.00121
Average (mean) rate of Calculated using excel
change of absorbance value/
Aus-1

Screen shot from excel showing calculations ---e.g. 0.0% NaCl:

9

Graph showing correlation between concentration of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8) /% and rate of
change of absorbance value/ Au s-1

0.00500

0.00450

Rate of change of absorbance value/ Au s-1
0.00400

0.00350

0.00300

0.00250

0.00200

0.00150

0.00100

0.00050 y = 0.0018x + 0.0012
R² = 0.97731
0.00000
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Concentration of NaCl/ % (+/-0.2)

Analysis of graph: The blue diamond point indicates anomalous data which is far off the linear regression trend,
thus is excluded from the results when drawing the linear regression line because it is evident that there may
be a systematic error in the experimentation e.g. wrong concentration of starch / amylase used since for all 15
repeats, when added 0.9% NaCl, the initial absorbance value/Au was very low, below any other experiments.
When other initial value usually ranges from 0.80000 to 1.32000, all the 15 data using 0.9%NaCl ranges in
between 0.25987 to 0.45000 which is significantly lower than other initial absorbance values/Au. (this will be
discussed further later in conclusion)

Error bars show the standard deviation of the rate of change of absorbance value /Aus-1 for each data point.
There is a different standard deviation for each concentration of NaCl (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )%, the
standard deviations are 0.00034, 0.00055, 0.00044, 0.00073, 0.00055 and 0.0054 (in increasing order of
concentration, standard deviation for data using 0.9% NaCl is eliminated from calculation as it is an anomaly.)

The processed data and graph shows that the hypothesis is correct. As concentration of NaCl increases, rate of
absorbance value increases. There is a positive, linear correlation between concentration of NaCl/% and rate of
change of absorbance value/Au s-1.

Statistical Test -Spearman’s rank correlation coefficient
Justification for use: Spearman’s Rank correlation coefficient tests if two variables are connected (correlated)
with each other. It will also measure the strength of any correlation, i.e. how close to a perfectly straight line it
is. (Ecofieldtrips Pte Ltd, 2015 )My experiment is testing if there is a correlation between concentration of
chloride ions/% and ∝-amylase activity measured by rate of change of absorbance value/ Aus-1 . Having two
variables predicted to correlate each other, Spearman’s Rank correlation seems the most suitable. Also, from
the graph, it is evident that there is a monotonic correlation (Laerd statistics) instead of other relationships such
10

as non-monotonic, and Spearman’s rank correlation is specifically suited to monotonic relationship between
two variables.

Null Hypothesis: There is no significant correlation between concentration of NaCl and average rate of change
in absorbance value.
Alternative Hypothesis: There is significant correlation between concentration of NaCl and average rate of
change in absorbance value.
Concentration of Average rate of change
NaCl/% (±𝟎. 𝟐) (A) Rank A absorbance value/ Aus-1 (B) Rank B Difference Difference squared
0 1 0.00121 1 0 0
0.3 2 0.00153 2 0 0
0.6 3 0.00219 3 0 0
0.9 - 0.000263 - - -
1.2 4 0.00362 4 0 0
1.5 5 0.00384 5 0 0
1.8 6 0.00414 6 0 0
Note: anomalous data is excluded from the statistical test

Formula to find the rs value, the closer it is to 1, the stronger the correlation

Result: Rs value is exactly one, which means there is
a very strong correlation between the two variables.

Null Hypothesis is rejected, Alternative Hypothesis
!(!) is accepted, there is significant positive and negative
rs= 1 – !(!! !!) = 1
correlation between concentration of NaCl and
average rate of change in absorbance value.
Conclusion
The finding answers my research question which is: “How does the concentration of sodium chloride ions
(0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )% affect the rate in which enzyme ∝ - amylase hydrolyses starch,
measured as the rate of change of absorbance values/ Au s-1(±0.001) of starch in iodine solution using a
spectrophotometer (light wavelength set at 430nm) in 300s?” As concentration of sodium chloride ions
(0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )/% (±𝟎. 𝟐) increases, rate in which enzyme ∝ - amylase hydrolyses starch
increases, as can be seen from the increase in rate of change of absorbance values/ Au s-1(±0.001) of starch in
iodine solution. This also supports the hypothesis, suggesting that the hypothesis is correct, can be accepted.

As concentration of NaCl increases from 0.0 % to 1.8%, the average rate of change of absorbance values
increases from 0.00121 to 0.00414 Au s-1. There is a 0.00293 Au s-1 increase in terms of rate, that means when
using 1.8% NaCl, the rate of change of absorbance value is almost 3 times faster compared to starch solution
with 0.0% NaCl added. This suggests, increase chloride ions concentration increases enzyme ∝ - amylase’s
activity so it is able to hydrolyse starch faster, allowing the colour of starch to turn from black/blue to yellow
faster, causing a faster rate in decrease of absorbance value/ Au s-1 . The qualitative observations also supports
the theory as the colour of starch solution is initially black/ dark blue but changes to light yellow after amylase
is added, indicating that hydrolysis of starch has occurred. After 300s, solution with 1.8% NaCl added appears
lightest yellow compared to 0.0% NaCl added, evidently, adding chloride ions which are activators speed up
this process.
Thus, a positive correlation is observed from the line graph plotted between concentration of NaCl /% and rate
of change of absorbance value/ Au s-1. The equation of the linear line y= 0.0018x +0.0012 has a positive
gradient of 0.0018, suggesting that as concentration of NaCl increases by every 0.1%, the rate of change of
absorbance value would increase by 0.0018 Au s-1 . Data shows that as concentration of NaCl increases from
0.3% to 0.6 %, the average rate of change of absorbance value increases from 0.00154 to 0.00219 Au s-1, that is
0.00065 Au s-1 increase in rate of change of absorbance value for 0.3% increase in concentration of HCl . When
divide 0.00065 by 3 (so meaning, every 0.1% increase in NaCl concentration), the increase in rate of change of
absorbance value is approximately 0.00022. This is quite close to the gradient of the equation, which is
11

0.00018. This shows that the equation generated is quite accurate and is a good model of the correlation,
further confirming the strong correlation and affirms that there is indeed an effect (positive correlation)
between the two variables.

Detailed Biological theory: ∝-amylase is an allosteric enzyme which means it mostly has two or more subunits
and can oscillate from active form to inactive form. The presence of an allosteric effector can change the affinity
of the enzyme for its substrate, or alter the maximal catalytic activity of the enzyme, or both. Moreover, the
binding of an activator at regulator site can stabilize the active form of that allosteric enzyme. T-state is known
to tense up the molecule and is less active. The R-state is known to be more relaxed and decreases the Km.
Chloride ion is suspected to be an allosteric effector- activator, which aids in the transformation of ∝-amylase
from T- state to R –state. Thus, increasing the enzyme ∝-amylase activity. (Berg, 2015 )
Reliability of data: The equation of the linear line y= 0.0018x +0.0012 has a R2 value of 0.977 which suggests a
strong correlation. The data points fit to the linear trend line more closely after eliminating the anomaly. The
results obtained from the experiment using 0.9% NaCl is eliminated as it is an outlier which is far off the linear
trend observed from other results. It is thought that the outlier is caused by a systematic error when
conducting the experiment. The initial absorbance value/Au of starch in iodine solution in addition of 0.9%
NaCl, was very low. Whilst other initial values range from 0.80000 to 1.32000, all the 15 data using 0.9%NaCl
range in between 0.25987 to 0.45000 only, which is significantly lower than other initial absorbance values/Au.
All procedures were conducted identically, following the methodology, thus the source of error may be the
materials or equipment. As the experiment was conducted over a span of a week, the starch solution and ∝-
amylase used were different each day. Hence, the starch solution used that day may be of wrong concentration,
below 1%, or it may be that it has been stored for too long that all the starch sinks to the bottom of the
container and what is poured out is only very diluted starch solution. The ∝-amylase may be a source of error
as well, it may have been stored for too long that it has become inactive. However, I feel the spectrophotometer
or the LoggerPro may have been the main contributor to the systematic error, the specific one used to conduct
the experiment with 0.9% NaCl may have been faulty, thus yielding wrong results. To avoid such problems
again, equipment should be checked thoroughly, run trials and compare results with other data sets obtained,
before experiments next time.

Eliminating the anomalous data, when: calculating average, standard deviation, plotting linear regression line,
allows the trend to be more accurate and has a better fit. In combination to the Spearman’s rank coefficient
statistical test, the data produced from this experiment is reliable and statistically valid. Since the rs value =1,
suggesting perfect positive correlation. However, only 6 data points are ranked, instead of the ideal 8(data
points), this may cause the statistical test to be less applicable to these results. On the other hand, R2 value
generated by excel from the graph is 0.977, this also suggests the data points are very close to the trend line,
not scattered, so again reaffirming the strong correlation between the two variables.

The error bars on my graph further show the data obtained are quite precise. The standard deviations are
relatively small. This suggests the results are reliable as it shows the ‘goodness of fit’ to the linear function (y=
0.0018x +0.0012) is relatively high, i.e. the function describes the data very well.
Reference from published research: it is

stated that Cl- is essential in the activity of

∝ −𝑎𝑚𝑦𝑙𝑎𝑠𝑒 , suggesting that this
experiment is true. “Activation experiments
of A. haloplanctis α-amylase by several
monovalent anions show that a negative
charge, not restricted to that of Cl−, is
essential for the amylolytic reaction.
Engineering of the chloride binding site
reveals that a basic residue is an essential
component of the site.” (Georges Feller‡)

As concentration of starch increases,
Figure 4 Graph showing Activation of starch and EPS velocity of ∝ −amylase activity increases.
hydrolysis by chloride obtained from published data. The graph also shows a linear trend just as

my results have shown.
12

Evaluation
Strength of investigation
- Logger Pro is used to record data, this eliminates human error and uncertainty of using time watch,
which greatly increase the accuracy of the results.
- Spectrophotometer is used, which enables calibration of 460nm, this is the wavelength for dark blue
wavelength, making the absorbance value more accurate.
- There are 7 data points for independent variables (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )% which suggests
sufficient data to test the research question.
- Spectrophotometer is calibrated using distilled water before experiment to correct for systematic error.
Limitations
Limitation Reasons/ significance Possible improvements
Small sample size, not Data unreliable/ not reliable enough Repeat methodology 50 times more to improve
enough repeats, only 15 reliability
times
Starch sink to bottom in Starch residues may not be poured Stir the starch solution with a glass rod, and
beaker when stored into the cuvette, the solution does swirl the beaker every time before pouring and
not contain as much starch, leading measuring.
to lower absorbance values.
Overall rate is calculated Not as accurate because Initial rate of reaction should be calculated, draw
concentration of amylase and starch curves using LoggerPro software(record data
would have decreased progressively every 10 s) and find gradient of tangent to the
as the reactants are used up initial part of the curve.
Temperature of amylase The amylase would not be working Place the amylase and starch in water bath at 36
and starch may have cooled at its optimum condition, which may ℃ ,when conducting the experiments. Only
down cause slower rate of reaction. extract the small volume needed for each repeat
at a time.
Starch sink to bottom of Absorbance value will decrease even Shake the cuvette by covering thumb over
cuvette before amylase is before the hydrolysis reaction as the opening it and invert 5 times, before placing into
even added black/ blue starch residues sink to the spectrophotometer. Then start the timer
the bottom of the cuvette. straight away as soon cuvette is in
spectrophotometer.
Not fresh enzyme amylase Chemical reactions may occur when Use fresh/new (unopened) amylase each day, or
/not stored properly amylase are stored in refrigerator complete experiments in a day.
overnight, affecting amylase activity
Extensions
- Conduct experiment using same procedure/ methodology but substitute ∝-amylase with 𝛽-Amylase,.
- Amylase from different sources such as fungi or animals can be used to investigate, as this would be
even closer to the real condition in the body.
- Conduct experiment using same procedure/ methodology but substitute Cl- with other halogen ions
such as F-, I-, Br- and see if they have similar activating effect as Cl-.
- Experiment with other allosteric protein/ enzyme such as e.g. animal hemoglobin, the binding of
oxygen may induce a conformational change in that subunit that interacts with the remaining active
sites to enhance their oxygen affinity.
Bibliography

The Editors of Encyclopædia Britannica. (1998, July 20). Amylase BIOCHEMISTRY. Retrieved January 2017, from ENCYCLOPAEDIA

BRITANNICA: https://global.britannica.com/science/amylase
Boundless. (2017, May 26). Control of Metablosim Through Enzyme Regulation. Retrieved January 16, 2017, from Boundless.com:

https://www.boundless.com/biology/textbooks/boundless-biology-textbook/metabolism-6/enzymes-72/control-of-metabolism-

through-enzyme-regulation-351-11577/

Senese, F. (2015, August 17). How does starch indicate iodine? Retrieved January 16, 2017, from General Chemistry Online!:

http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch-as-redox-indicator.shtml

Lee, S. S. (2011, March 31). Slideshare. Retrieved January 16, 2017, from Slideshare: http://www.slideshare.net/wkkok1957/effect-

of-on-the-activity-of-amylase-using-visible-spectrophtometer

Georges Feller‡, O. l. (n.d.). Structural and Functional Aspects of Chloride Binding to Alteromonas haloplanctis α-Amylase*. Retrieved

January 2017, from ibc The Journal of Biological Chemistry: http://www.jbc.org/content/271/39/23836.long

Aghilan, Chaeroseshinie, Aishah Dewi. Effect of salt concentration on salivary amylase on the breaking down of starch. Present.me.
Ecofieldtrips Pte Ltd. (2015 ). MANGROVE-STATISTICS. Singapore: ECO FIELD TRIPS.
Laerd statistics. (n.d.). Spearman's Rank-Order correlation. Retrieved February 9, 2017, from Laerd statistics:

https://statistics.laerd.com/statistical-guides/spearmans-rank-order-correlation-statistical-guide.php 13

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