Department of Chemical and Biomolecular Engineering

NATIONAL UNIVERSITY OF SINGAPORE

Chemical Engineering Process Laboratory I

AY2014/15 Semester 2
Experiment B1
Protein Quantification, Activity and Specific Binding Constant

Group Members:

Eric Poon Chinn Wey

A0112187L

Foo Chee Wei Nicholas

A0110525X

Teng Ming Xiang Wilson

A0110392U

Group Number:

T14

Date of Experiment:

31 March 2015

SUMMARY
In the first part of the experiment (CI), a High Performance Liquid Chromatography (HPLC)
system was used to determine the concentration of 2 unknown samples of -amylase. The 2
samples were labelled as S25 and S60, which had been stored for 2 hours at 25C and 60C
respectively prior to the start of the experiment. The determination of concentration was done
by analysing the chromatogram generated by the HPLC system for the 2 unknown samples.
The calibration curves for each of the samples on the chromatogram were plotted with
maximum absorbance (A280) against eluent volume (mL). The y-axis was defined as
absorption at 280nm wavelength as the amino acids with aromatic groups in the enzyme
absorb most strongly at that wavelength. The concentrations for S25 and S60 were found to
be 0.133 mg/ml and 0.129 mg/ml respectively.
In the second part of the experiment (CII), various S25 and S60 samples were subjected to
the addition of starch and quenched at regular time intervals by the addition of iodine. Iodine
stops the enzymatic action of -amylase on the -1, 4-glucose linkages of starch by reacting
with the unreacted starch. Subsequently, the absorbance and concentration for the different
S25 and S60 samples could be analysed using a spectrophotometer with wavelength set at
600nm.
In the third part of the experiment (CIII), samples with varied starch concentrations were
added to iodine solutions of known volume and concentration and quenched after 5 minutes.
The end concentrations were recorded using the spectrophotometer set at 600nm and used in
the Michaelis-Menten relation. The Lineweaver-Burke equation was selected for linear
regression analysis to find the specific binding constant, K m. The value for Km was found to
be 19.13.

TABLE OF CONTENTS
SUMMARY 2
1. RESULTS AND CALCULATIONS
2. DISCUSSION

3. ERROR ANALYSIS
4. CONCLUSION

13

5. NOTATIONS

13

6. REFERENCES

14

7. APPENDICES

15

12

1. RESULTS AND CALCULATIONS

1.1 Determination of protein concentration in two unknown samples
The two -amylase samples, S25 and S60 were injected into the High Performance Liquid
Chromatography (HPLC) system in 20 L portions each. The analysis report for both samples
are attached in Appendix A and the concentrations in parts per million (ppm) and mass per
unit volume (mg/ml) are indicated below. The latter is obtained by converting the former with
the appropriate conversion factor that is

S25
132.855
0.132855

Concentration (ppm)
Concentration (mg/ml)

S60
128.704
0.128704

Table 1 Concentrations of S25 & S60

1.2. Determination of protein activity of S25 and S60

Sample
1
2
3
4
5
6
7
8
9
10
11
12
13

S25
S60
Starch Concentration
ABS
Starch Concentration
wt %
mg/ml
wt %
mg/ml
0
0.755
0.3848
3.848
0.755
0.3848
3.848
0.5
0.710
0.3620
3.620
0.690
0.3517
3.517
1.0
0.637
0.3247
3.247
0.623
0.3174
3.174
1.5
0.571
0.2910
2.910
0.591
0.3012
3.012
2.0
0.635
0.3234
3.234
0.547
0.2787
2.787
2.5
0.521
0.2657
2.657
0.516
0.2631
2.631
3.0
0.508
0.2590
2.590
0.476
0.2424
2.424
3.5
0.457
0.2329
2.329
0.447
0.2280
2.280
4.0
0.383
0.1950
1.950
0.435
0.2217
2.217
4.5
0.357
0.1819
1.819
0.402
0.2048
2.048
5.0
0.349
0.1781
1.781
0.381
0.1940
1.940
5.5
0.311
0.1583
1.583
0.373
0.1899
1.899
6.0
0.306
0.1557
1.557
0.350
0.1782
1.782
Table 2 Absorbance and Concentration of S25 & S60 at various time

Time

ABS

At t=0, 1mL of sample S25 was added to the prepared 3mL starch solution that has
concentration of 0.4% starch. At 0.5 minute intervals up to t=6, 0.2mL of this reaction
mixture was added into the iodine solutions to obtain 12 solution samples. These samples
were placed in a UV visible spectrophotometer to detect the intensity of light absorbed by
each sample as well as the concentration of starch (ppm) in it at different time point. The
entire procedure is repeated for the S60 enzyme sample and the results obtained is tabulated
and shown below. To convert the units of concentration to milligram per millilitre (mg/mL),
the following calculation, using the data for sample 2 as example, can be performed.

Assuming

density

of

solution

to

be

same

as

water,

i.e.

1000

mg/mL,

With the above data, absorbance of the 600 nm light by the solution samples can be plotted as
a function of time and starch concentration. The resultant plots are shown below

Figure 1 Absorbance (A) at 600 nm as a

function of (a) time, t (b) concentration, c for S25

Figure 2 Absorbance (A) at 600 nm as a function of (a) time, t (b) concentration, c for S60

Through graphical analysis of the above figures, protein activity for sample S25 and S60 can
be determined. For this experiment, one unit of -amylase activity has been arbitrarily
defined as the amount of enzyme needed to hydrolyse 10 mg of starch per 30 minutes.
The end point of starch hydrolysis is defined to as the time to reach A 600=0.4
Activity for S25
From Figure 1(a),
Rearranging,

From Figure 1(b),

Rearranging,

Since it took 4.29 minutes to hydrolyse 5.8869 mg of starch, the number of units activity
based on the earlier definition for one unit of activity is as such

The amount of -amylase in the withdrawn 0.2 mL of reaction mixture that was added in the
iodine mixture is

. Since it is known that this

amount of -amylase brought about 4.1167 units of protein activity,

1.3 Determination of the specific binding constant for starch and -amylase
The reaction kinetics for this reaction can be represented using the Michaelis-Menten
approach which is represented by:

(where q is the reaction rate, qm is the maximum reaction rate, and Km is the binding constant,
in units consistent with that of S.)
The above equation can be linearised in several forms to obtain the kinetic parameters,
namely Lineweaver-Burke, Langmuir and Eadie-Hofstee. All 3 forms will be presented in the
rest of this section. The detailed calculations involving the manipulation of the experimental
data variables into the appropriate forms to fit into the three linear models with governing
equation Y= mX + C along with the derivation of the K are shown in Appendix C.

2. DISCUSSION
2.1 Briefly describe the experiment that you designed in CI. What are the protein
concentrations in S25 and S60 in mg/mL?
The experiment designed in part CI was to determine the concentration of the 2 protein
samples in S25 and S60 using the HPLC system. The protein samples being used in the
experiment were -amylase incubated at two different temperature, 25C and 60C
respectively. 1 mL of the protein samples were purified using a syringe filter and run through
7

the HPLC system. The HPLC system was set at 280nm as the enzyme -amylase contains
aromatic amino acid residues which absorb ultraviolet (UV) light at this wavelength. The
sample were drawn and sent to the column in HPLC through the stationary phase by
dissolving the sample in the mobile phase which consists of water and acetonitrile. The
stationary phase usually are made from silica-based resins in the form of tiny spheres. They
are packed in a column orderly. When proteins mixed with the mobile phase are being run
through the column, the proteins are retarded to differential extents by their chemical
interaction with the stationary phase. The proteins are separated as they flow through the
column with stationary phase. Each protein has a unique retention time depends on the type
of stationary phase used. Retention time is the time the protein spent in travelling through the
column. The type of proteins involved is reflected in the chromatogram graph as different
peaks at particular time. However, since only one protein, -amylase was involved in the
experiment, it could be observed only one peak appeared in the chromatogram graph.
Although the interaction of the stationary phase used in the experiment had denatured the
protein, this is not of the concern of the experiment because the aim was solely to determine
the concentration of -amylase, not to separate and identify the proteins.
By assuming the HPLC system utilises the Beer-Lambert law, the concentrations of the two
samples could be determined from the chromatogram graph which was generated by the
computer programme in HPLC system. The concentrations of the two samples were
represented by the area under the chromatogram graph.
A = cl

for A<1

where A is a dimensionless parameter that represents the absorbance of a given wavelength,

is the molar absorptivity coefficient, c is the concentration of the compound in the solution
and l is the path length travelled by the wave through the sample (Sheffield Hallam
University, n.d.).
The concentrations of the samples were interpreted by the computer system in terms of mass
fraction in the unit of parts per million (ppm). In this case, we assume 1ppm to be
0.001mg/mL because the sample solutions are very dilute and could almost be assumed as
water, i.e. the contribution of weight from -amylase is assumed to be negligible.
Reading from the graph, the concentration of S25 was 0.132mg/mL and the concentration of
S60 was 0.128mg/mL.
In actuality, both the samples S25 and S60 were prepared from the same -amylase. Hence
their concentration should be identical. When an enzyme denatured, its tertiary structure that
serves as its function is altered. However, the primary structure should be maintained.
Therefore, the concentration does not change. The slight deviation we see from the
experimental results is very likely caused by experimental errors. The analysis of the errors
will be discussed in the later section of this report.

2.2 Determine protein activity present in S25 and S60, accounting for any
differences.
In this experiment, protein activity refers to the reaction of -amylase with starch. Starch is a
carbohydrate comprised of unbranched amylose and branched amylopectin components.
Iodine reacts with amylose in starch to form a deep blue complex which absorbs light at
600nm (UC Davis Chemwiki, n.d.). Since -amylase in S25 and S60 attacks only the -1,4
bonds in amylose but not the -1,6 bonds in amylopectin, iodine can be used to determine the
amount of amylose which has not been broken down by starch.
A sample containing a larger amount of amylose which has not been broken down by the
enzyme indicates lower protein activity. The protein activity is inversely proportional to
intensity of the deep blue complex produced upon addition of iodine, which can be detected
by the UV spectrophotometer set at 600nm.

Sample
Concentration (mg/ml)
Protein Activity (units)
S25
0.132855
4.1167
S60
0.128704
3.7908
Table 3 Concentration and Protein Activity for S25 and S60
However, the table above indicates that S60 has a lower protein activity despite its higher
concentration of -amylase. This can be attributed to the higher temperature which S60
sample was stored at, compared to that of S25. In enzymatic operations, protein activity does
not increase indefinitely with rising temperature. After an optimum temperature is reached
where protein activity is at its highest, the protein activity starts to decrease thereafter as
temperature is raised further. This is due to denaturation of the enzyme.
-amylase, not unlike other enzymes, has a very specific quaternary structure which allows it
to interact with amylose as a substrate. Most enzymes operate using the lock and key model,
where substrate and enzyme share an exclusive set of complementary geometric structures
which allow them to fit compatibly into each other. The induced fit model is also commonly
used to describe enzyme activity, which involves active sites on the enzyme making minor
shape changes to accommodate the substrates geometric structure.

Figure 6
From the figure above, -amylases protein activity with time starts to decrease after
temperature is raised beyond 45C. This indicates the occurrence of denaturation in the
enzyme, which causes weak non-covalent interactions such as hydrogen bonds and dipole
moment attractions to be broken. This causes the enzyme to unfold, which would lead to
changes in the specific quaternary structure of the enzyme, as well as the shape of the active
sites. With a loss in effective active sites, -amylase is not able to properly bind to its
substrates and a decrease in protein activity on amylose in starch ensues. Since S25 was kept
at 25C, it was not denatured and exhibited better protein activity compared to S60, which
was incubated at 60C and had been denatured, as indicated by the exponential decrease in
protein activity with time.

Significance with regards to Pharmaceutical Drug Assays

There are many similarities between enzyme activity and drug delivery in pharmaceutical
industries. Drugs administered to patients need to be highly specific in targeting only the
intended cells in the human body and not healthy cells to prevent side effects. Therefore, a
large number of drugs issued from pharmacies today are protein-based, employing the usage
of enzymes with appropriate active sites to react with ailment-causing substrates and convert
them into less harmful substances.
Drug assays are used in pharmaceutical industries to monitor protein activity and quantify
important parameters to maximise efficacy of drugs, akin to how the S25 and S60 samples
were used in the spectrophotometer to quantify the protein and analyse protein activity.
Moreover, measurements of drug assays aids in drug testing when determining the optimal
temperature to maximise the drug delivery effects in the human body for instance.

2.3.1 Choice of linearised form of the Michaelis-Menten relation in CIII

The following table summarised the key findings from the graphical analysis performed in
Section 2.3

10

Lineweaver-Burke

Langmuir

Eadie-Hofstee

19.13
2.813
0.9934

22.15
3.111
0.9421

19.599
2.8644
0.9003

Equation
Km (mg/mL)
qm (mg/mL min)
R2

Table 4 Equation, Km, qm & R2 values for the 3 linearised forms of the Michaelis-Menten approach

Lineweaver-Burke plot is chosen as the linearised model to determine the K m and qm values
for the Michaelis-Menten approach. This is so as it can be seen that the correlation
coefficient, R2, for the Lineweaver-Burke model is the closest to 1, which indicates that the
discrepancies between the data points and the best fit line is the smallest. Consequentially the
gradient and Y-intercept values would also be more accurate and since they are a function of
Km and qm, the values for Km and qm would be more accurate as well. The Langmuir and
Eadie-Hofstee model has less linear properties as the ratio S/q and q/S is one of their
independent variable groups respectively. q is an expression containing S and S f, which are
the error containing starch concentration data. In particular, S f would contain higher
percentage error as it is calculated from Equation 4, a linear fitting model, using the
experimentally determined absorbance (A). As such, the various percentage errors are
compounded to give less accurate q, and subsequently Km & qm values.

2.3.2 Experiment design

The objective of the experiment is to determine the reaction rate q in order to deduce the
binding constant Km. In order to do so, the Michaelis-Menten is called upon to provide an
equation that would allow the determination of Km. The equation has two variable unknown,
namely S, which is the substrate (-amylase) concentration with q being the reaction rate.
When the Michaelis-Menten equation is obeyed, the initial rate of an enzyme-substrate
reaction can be determined from the slope of a chord joining two points on a typical
concentration-time plot. Hence the simple relation of
may be used. It is clear from here that what is required is an experiment with differing initial
starch concentrations. Also, it is known that the Michaelis-Menten can be linearised to 3
different forms and only 3 or more data points would be needed. As such 5 test tubes of 3 mL
starch solutions each with differing initial starch concentrations, within the range of 0.5-2%
from the 3% starch provided, are prepared. At t=0, 1 mL of sample S25 was pipetted into all
the solutions to catalyst the starch hydrolysis reaction .At t=5, 0.2 mL of the reaction
mixtures are pipetted into iodine solutions to quench the reaction. The resultant solutions are
placed in the spectrophotometer to determine the absorbance from which the final
concentration of unreacted starch may be determined. 5 sets of q values with their
corresponding S values were obtained and tabulated in Table 4. The Lineweaver-Burke plot
can then be established as shown in Figure 3. As detailed in section 2.3, the binding constant
between starch and -amylase, Km, can be determined to be 19.13 mg/mL

11

3. ERROR ANALYSIS
1. In Part I of the experiment, the homogeneity of the sample solutions of S25 and S60 were
compromised as the concentrations of both samples differ slightly when they are prepared
from the same source. The reason being that the samples were left incubated at their
respective temperatures for 2 hours before being extracted. Within 2 hours, sedimentation of
the enzyme took place. Hence, the concentration of the samples at the top can vary
significantly from the bottom. Accurate concentration cannot be determined even though
extraction of the samples was done at the same level for both samples before analysing with
HPLC. To minimize this source of error, the samples should be shaken thoroughly prior to
extraction to ensure the sample solutions were homogenized.
2. The reaction may not be completely quenched upon adding the reaction mixture to iodine
at regular time intervals. This is because the absorbance measurement was not conducted
immediately after the quenching, regions in reaction mixture which is less accessible by
iodine molecules may have reaction proceeding on despite quenching was done. Even though
shaking of the reaction mixture with iodine was done after quenching, the results obtained
still have anomaly as reflected on Appendix A CI. Furthermore, this quenching process is also
highly subjected to human reaction time which may contribute to a large percentage error.
Hence, a better method to reduce the error from this is to use Maltosylamine, a specific
inhibitor of -amylase to quench the reaction (Walker & Axelrod, 1979). It acts by blocking
the active site and reducing the reaction rate at a higher rate.
3. There was only one cuvette provided to carry out all the absorbance measurements. Hence,
the absorbance readings might be subjected to deviation as well because the cuvette may be
stained upon being reused. The cleaning of the cuvette was not done thoroughly, hence the
reaction mixture may be contaminated as well. To minimize the error from this source, new
cuvette should be used for each absorbance reading measurement.
4. The sample solution S60 was not immediately extracted after being taken out from the
incubator. The remaining active sites on the enzyme may still display various rates of reaction
subjected to the ambient temperature, even though the enzyme had already been denatured.
The decrease in temperature causes the deviation in the activity measured, hence affecting the
absorbance value. To reduce this source of error, the sample solution S60 should be used
immediately after removal from incubator. Otherwise, the experiment for S60 should be
conducted in a water bath maintained at 60C to ensure a more reliable results.

4. CONCLUSION
For part CI, the concentrations of -amylase in 2 samples, S25 and S60, incubated for 2 hours
at 25C and 60C respectively were assessed using the HPLC system and evaluated to be
0.133 mg/ml and 0.129 mg/ml respectively. In part CII, the intensity of the deep blue
complex formed when iodine was added to starch was used to analyse the protein activity of
-amylase on starch through the use of a UV-visible spectrophotometer with wavelength set
at 600nm. The enzyme activities of S25 and S60 were determined to be 4.12 and 3.79 units of
-amylase required per unit of activity respectively. It was also deduced that the lower protein
12

activity for S60 was most likely due to denaturation of the enzyme from exceeding the
optimal temperature. For part CIII, the Lineweaver-Burke equation was chosen amongst the
other Michaelis-Menten relations as it had the correlation coefficient value R closest to 1.
After plotting, the binding constant between -amylase and starch had a value of 19.13 A600
and the highest reaction rate, qm had a value of 2.813 A600/min.

5. NOTATION
6. REFERENCE
Sheffield Hallam University. (n.d.) Beer's law - Theoretical principles. Retrieved from
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
Walker, D.E., Axelrod, B. (1979). Maltosylamine, a specific inhibitor of beta-amylase. Arch
Biochem Biophys, 195(2), 392-935. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/157719
UC Davis Chemwiki. (n.d.). Starch and iodine - Chemwiki. Retrieved from
http://chemwiki.ucdavis.edu/Biological_Chemistry/Carbohydrates/Case_Studies/Starc
h_and_Iodine

7. APPENDICES
Appendix A: Experimental data sheet
CI: Protein Quantification (HPLC)
Concentration of sample S25 -amylase stock solution: 132.855 ppm
Concentration of sample S60 -amylase stock solution: 128.704 ppm
S25
Sample

Time

ABS

1
2
3
4
5
6
7
8
9
10
11
12
13

0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0

0.755
0.710
0.637
0.571
0.635
0.521
0.508
0.457
0.383
0.357
0.349
0.311
0.306

Starch
Concentration
wt %
mg/ml
0.3848
3.848
0.3620
3.620
0.3247
3.247
0.2910
2.910
0.3234
3.234
0.2657
2.657
0.2590
2.590
0.2329
2.329
0.1950
1.950
0.1819
1.819
0.1781
1.781
0.1583
1.583
0.1557
1.557

S60
ABS
0.755
0.690
0.623
0.591
0.547
0.516
0.476
0.447
0.435
0.402
0.381
0.373
0.350

Starch
Concentration
wt %
mg/ml
0.3848
3.848
0.3517
3.517
0.3174
3.174
0.3012
3.012
0.2787
2.787
0.2631
2.631
0.2424
2.424
0.2280
2.280
0.2217
2.217
0.2048
2.048
0.1940
1.940 13
0.1899
1.899
0.1782
1.782

Table 2 Absorbance and Concentration of S25 & S60 at various time

Sample No
1
2
3
4
5
CII: Specific Binding Constants

Initial Starch Concentration, S

wt %
0.50
0.75
1.00
1.25
1.50

mg/mL
5
7.5
10
12.5
15.0

Absorbance, A600 (600 nm)

0.406
0.691
1.056
1.361
1.678

Table 4 Determination of Specific Binding Constant from S25

14

Appendix B: Risk Assessment Form

15

Appendix C: Calculations for Activity of sample S60 and

Derivations of Lineweaver-Burke, Langmuir & EadieHofstee plots
Calculations for Activity of S60
From Figure 2(a),
Rearranging,

From Figure 1(b),

Rearranging,

Since it took 4.66 minutes to hydrolyse 5.8884 mg of starch, the number of unit activity
based on the earlier definition for one unit of activity is as such

The amount of -amylase in the withdrawn 0.2 mL of reaction mixture that was added in the
iodine mixture is

. Since it is known that this

amount of -amylase brought about 3.7908 units of protein activity,

16

Derivations of Lineweaver-Burke, Langmuir & Eadie-Hofstee plots

Based on Equation (1), the concentration of starch after 5 minutes, Sf, can be determined
from the absorbance values found in Table 4
q can be deduced by calculating the average rate of change of concentration of starch over the
reaction time of 5 minutes

Sample calculation of sample no.1 from Table 4

Sample

S (mg/mL)

Sf (mg/mL)

q (mg/mL min)

1
2
3
4
5

5.00
7.50
10.0
12.5
15.0

2.0683
3.5217
5.3830
6.9383
8.5548

0.5863
0.7957
0.9234
1.112
1.289

The calculations were iterated for other samples and the results are tabulated below
Table 5 Initial starch concentration (S), final starch concentration (Sf) and reaction rate (q)
for the 5 samples

Lineweaver-Burke
Sample calculation of sample no.1 with data from Table 5

17

The calculations were iterated for other samples and the results are tabulated below
Sample

S (mg/mL)

1
2
3
4
5

5.000
7.500
10.00
12.50
15.00

q (mg/mL
min)
0.5863
0.7957
0.9234
1.112
1.289

1/S (mL/mg)
0.2000
0.1333
0.1000
0.08000
0.06667

1/q (mL
min/mg)
1.706
1.257
1.083
0.8993
0.7758

Table 6 Relevant data for Lineweaver-Burke plot

The equation is given as

Using data from Table 6, a plot of 1/q against 1/S can be derived as shown below

Figure 3 Lineweaver-Burke plot (Y=1/q; X=1/S)

From Figure 3,

18

Langmuir
Sample calculation of sample no.1 from Table 5

The calculations were iterated for other samples and the results are tabulated below
Sample

S (mg/mL)

q (mg/mL min)

S/q (mL/mg)

1
2
3
4
5

5.000
7.500
10.00
12.50
15.00

0.5863
0.7957
0.9234
1.112
1.289

8.528
9.428
10.83
11.24
11.64

Table 7 Relevant data for Langmuir plot

Using data from Table 7, a plot of S/q against S can be derived as shown below

19

Figure 4 Langmuir plot (Y=S/q; X=S)

From Figure 4,

Eadie-Hofstee
Sample calculation of sample no.1 from Table 5

The calculations were iterated for other samples and the results are tabulated below
Sample

S (mg/mL)

q (mg/mL min)

q/S (mL/mg)

1
2
3
4
5

5.000
7.500
10.00
12.50
15.00

0.5863
0.7957
0.9234
1.112
1.289

0.1173
0.1061
0.09234
0.08896
0.08593

Table 8 Relevant data for Eadie-Hofstee plot

Using data from Table 8, a plot of q against q/S can be derived as shown below

20

Figure 5 Eadie-Hofstee plot (Y=q; X=q/S)

From Figure 5,

21