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Department of Biochemistry

Faculty of Pharmacy
University of Santo Tomas

LABORATORY MANUAL IN

BIOCHEMICAL
CATALYSIS

Group 5 3BBC
Josol, Sheena C.
Jugno, Catherine C.
Manalo, Micah Kesiya
M.
Manipol, Erwin T.
TABLE OF CONTENTS

CONTENTS

Determination of Different Parameters Influencing Invertase


Activity

PAGE
Experiment 1A: Effect of Sucrose Concentration on Invertase
Activity
3

Experiment 1B: Determination of Optimal Incubation Time


6

Experiment 1C: Determination of Optimal Incubation


Temperature 10

Experiment 1D: Effect of pH on Invertase Activity 12

Experiment 2: Substrate Specificity 14

Experiment 3: Isolation and Purification of Invertase 16

Experiment 4: Effect of Inhibitors on Invertase Activity 20

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DETERMINATION OF DIFFERENT PARAMETERS INFLUENCING
INVERTASE ACTIVITY

THEORETICAL BACKGROUND
Invertase

The official name for Invertase is beta-fructofuranosidase (EC.3.2.1.26), which


implies that the reaction catalyzed by the enzyme, is the hydrolysis of the terminal non-
reducing beta-fructofuranoside residues in beta-fructofuranosides.

In contrary to most other enzymes, invertase exhibits relatively high activity over
a broad range of pH (3.54.5) with the optimum near pH of 4.5. The enzyme activity
reaches its maximum catalytic activity at 60 C.

Factors affecting Enzyme activity

In order to understand the basic enzymatic mechanism and to select a method


for enzyme analysis, one must have a basic knowledge about the enzymes kinetic
theory. The conditions selected to measure the activity of an enzyme would not always
be the same as those selected to measure the concentration of its substrate. Several
factors affect the rate at which enzymatic reactions occur like temperature, pH, enzyme
concentration, substrate concentration, and the presence of any inhibitors or activators.

Substrate Concentration

When enzyme is mixed with excess of substrate, the initial rate varies
hyperbolically with substrate concentration, [S], for a fixed concentration of enzyme. At
low substrate concentrations, the occupancy of the active sites on the enzyme
molecules is low and reaction rate is related directly to the number of sites occupied.
This approximates to first order kinetics in that the rate is directly proportional to the
substrate concentration. At high substrate concentrations, effectively all of the active
sites are occupied and the reaction becomes independent of the substrate
concentration, since no more enzyme-substrate (ES) complex can be formed and zero-
order or saturation kinetics are observed. Under these conditions, the reaction rate is
dependent upon the conversion of ES complex to products and the diffusion of products
from the enzyme. The mathematical equation expressing this hyperbolic relationship
between initial rate and substrate concentration is known as the Michaelis-Menten
equation.

Temperature

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Temperature affects enzyme activity in much the same way as it affects other
chemical reactions. Rates increase by between 4 and 8% per degree C, although at high
temperatures denaturation of the enzyme protein decreases product formation. Thus it is
important when carrying out an enzyme assay to ensure that the temperature remains
constant, and also that you know exactly what it is. The rates of enzyme-catalyzed
reactions also increase with increasing temperature. However, enzymes are proteins
that become denatured at high temperatures. Each enzyme has an optimum
temperature, at which it operates at maximal efficiency. If the temperature is raised to a
point somewhat beyond the optimal temperature, the activity of many enzymes decline
abruptly. An enzyme optimum temperature is usually close to the normal temperature of
the organism it comes from. For example, most human enzymes have temperature
optima close to 37C

Incubation Time
Enzyme catalyzed reactions are reversible. Initially, there is little or no product
present, and therefore the reaction proceeds only in the forward direction. However, as
the reaction continues, so there is a significant accumulation of product, and there is a
significant rate of back reaction. As a result, the rate of formation of product slows down
as the incubation proceeds, and if the incubation time is too long, then the measured
activity of the enzyme is falsely low. The longer an enzyme is incubated with its
substrate, the greater the amount of product that will be formed. However, the rate of
formation of product is not a simple linear function of the time of incubation.

pH
Enzymes are active only within a limited range of pH. Changes in ionizable
groups may result in changes in the tertiary structure of the enzyme. Drastic changes in
pH often lead to denaturation. Although a few enzymes tolerate large changes in pH,
most enzymes are active only within a narrow pH range. For this reason, living
organisms employ buffers that closely regulate pH. The pH value at which an enzymes
activity is minimal is called pH optimum.

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EXPERIMENT 1A
EFFECT OF SUCROSE CONCENTRATION ON
INVERTASE ACTIVITY

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Determine the effect of sucrose concentration on invertase activity;
2. Identify optimum concentration of substrate;
3. Generate a graph showing the effects of incubation time to the invertase activity.

MATERIALS AND METHODS


Materials
Sucrose 0.1M Acetate buffer Microcentrifuge
(pH 4.5) tubes
DNS reagent
Micropipettes Microwell plate
2% TCA solution
Spectrophotometer

Procedure
Reagent Preparation
1. DNS reagent: Mix 300 g of potassium sodium tartrate tetrahydrate and 16 g of
sodium hydroxide with 500 mL of distilled water. Add slowly 10 g of 3,5 DNS acid.
Dilute with distilled water to make 1 L solution. Transfer to amber colored
container.
2. 2% TCA: Accurately weigh 0.02 g of tricholoacetic acid and dissolve in 10 mL
distilled water.
Enzyme-Substrate complex
1. Substrate Preparation: Prepare five different substrate concentrations
derived from km based calculations. For sucrose, substrate
concentrations to be prepared were 7.5, 15, 30, 60 and 90 mM.
2. Repeat procedure on substrate preparation using glucose. Prepare
glucose solution of 1, 0.500, 0.250, 0.125, 0.0625 mg/mL
concentrations. Generate glucose standard curve by graphing
concentration versus absorbance reading.
3. Enzyme Preparation: Make a standard working invertase solution 0.1% (w/v) by
dissolving 1 gram of standard invertase in 10 mL of acetate buffer pH 4.5.
4. Mix the prepared substrates and enzymes by adding 75 l of invertase solution to
each of the substrate concentrations prepared.

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5. Incubate it for 5 minutes at 60C and add 50 L of DNS reagent. Boil
solution until color develops into bloody red.
6. If immediate transfer of the solution to the microwell plate is not
possible, add 20 L TCA solution, transfer and subject it to
spectrophotometer for absorbance reading at 540 nm.
7. Note substrate concentration which yielded the highest absorbance.

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EXPERIMENT 1A
EFFECT OF SUCROSE CONCENTRATION ON
INVERTASE ACTIVITY

RESULTS
Effect of Substrate Concentration
Well Sucrose concentration Absorbance
Number (mM) 540 nm
1 7.5 0.062
2 15 0.067
3 30 0.099
4 60 0.138
5 90 0.122

Glucose Standard Curve

Glucose concentration Absorbance


(g/mL) 540 nm
62.5 0.051
125 0.065
250 0.121
500 0.216
1000 0.338

Glucose Standard Curve


0.4

0.3 f(x) = 0x + 0.04


R = 0.99
0.2
Absorbance at 540nm
0.1

0
0 200 400 600 800 1000 1200
[Glucose] g/ml

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The equation of the line from the standard curve was used to compute for the
concentrations of the glucose formed. The rate of the formation of glucose was then
graphed with respect to the concentration of the substrate.
Sucrose-Invertase Assay
Glucose Formed (g /mL) Vo
(g /mL-min)
81.67 8.17
98.33 9.83
205.00 20.50
335.00 33.50
281.67 28.17

CONCLUSIONS
It was shown in the graph generated that the rate of formation of glucose
increased as the concentration of the substrate is increased until it reached a maximum
velocity and slowly diminishes as the substrate concentration is increased.

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EXPERIMENT 1B
DETERMINATION OF OPTIMAL INCUBATION TIME

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Determine the effect of incubation time on invertase activity;
2. Identify optimum incubation time of substrate;
3. Generate a graph showing the effects of incubation time on invertase activity.

MATERIALS AND METHODS


Materials
Sucrose 0.1M Acetate buffer Microwell plate
(pH 4.5)
DNS reagent Timer
Micropipettes
2% TCA solution Spectrophotometer
Microcentrifuge
tubes

Procedure
1. Prepare enzyme-substrate solution utilizing the optimal substrate concentration
determined from the previous experiment.
2. Incubate solutions at five minutes interval, followed by addition of DNS reagent and
boil until red color development.
3. Add 2% TCA solution to stop further reaction as described in the first experiment.
4. Read absorbance at 540 nm.
5. Note incubation time which yielded the highest absorbance.

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EXPERIMENT 1B
DETERMINATION OF OPTIMAL INCUBATION TIME

RESULTS
Incubation time

Well Incubation Time Absorbance


number (min) 540 nm
1 5 0.048
2 10 0.048
3 15 0.052
4 20 0.047

CONCLUSION
The optimum incubation time for maximum rate of glucose formation was
illustrated in graph above. It was shown that the velocity of glucose formation was at its
maximum at 5 minutes and slowly decreases as the time of incubation is increased.
Thus, the optimal incubation time for the invertase is 5 minutes.

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EXPERIMENT 1C
DETERMINATION OF OPTIMAL INCUBATION TEMPERATURE

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Determine the effect of incubation time on invertase activity;
2. Identify optimum incubation time of substrate;
3. Generate a graph showing the effects of incubation time on invertase activity.

MATERIALS AND METHODS


Materials
Sucrose 0.1M Acetate buffer Microwell plate
(pH 4.5)
DNS reagent Timer
Micropipettes
2% TCA solution Thermometer
Microcentrifuge
Spectrophotometer
tubes

Procedure
1. Prepare enzyme-substrate solution using the predetermined conditions identified
from the previous experiment.
2. Subject prepared solutions to incubation at different temperature range from 30-
70 C
3. Repeat procedures from previous experiment specifying addition of reagents and
absorbance reading.
4. Note incubation temperature which yielded the highest absorbance.

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EXPERIMENT 1C
DETERMINATION OF OPTIMAL INCUBATION TEMPERATURE

RESULTS
Incubation Temperature

Well Incubation Absorbance


number Temperature 540 nm
C

1 30 0.048
2 40 0.051
3 50 0.051
4 60 0.111
5 70 0.053

CONCLUSION
Based from the graph generated, the optimum incubation temperature to achieve
the maximum rate of glucose formation is 60 C. The invertase is active and catalyses
the hydrolysis of sucrose to form glucose and fructose at this temperature.

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EXPERIMENT 1D

EFFECT OF pH ON INVERTASE ACTIVITY

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Determine the effect of incubation time on invertase activity;
2. Identify optimum incubation time of substrate;
3. Generate a graph showing the effects of incubation time on invertase activity.

MATERIALS AND METHODS


Materials
Sucrose pH meter Microwell plate
DNS reagent Micropipettes Timer
2% TCA solution Microcentrifuge Spectrophotometer
tubes
Buffers

Procedure
1. Prepare enzyme-substrate solution using the predetermined conditions identified
from the previous experiment.
2. Subject prepared solutions to different pH ranging from pH 2-8.
3. Repeat procedures from previous experiment specifying addition of reagents
followed by absorbance reading at 540 nm.
4. Note pH which yielded the highest absorbance.

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EXPERIMENT 1D

EFFECT OF pH ON INVERTASE ACTIVITY

RESULTS
Effect of pH

Well pH Absorbance
number 540 nm
1 3.5 0.046
2 4.5 0.217
3 6.5 0.049
4 8.0 0.093
5 11.0 0.0188

CONCLUSION
As illustrated by the points in the graph, it can be concluded that the optimum pH
for invertase activity is at pH 4.5. However, there is an ambiguous data found at pH 11
where it shows a very high enzymatic activity yet according to several published studies
the activity of invertase must be around the acidic pH to neutral. This may be a result of
wrong buffer preparation or might be because of any misstep in one of the procedure
used.

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EXPERIMENT 2
SUBSTRATE SPECIFICITY

THEORETICAL BACKGROUND
Enzyme specificity is the ability of an enzyme to choose exact substrate from a
group of similar chemical molecules. It is a molecular recognition mechanism and it
operates through the structural and conformational complementarity between enzyme
and substrate. Enzymes show different degrees of specificity towards their substrate.

Generally, there are four distinct types of specificity:

Absolute specificity - the enzyme will catalyze only one reaction.

Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.

Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.

Stereochemical specificity - the enzyme will act on a particular steric or optical


isomer.

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Compare substrate specificity of invertase to substrates sucrose, maltose and
glucose;
2. Generate lineweaver burk plot.

MATERIALS AND METHODS


Materials
Sucrose 0.1M Acetate buffer Microwell plate
(pH 4.5)
DNS reagent Digital Dry Bath
Micropipettes
2% TCA solution Spectrophotometer
Microcentrifuge tubes

Procedure
1. Prepare five different concentrations of maltose and lactose following the
observed concentrations as in experiment 1D.

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2. Follow succeeding procedures from the aforementioned experiment.

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EXPERIMENT 2
SUBSTRATE SPECIFICITY

RESULTS
Maltose

Substrate concentration Absorbance


mM 540 nm 1/[Substrate] 1/VO
7.5 0.046 0.133333333 0.352945
15 0.044 0.066666667 0.4
30 0.045 0.033333333 0.222222
60 0.048 0.016666667 0.146342
90 0.052 0.011111111 0.075949

Lineweaver-Burk Plot of Maltose

Lineweaver-Burk Plot
[Maltose]
0.5
0.4
f(x) = 2.17x + 0.13
0.3 R = 0.64
1/Vo 0.2
0.1
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
1/[Maltose]

Lactose

Substrate concentration Absorbance


mM 540 nm 1/[Substrate] 1/VO
7.5 0.049 0.133333333 0.352945
15 0.044 0.066666667 0.461531
30 0.045 0.033333333 0.4
60 0.050 0.016666667 0.260872
90 0.043 0.011111111 0.285714

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Lineweaver-Burk Plot of Lactose

Lineweaver-Burk Plot
[Lactose]
0.6

0.4
f(x) = 0.67x + 0.32
1/Vo R = 0.17
0.2

0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
1/[Lactose]

The data and graph for sucrose-enzyme activity from the previous experiment
was used and from the individual data, the Michaelis-Menten graph was generated. It
explains the specificity of invertase to various substrate.

Substrate Specificty of Invertase


40

30
[Maltose]
20 [Lactose]
Vo (g/min-mL)
[Sucrose]
10

0
0 20 40 60 80 100

Substrate concentration (mM)

It was evident that the invertase is specific to the hydrolysis of sucrose to form
glucose and fructose. Since maltose and lactose are not hydrolyzed to monomers, they
produced a lower absorbance thus a very low rate of reaction.

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Km and Vmax of Different Substrates

Substrate Km (mM) Vmax (g/mL-min)


Sucrose 29.7396 37.7358
Maltose 17.1561 7.9177
Lactose 2.0951 3.1496

Using the Lineweaver-Burk equation, the Km and the Vmax were computed for
each of the substrate. As shown in the table, Sucrose has the highest Km and Vmax.
This proves that the activity of the invertase is specific to the hydrolysis of sucrose.

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EXPERIMENT 3

ISOLATION AND PURIFICATION OF INVERTASE


THEORETICAL BACKGROUND
Invertase, also called beta-fructofuranosidase (EC.3.2.1.26), is a glycoprotein
with an optimum pH 4.5 and stability at 60 C. According to Samarth Kulshrestha et al.,
invertase exists in more than one form in yeasts. It can either be intracellular or
extracellular. The extracellular invertase has a molecular weight of 135 KDa whereas the
intracellular invertase has a molecular weight of 270 KDa. Saccharomyces cerevisiae
(baker's yeast) is the chief strain used for the production and purification of the enzyme.

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Isolate and purify invertase from dry yeast.
2. Rationalize the steps in purifying an enzyme.
3. Skillfully perform gel filtration chromatography and SDS-PAGE.
4. Construct a purification table.

MATERIALS AND METHODS


Materials
Dry Yeast Polyacrylamide gel Gel filtration column
70% (NH4)2SO4 Sephadex G-100 Centrifuge
solution
250mL Erlenmeyer Hot plate
0.1M Acetate Buffer flask
Digital Dry Bath
(pH 4.5)
Conical tubes
Spectrophotometer
0.1M NaHCO3
Micro well plate
Electrophoresis Set-
Bradford Reagent
Micro centrifuge up
0.1% DNS Reagent tubes

Procedure
1. Dissolve 28.25g of dry yeast in 100mL of 0.1M NaHCO3 in a 250mL Erlenmeyer
flask.
2. Plug the mouth with cotton and cover with aluminum foil. Incubate the mixture at
37C for 24 hours.
3. Subject the mixture to sonication (if applicable) or homogenize using a
blender/vortex.

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4. Transfer the mixture in conical tubes and centrifuge at 4500 rpm for 90 minutes
at 4C.
5. Discard the precipitate and collect the supernatant.
6. Slowly add 70% solution of (NH4)2SO4 to the supernatant until the appearance
of precipitates.
7. Centrifuge the solution at 4500 rpm for 90 minutes at 4C.
8. Discard the supernatant and collect the precipitate.
9. Dissolve the precipitate in enough acetate buffer and filter the solution using a
filter paper.
10. Prepare the gel filtration column using the sephadex G-100.
11. Wash the column with acetate buffer for 8-10 minutes.
12. Run the samples (must be <5% of the bed volume) in the column and collect
1mL fractions in micro centrifuge tubes.
13. Perform enzymatic assay on all the gel fractions and read the absorbance at
540nm.
14. Read also the absorbance of the gel fractions at 280nm.
15. Graph the absorbance at 280nm and 540nm and pool the fractions which exhibit
high protein content and enzymatic activity.
16. Perform SDS-PAGE to determine the purity of the isolated enzyme.

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EXPERIMENT 3

ISOLATION AND PURIFICATION OF INVERTASE

RESULTS

The equation of the line from the standard curve was used to compute for the
total activity of the crude, (NH4)2SO4 precipitate, and the gel filtration fractions. The BSA
standard curve was used to determine the protein content of each purification step.

Fraction numbers 7, 9, and 11 were pooled together as they exhibited


the highest enzymatic activity of all the fractions.

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As shown in the SDS-PAGE result, no bands can be seen in both the crude
sample and the gel fraction in the resolving gel. However, a faint band in the well of the
crude sample can be seen which indicates that the molecular weight of the protein is
larger than the ladder used.
Purification Table

Total Total Specific


Purification
Step protein activity activity % yield
fold
(mg) (units) (units/mg)
Homogenization 4.26 35669 8364 100 1
Ammonium
sulfate 2.03 19973 9839 56 1.18
precipitation
Sephadex G-100 0.59 15540 26242 44 3.14

Based from the results, the percent yield of the enzyme after the purification is
found to be 44% and the purification fold increases to 3.14. These numerical results
show that the enzyme was purified but the purification procedure can be considered
inefficient in producing a pure invertase because of a low yield and a very low
purification fold.

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EXPERIMENT 4

EFFECT OF INHIBITORS ON INVERTASE ACTIVITY


THEORETICAL BACKGROUND
The activity of enzymes can be inhibited. Studies of the methods by which
enzymes are inhibited have practical applications. Inhibition may be reversible or
irreversible. Irreversible inhibitors usually bond covalently to the enzyme, often to a side
chain group in the active site. In reversible inhibition, the inhibitor can dissociate from the
enzyme because it binds through non covalent bonds. Most common forms of reversible
inhibition are competitive, non-competitive and uncompetitive inhibition
In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at
the same time. This usually results from the inhibitor having an affinity for the active site
of an enzyme where the substrate also binds; the substrate and inhibitor compete for
access to the enzyme's active site. This type of inhibition results in an increase in Km but
still retains the Vmax . In uncompetitive inhibition, the inhibitor binds only to the
substrate-enzyme complex. This type of inhibition causes Vmax to decrease and Km to
decrease. In non-competitive inhibition, the binding of the inhibitor to the enzyme
reduces its activity but does not affect the binding of substrate. As a result, the extent of
inhibition depends only on the concentration of the inhibitor. Vmax will decrease due to
the inability for the reaction to proceed as efficiently, but Km will remain the same as the
actual binding of the substrate, by definition, will still function properly.

OBJECTIVES
At the end of the experiment, the students should be able to:
1. Determine if copper sulfate and sucralose inhibits invertase activity;
2. Identify the nature of inhibition;
3. Generate a lineweaver burk plot of the inhibition.

MATERIALS AND METHODS


Materials
DNS reagent Copper (II) sulfate 25 mL volumetric
flask
0.1M acetate buffer Sucralose (Splenda)
(pH 4.5) Microcentrifuge
Micropipettes tubes
Sucrose
microwell plate

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Procedure
A. Reagent Preparation
1. DNS reagent: Mix 300 g of potassium sodium tartrate tetrahydrate and 16 g
of sodium hydroxide with 500 mL of distilled water. Add slowly 10 g of 3, 5
DNS acid.
2. Dilute with distilled water to make 1 L solution. Transfer to amber colored
container.
B. Inhibitor Preparation
1. 0.001 M CuSO4: Accurately weigh 0.004 g of copper (II) sulfate and dissolve
with 25 mL distilled water.
2. 0.005 M CuSO4: Weigh 0.02 g of copper (II) sulfate accurately and dissolve
with 25 mL distilled water.
3. 16.2 M Sucralose: Weigh 0.16 g of sucralose accurately and dissolve with 25
mL distilled water.
C. Enzyme-Substrate-Inhibitor complex
1. Substrate Preparation: Prepare three sets of five different substrate
concentrations derived from km based calculations. For sucrose, substrate
concentrations to be prepared were 7.5, 15, 30, 60 and 90 mM.
2. Enzyme Preparation: Make a standard working invertase solution 0.1 %(w/v)
by dissolving 1 gram of standard invertase in 10 mL of acetate buffer ph 4.5.
3. Mix the prepared substrates and enzymes by adding 60 l of invertase
solution to each of the substrate concentrations prepared.
4. Add 60 l of 0.001 M CUSO4, 0.005 M CuSO4, 16.2 M Sucralose separately
to each of the enzyme-substrate mixture prepared at different concentrations.
5. Incubate it for 10 minutes at 60C and add 60 L of DNS reagent. Boil
solution until color develops into bloody red.
6. If immediate transfer of the solution to the microwell plate is not possible, add
TCA solution, transfer and subject it to spectrophotometer for absorbance
reading at 540nm.
EXPERIMENT 4

EFFECT OF INHIBITORS ON INVERTASE ACTIVITY

RESULTS
Without Inhibitor (Control)
S ABSORBANCE V 1/S 1/V
7.5 0.789 250.5 0.13333333 0.003992016
15 1.592 518.1666667 0.066666667 0.001929881
30 2.904 955.5 0.033333333 0.001046572
60 2.566 842.8333333 0.016666667 0.001186474
90 3.831 1264.5 0.011111111 0.000790826

Competitive Inhibitor: 0.001 M CuSO4


S ABSORBANCE V 1/S 1/V
7.5 0.464 142.1666667 0.133333333 0.007033998
15 1.059 340.5 0.066666667 0.002936858
30 1.630 530.8333333 0.033333333 0.00188383
60 2.638 866.8333333 0.016666667 0.001153624
90 3.047 1003.166667 0.011111111 0.000996843

Noncompetitive Inhibitor: 0.005 M CuSO4


S ABSORBANCE V 1/S 1/V
7.5 0.624 195.5 0.133333333 0.00511509
15 1.101 354.5 0.066666667 0.002820874
30 3.036 693.5 0.033333333 0.001441961
60 3.580 999.5 0.016666667 0.0010005
90 3.58 1180.833333 0.011111111 0.00084686

Uncompetitive Inhibitor: Sucralose


S ABSORBANCE V 1/S 1/V
7.5 1.072 344.8333333 0.133333333 0.002899952
15 1.343 435.1666667 0.066666667 0.00229797
30 1.994 652.1666667 0.033333333 0.00153335
60 2.344 768.8333333 0.016666667 0.001300672
90 2.421 794.5 0.011111111 0.001258653

Lineweaver-Burk Plot of Enzyme Inhibition


Km and Vmax values of inhibitors

Km Vmax
Without Inhibitor 12.72727273 909.0909091
0.001M CuSO4 245.5 5000
0.005M CuSO4 88.75 2500
Sucralose 51 2000

The resulting values for the Km and the Vmax for the different types of inhibition
shows an increase of the Km and the Vmax. With that data acquired, the identification of
the type of inhibition is inconclusive. However, with the Lineweaver-Burk plot generated,
it can be categorized.
Both 0.01 M and 0.005 M of CuSO4 act as an competitive inhibitor. Their Km
values increased and their Vmax is the same but it lightly deviated from the y-axis. The
sucralose on the other hand displays a decrease on the Km value which makes it
possible to considered as an uncompetitive inhibitor.
The concentration of the inhibitors is a factor of their identity as an inhibitor.
According to other studies, CuSO4 serves as a non-competitive inhibitor of invertase at
concentrations below 0.0022M, and CuSO4 is a competitive inhibitor of invertase at
concentrations above 0.0044 M.