Viacrucis, Jose Dale III L.

Date Performed: October 10, 2018

CHEM 160.1 – 6L Date Submitted: October 17, 2018

Exercise 5
Determination of the Activity of Amylase

I. Results

Table 5.1. Absorbance of standard starch solutions.

Test tube no. [starch] Amount of starch A620
(µmol/mL) (µmol)
1 0 (Blank) 0 0
2 0.0417 0.0834 0.468
3 0.1043 0.2086 0.816
4 0.2086 0.4171 1.39
5 0.4243 0.8486 2.157
6 0.6257 1.2514 2.395
7 0.8343 1.6686 2.436

Table 5.2. Absorbance at 620 nm of the different reaction mixtures

Test tube no. Incubation time H2O Urea NaCl
(min)
1 0 0.874 0.910 0.968
2 2 0.780 0.903 0.503
3 4 0.736 0.764 0.228
4 6 0.558 0.655 0.155
5 8 0.480 0.598 0.092
6 10 0.355 0.491 0.054

II. Sample Calculations

2.92 µ𝑚𝑜𝑙
[starch] = × (𝑣𝑜𝑙𝑢𝑚𝑒) × (𝐷𝐹)
𝑚𝐿

Example:
2.92 µ𝑚𝑜𝑙 1
[starch]test tube no. 2 = × 0.10𝑚𝐿 × 7 = 0.0417 µ𝑚𝑜𝑙/𝑚𝐿
𝑚𝐿
 Amount of starch (µ𝑚𝑜𝑙) = [𝑠𝑡𝑎𝑟𝑐ℎ] × 2 𝑚𝐿
Example:
µ𝑚𝑜𝑙
Amount of starch (µ𝑚𝑜𝑙)test tube no 2 = 0.0417 × 2𝑚𝐿 = 0.0834 µ𝑚𝑜𝑙
mL

Interpolation sample calculations:

µ𝑚𝑜𝑙 𝑠𝑡𝑎𝑟𝑐ℎ 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 = A620 x̂

𝑎𝑐𝑡𝑢𝑎𝑙 µ𝑚𝑜𝑙 𝑠𝑡𝑎𝑟𝑐ℎ 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 = µ𝑚𝑜𝑙 𝑠𝑡𝑎𝑟𝑐ℎ 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 x DF (7)

𝑈𝑛𝑖𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈)

Specific Activity = 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒

Example:
−0.177
Specific Activity of Reaction Mixture 1 (with H2O) = 𝑚𝑔 = 0.059 U/mg
(1.5 )(0.5 𝑚𝐿)
𝑚𝐿

III. Discussion

The experiment on determination of the activity of amylase started with the preparation
of different reagents needed. First, 0.1% starch solution was prepared by dissolving 0.1 g of
starch in 100 mL of 50 mM phosphate buffer (pH = 6.7). The starch was fully dissolved by
placing it in low heat. Second, I2/KI solution was then prepared by dissolving 10.0 g. of
potassium iodide in 250 mL distilled water before adding 5 g. of iodine. The solution was
made sure to be protected from light. Lastly, 0.02 M NaCl was prepared by dissolving 0.117
g. of NaCl in 100 mL distilled water.
Aliquots of 0.10, 0.25, 0.50, 1.00, 1.50, and 2.00 mL 0.1% starch solution was transferred
in test tubes which were labeled accordingly. Each of the standard solution was brought to
2.00 mL with distilled water. A blank of 2.00 mL pure water was also prepared. Before the
absorbance was read at 620 nm, the solutions were added with 1 drop of I2/KI solution was
made and mixed using vortex.
Moreover, salivary amylase was prepared by collecting 5 mL saliva in a beaker. It was
diluted to 100 mL using 50 mM phosphate buffer, with an addition of 0.5 mM CaCl2. Next,
 the solution was centrifuged for 10 minutes at 3000 x g. Supernatant, containing the enzyme
was obtained.
Furthermore, reaction mixtures were prepared in three separate beakers. Reaction was
started by adding 0.5 mL amylase solution to each beaker. Samples with volume of 1.0 mL
was collected from each reaction mixture at 0, 2 , 4 , 6, 8, and 10 minute intervals before
diluting it in 1.0 mL water. The enzyme reaction was stopped by heating the solution, then
cooled. A drop of I2/KI solution was added before absorbance at 620 nm. was measured.

2.5
y = 4.8528x + 0.2102
R² = 0.9651
2

1.5
A620

0.5

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
[starch]

Figure 1. Standard curve of the starch standard solutions

Concentrations of the standard starch solutions in µmol/mL was calculated (see Sample
Calculations). Values obtained were used to plot the standard curve of the standard solutions as
seen in Figure 1. As calculated, the linear expression of the standard curve is y = 4.8528x +
0.2102 where y is the absorbance value at A620, while x is the starch concentration (µmol). The
slope of the equation determines the enzyme activity, thus in the case of the starch standard
solutions, the enzyme activity (U) is 4.8528 (U) or 4.8528 µmol of starch per minute. The µmol
starch remaining after each time intervals for the different reaction mixtures were interpolated
using the standard curve of the standard solutions. The obtained values were then used to
obtained the actual umol starch remaining (see sample calculations). Actual µmol starch
remaining vs. incubation time of each reaction mixture were then plotted to obtain the unit
activities and the specific activities (see sample calculations). Data obtained is tabulated in Table
5.3.
Table 5.3. Summary of the µmol starch remaining, slope, and the specific activity of the different
reaction mixtures
actual µmol starch remaining
Test tube no. Incubation time H2O Urea NaCl
(min)
1 0 0.9576 1.0094 1.0934
2 2 0.8218 0.996 0.4221
3 4 0.7581 0.7987 0.0256
4 6 0.5012 0.6419
5 8 0.3892 0.5593
6 10 0.2086 0.4053
Slope (U) 0.177 0.1778 0.5339
Specific Activity 0.059 0.0593 0.1780
(U/mg)

1.2

1
µmol starch remaining

0.8

0.6

0.4

0.2
y = -0.177x + 1.1957

0
0 2 4 6 8 10
Incubation time (min)

Figure 2. Plot of the actual µmol starch remaining vs. incubation time of reaction mixture 1 (with
H2O)
 1.2

1
µmol starch remaining

0.8

0.6

0.4

y = -0.1778x + 1.2996
0.2

0
0 2 4 6 8 10
incubation time (min)

Figure 3. Plot of the actual µmol starch remaining vs. incubation time of reaction mixture 2 (with
urea)

1.2

0.8
µmol starch remaining

0.6
Y-Values
0.4 Linear (Y-Values)

0.2

y = -0.5339x + 1.5815
0
0 2 4
-0.2
incubation time (min)

Figure 4. Plot of the actual µmol starch remaining vs. incubation time of reaction mixture 2 (with
NaCl)
 As seen in the data obtained, reaction mixture 1 and 2 have almost the same specific
activity value and enzyme activity value which could mean that the presence of urea does not
really affect the enzymatic activity of amylase. However, reaction mixture 3 with NaCl showed a
double amount of value of specific activity. This means that the presence of NaCl hasten the
enzymatic reactions of amylase.
The reason why the graph is not linear throughout the incubation time because the
enzyme may already become saturated and the maximum velocity of the enzymatic reaction will
be obtained at a certain point of substrate concentration. Level-off appearance of the graph will
be observed and the plateau portion indicates enzyme saturation.
Catalytic actions of the enzymes are altered by chemical substances such as enzyme
inhibitors. They consequently slow down or stop the catalysis reaction. Inhibitions of the
enzymes can be reversible or irreversible. There are two types of reversible inhibitions. One type
is competitive inhibition which occurs when the substrate and a substance resembling the
substrate are both added to the system, binding to the active site of the enzyme. The presence of
the substance that resembles the appropriate substrate will lead to prevention of the binding of
the appropriate substrate to the active site. This type of inhibition decreases the affinity of the
enzyme for the substrate by adding structural analogues of the substrate suitable for the active
site of the enzyme. On the other hand, non-competitive inhibition occurs when non-competitive
inhibitors do not attach to the active site but rather to the other sites or portions of the enzyme
surface. Occurrence of this alters the tertiary structure of the enzyme thus reducing its catalytic
effectiveness. Unlike in the competitive inhibition, this type of inhibition does not affect the
affinity of the enzyme for the substrate, but it lowers the maximum rate of reaction. Moreover,
irreversible inhibitions result from some compounds altering the enzyme structure permanently.
Enzyme activity is affected by number of factors which would consequently alter the rate
of reaction by the enzyme. Factors include temperature, pH, and concentration (substrate-enzyme
concentration). Temperature increase also increases the kinetic energy of the molecules. In fluid,
more random collisions occur between molecules per unit time. Enzymes catalyse reactions by
random collision with substrate molecules, thus increase in temperature increases the rate of
reaction. This would result to more products formed (Reece et al., 2011). However, an increase
in temperature also means an increase in the vibrational energy of the molecules, putting strain
on the bonds that hold them together. This could result to bond breakage, affecting the shape or
the conformation of the enzyme which consequently decreases the rate of reaction. Moreover,
different enzymes have varying optimum pH levels. A change in pH above or below the
optimum levels will immediately decrease reaction rate. Extreme changes in pH causes
denaturation and will cause permanent loss of function of the enzymes (Reece et al., 2011).

IV. References