Post-laboratory Report on

Exercise 5
Determination of the Activity of Invertase

Vikki Anne R. Cedo

CHEM 160.1 - 3L
2nd Semester 2014-2015

Groupmates:
Desiree Joy Cerico
Ma. Kriselle Ornales
Mary Ranzelle Pasang

Ms. Korina Vida G. Sinad

Laboratory Instructor

Enzymes are large molecules that increase the rates of chemical reactions
without themselves undergoing any change; they are not consumed in the reaction
that they catalyze (Bettelheim, 2007). Without enzymes to serve as biological
catalysts, life as we know it would not be possible. Enzymes lower the activation
energy of the reaction and as a result, the reaction proceeds in a shorter time.
Invertase (beta-fructofuranoside) is an enzyme that is mainly used in the food
industry where fructose is preferred over sucrose because it is sweeter and does not
crystallize easily. Invertase catalyzes the cleavage of sucrose into two
monosaccharides: glucose and fructose. Carbohydrates, being the primary source of
energy in all living organisms, are very significant given that some of them also
have other roles aside from supplying energy like signalling and serving as stress
protectants. Invertase plays a central role as it is a sucrose hydrolyzing enzyme, so
named because of the inversion that happens in the optical rotation during the
hydrolysis of sucrose.
This exercise was performed to determine the effect of activator and inhibitor
on enzyme activity.
Table 5.1. Absorbance of standard glucose solutions.
Test tube no.
1
2
3
4
5
6
7
8

[Reducing sugar]
(mol/mL)
Blank
0.05
0.10
0.20
0.30
0.40
0.50
1.0

Amount of reducing
sugar (mol)
0
0.05
0.10
0.20
0.30
0.40
0.50
1.0

A510
0
0.026
0.134
0.128
0.196
0.264
0.322
0.611

0.7
0.6
0.5
0.4
Absorbance at A510

0.3
0.2
0.1
0
0

0.2

0.4

0.6

0.8

1.2

Reducing sugar concentration (mol/mL)

A510

Linear

Figure 5.1. Absorbance of the different concentrations of the reducing sugar at

510nm.

Linear Regression values:

y-int = 0.020138149, slope = 0.596037177, r=0.992230854, R2 = 0.984522069
Linear equation: Y = 0. 596037177x + 0.020138149

Interpolation sample calculations:

Sample + H2O
At 2 minutes, Y = 0. 596037177 (0.263) + 0.020138149 = 0.177 mol of reducing
sugar / mL
Sample + Urea
At 2 minutes, Y = 0. 596037177 (0.253) + 0.020138149 = 0.171 mol of reducing
sugar / mL
Sample + BSA

At 2 minutes, Y = 0. 596037177 (0.311) + 0.020138149 = 0.206 mol of reducing

sugar / mL

Table 5.2. Absorbance at 510nm of the different reaction mixtures.

Test tube no.

Incubation

H 2O

Urea

BSA

1
2
3
4
5
6

time (min)
0
2
4
6
8
10

0.220
0.263
0.278
0.290
0.319
0.290

0.241
0.253
0.249
0.269
0.277
0.290

0.249
0.311
0.304
0.334
0.347
0.394

Table 5.3. Concentration of samples and incubation time.

Test tube no.

1
2
3
4
5
6
Slope (U)
Specific
Activity
(U/mg)

Incubation
time (min)
0
2
4
6
8
10

mol glucose/mL
H 2O
Urea
0.151
0.177
0.186
0.193
0.210
0.192
4.44 x 10-3
2.96 x 10-3

0.163
0.171
0.169
0.180
0.185
0.193
2.9 x 10-3
1.93 x 10-3

BSA
0.169
0.206
0.201
0.219
0.227
0.255
7.34 x 10-3
4.89 x 10-3

0.3
0.25
0.2

Reducing sugar concentration (mol/mL)

H2O

0.15

Urea

BSA

0.1
0.05
0
0

10

Incubation Time (min)

Figure 5.2. Different concentrations of reducing sugar and incubation time.

Specific activity is used in measuring enzyme kinetics (the rate of reaction of
an enzyme with a particular substrate). It is the amount of substrate the enzyme
converts per mg of protein in the enzyme preparation per unit of time (Nelson &
Cox, 2000). It is also a measure of enzyme purity. The larger the value of the SA,
means the preparation is more pure; this is because the amount of protein is
typically less (in mg), but the rate of reaction stays the same (but may also increase
due to reduced interference or removal of inhibitors). The specific activity in the
presence and absence of inhibitors and regulators were computed as follows:
SA of sample+ water: U / mg of protein
= 4.44 x 10-3 / 1.5 mg
= 2.96 x 10-3 U/mg
SA of sample + urea: U / mg of protein
= 2.9 x 10-3 / 1.5 mg
= 1.93 x 10-3 U/mg
SA of sample + BSA: U / mg of protein
= 7.34 x 10-3 / 1.5 mg
= 4.89 x 10-3 U/mg

The graph is not linear throughout the incubation time because at a certain
point of substrate concentration, the enzyme may already be saturated and the
enzymatic reaction's maximum velocity can be obtained. The graph would then
appear to level off and the plateau portion indicates enzyme saturation. Urea has
been reported as one of the substances which can be a potential non-specific
protein denaturant; it is also a non-competitive inhibitor. Both urea and BSA are
inhibitors that theoretically, should lower enzyme activity for invertase.
Enzyme inhibitors are chemical substances which alter the catalytic action of
the enzymes and consequently slows down or stops the catalysis. Enzyme
inhibitions can be reversible or irreversible. Reversible inhibitions are competitive
and non-competitive. Competitive inhibition occurs when the substrate and a
substance that resembles the substrate are both added to the enzyme and then
they bind to the active site of the enzyme, preventing the binding of the appropriate
substrate. Competitive inhibitors decrease the affinity of the enzyme for the
substrate by adding structural analogs of the substrate that is suitable for the active
site of the enzyme. Non-competitive inhibitors on the other hand do not attach to
the active site of the enzyme but attaches to other sites or portion of the enzyme
surface. Even so, they may sufficiently alter the tertiary structure of the enzyme so
that its catalytic effectiveness is reduced. It does not affect the affinity of the
enzyme for the substrate but it lowers the maximum rate of reaction. Irreversible
inhibitions are a result of some compounds altering the structure of the enzyme
permanently.
There are a number of factors that affect enzyme activity. Changing these
alter the rate of reaction caused by the enzyme. Some of these factors are
temperature, pH, and concentration (substrate and enzyme concentration). An
increase in the temperature increases the kinetic energy that the molecules have.
In fluids, this means that more random collisions may occur between molecules per
unit time. Enzymes catalyze reactions by random collision with substrate molecules,
thus temperature increases the rate of reaction, and as a result, forms more
products (Reece et al., 2011). However, increasing temperature also means an
increase in the vibrational energy that the molecules have (enzyme molecules),
which puts strain on the bonds that hold them together. Hydrogen and ionic bonds
break as a result of that strain. Bond breakage within the enzyme means the
change of conformation or shape, affecting the shape of the active site rendering it
uncomplementary to the shape of the substrate and therefore decreases the rate of
reaction. Different enzymes have varying optimum pH values. This is the pH value
at which the bonds within them are influenced by H+ and OH- ions in such a way
that the shape of their active site is most complementary to the shape of their
substrate. A change in pH above or below the optimum will immediately cause a
decrease in the rate of reaction because more enzyme molecules will have active

sites whose shapes aren't complementary to the shape of their substrate. Small
changes in pH, be it above or below the optimum, do not cause permanent or
irreversible change to the enzyme, since the bonds can be reformed. However,
extreme changes in pH can cause denaturation and this will cause permanent loss
of function.
Enzyme and substrate concentrations affect the rate of reaction of an
enzyme catalyzed reaction. When the substrate concentration is kept constant, and
the enzyme concentration is increased, the rate of reaction increases linearly. If the
enzyme concentration doubles, the rate doubles as well. Increasing the substrate
concentration increases the rate of reaction because more substrate molecules will
be colliding with enzyme molecules, so more product will be formed. However, after
a certain concentration, any increase will have no effect on the rate of reaction,
since it will no longer be the limiting factor. If the enzyme concentration is kept
constant and the substrate concentration is increased, a saturation curve results.
The rate does not raise continuously. Instead, a point is reached after which the rate
stays the same even if the substrate concentration is increased further (Bettelheim,
2007). The enzymes will be saturated. This occurs because at the saturation point,
substrate molecules are bound to all available active sites of the enzymes. The
reaction takes place at the active sites, once they are all occupied, the reaction
proceeds at its maximum rate. Increasing substrate concentration will no longer
increase the rate of reaction for the excess substrate cannot find any active sites to
which they can bind. Enzyme concentration, when increased, will increase the rate
of reaction, because more enzymes will be colliding with substrate molecules.

Literature Cited:
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry.
Brooks/Cole by Thomson Learning.
Nelson, D. and Cox, M. 2000. Lehninger Principles of Biochemistry, 3rd Edition.
Worth Publishers, New York, NY, USA.
Reece, Jane B., Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V.
Minorsky, and Robert B. Jackson. Campbell Biology. 9th ed. Boston: Benjamin
Cummings/ Pearson Education, 2011. Print