Professional Documents
Culture Documents
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
PAGE
Experiment 1A: Effect of Sucrose Concentration on Invertase
Activity
3
24
2 | Page
DETERMINATION OF DIFFERENT PARAMETERS INFLUENCING
INVERTASE ACTIVITY
THEORETICAL BACKGROUND
Invertase
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.
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
3 | Page
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.
4 | Page
5 | Page
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.
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.
6 | Page
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.
7 | Page
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
0
0 200 400 600 800 1000 1200
[Glucose] g/ml
8 | Page
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.
9 | Page
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.
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.
10 | P a g e
EXPERIMENT 1B
DETERMINATION OF OPTIMAL INCUBATION TIME
RESULTS
Incubation time
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.
11 | P a g e
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.
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.
12 | P a g e
EXPERIMENT 1C
DETERMINATION OF OPTIMAL INCUBATION TEMPERATURE
RESULTS
Incubation Temperature
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.
13 | P a g e
EXPERIMENT 1D
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.
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.
14 | P a g e
EXPERIMENT 1D
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.
15 | P a g e
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.
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.
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.
Procedure
1. Prepare five different concentrations of maltose and lactose following the
observed concentrations as in experiment 1D.
16 | P a g e
2. Follow succeeding procedures from the aforementioned experiment.
17 | P a g e
EXPERIMENT 2
SUBSTRATE SPECIFICITY
RESULTS
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
18 | P a g e
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.
30
[Maltose]
20 [Lactose]
Vo (g/min-mL)
[Sucrose]
10
0
0 20 40 60 80 100
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.
19 | P a g e
Km and Vmax of Different Substrates
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.
20 | P a g e
EXPERIMENT 3
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.
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.
21 | P a g e
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.
22 | P a g e
EXPERIMENT 3
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.
23 | P a g e
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
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.
24 | P a g e
EXPERIMENT 4
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.
25 | P a g e
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
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
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.