exp 3 biochemistry enzyme activity

8/10/2019 Exp 3 Biochemistry Enzyme activity

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EXPERIMENT 3: INVESTIGATION ON ENZYME ACTIVITY

AND THE FACTORS THAT AFFECTS IT

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1.0Introduction

Enzymes are globular proteins which act as biological catalysts. This means that they speed

up the rate of reaction by lowering the activation energy, which is the energy required to break

bonds. Enzymes are a complex tertiary and sometimes quaternary shape and catalyse reactions

by forming a complex (known as the enzyme substrate complex) at a specific region of the

enzyme called the active site.

Enzymes are specific; any individual enzyme can usually only catalyze one particular

reaction. The rate of enzyme activity is influenced by time, enzyme concentration, temperature,

pH, and the presence of inhibitors.The first factors that affect the rate of enzyme activity are

temperature. As the temperature rises, reacting molecules have more and more kinetic energy.

This increases the chances of a successful collision and so the rate increases. There is a certain

temperature at which an enzyme's catalytic activity is at its greatest (see graph). Above this

temperature the enzyme structure begins to break down (denature) since at higher temperatures

intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic

energy.

Figure 1: The effect of temperature on enzyme activity.

Enzyme + substrate enzyme substrate complex product


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The next factors are pH. Each enzyme works within quite a small pH range. There is a pH at

which its activity is greatest (optimum pH). This is because changes in pH can make and break

intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its

effectiveness.

Figure 2: The effect of pH on enzyme activity.

Other factors are inhibitors. Some substances reduce or even stop the catalytic activity of

enzymes in biochemical reactions. They block or distort the active site. These chemicals are

called inhibitors, because they inhibit reaction. Inhibitors that occupy the active site and prevent

a substrate molecule from binding to the enzyme are said to be active site-

directed (or competitive, as they 'compete' with the substrate for the active site). Inhibitors that

attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non-

active site-directed (or non-competitive).

Next one is concentrations. Changing the enzyme and substrate concentrations affect the

rate of reaction of an enzyme-catalysed reaction. Increasing substrate concentration will

increases the rate of reaction. This is 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 substrate concentration will no

longer be the limiting factor.The enzymes will effectively become saturated, and will be working

at their maximum possible rate. While, by increasing the enzyme concentration, it will increase

the rate of reaction which as more enzymes will be colliding with substrate molecules.


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However, this too will only have an effect up to a certain concentration, where the enzyme

concentration is no longer the limiting factor.

Figure 3 & 4: The effect of concentration on enzyme activity.

In regards to substrate concentration, enzyme kinetics follow the Michaelis-Menton Model. The

model takes the form of an equation describing the rate ofenzymatic reactions, by

relatingreaction rate to , theconcentration of asubstrate S. Its formula is given by :

.

Here, represents the maximum rate achieved by the system, at maximum (saturating)

substrate concentrations. The Michaelis constant is the substrate concentration at which the

reaction rate is half of .

Figure 5: Example of Michaelis-Menton graph.
http://en.wikipedia.org/wiki/Enzymatic_reactionhttp://en.wikipedia.org/wiki/Reaction_ratehttp://en.wikipedia.org/wiki/Concentrationhttp://en.wikipedia.org/wiki/Enzyme_substrate_(biology)http://en.wikipedia.org/wiki/Enzyme_substrate_(biology)http://en.wikipedia.org/wiki/Concentrationhttp://en.wikipedia.org/wiki/Reaction_ratehttp://en.wikipedia.org/wiki/Enzymatic_reaction


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1.1Objective

1) To determine the effects of temperature on the enzymatic activity and changes in enzyme

concentration of an enzyme-catalysed reaction.

2) To describe the relationship between substrate concentration and the maximum velocity

of an enzyme.

3) To estimate the Michaelis-Menten parameters, effect of pH and temperature on enzyme

activity and kinetics of inhibition.

2.0

Material

1. 1% starch solution

2. Amylase solution

3. 0.2 M phosphate buffer

4. DNS reagent

5. Glucose standard

6. 0.1 M Lead (II) nitrate Pb(NO3)2

7.

Distilled water

2.1Procedure

2.1.1 Determination of time and enzyme concentration

Enzyme was prepared into 0.2 ml and mixed with 0.3 ml of 0.2 M phosphate buffer at pH

7.0 followed by 0.5 ml of 1 % (w/v) of starch solution.

The similar sample was prepared as step (a) according to the table 1 for determination of

optimum time for amylase activity.


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Table 1: Determination of time and enzyme concentration

Test tube no Amylase solution(ml)

Phosphate buffer(0.2 M), ml

1 % starchsolution, ml

Duration (min)

1 0.2 0.3 0.5 30

2 0.2 0.3 0.5 60

3 0.2 0.3 0.5 90

4 0.2 0.3 0.5 120

5 0.2 0.3 0.5 150

Allowed the mixture for duration according in Table 1 at 30C using an incubator with

shaking at 150 rpm.

After 30 min, glucose concentration was determined by using DNS method.

The optical density was determined at 540 nm.

The standard curve was prepared for glucose by using the concentration of pure glucose in

the range of 0-1000 mg/L.

1U (unit) of amylase activity is defined as the amount of enzyme required to release 1g of

reducing sugar per min under the conditions stated.


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2.1.2 Effect of temperature on the amylase activity

Table 2: Determination effect of temperature on the amylase activity

Test tube no Amylase solution

1-5% (ml)

Phosphate buffer

(0.2 M), ml

1 % starch

solution, ml

Temperature OC

1 0.2 0.3 0.5 Ice water Bath

2 0.2 0.3 0.5 30

3 0.2 0.3 0.5 40

4 0.2 0.3 0.5 50

5 0.2 0.3 0.5 100

The test tube was prepared according to the Table 2 and duplicate for each test tube. Incubate thesample from 30 to 60 minutes.

Then, repeat the same analysis for second sample which is at 60 minutes.

Allow the mixture for 30 minutes at temperature based on Table 3. After 30 minutes, the glucoseconcentration was determined by using DNS method at 540 nm.


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2.1.3 Effect of pH on the amylase activity

mined

Table 3: Determination effect of pH on the amylase activity

Test tube no Amylase solution1-5% (ml)

Phosphate buffer(0.2 M), ml

1 % starchsolution, ml

Temperature OC

1 0.2 2 0.5 Ice water Bath

2 0.2 4 0.5 30

3 0.2 7 0.5 40

4 0.2 9 0.5 50

5 0.2 12 0.5 100

The optimum pH for the enzyme was determined by incubating the reaction mixtures prepared using

the buffer solution of pH from 5 to pH 9. The buffer systems (0.2 M) used in the experiment is stated

in Table 3.

Make a duplicate for each of the test tube. Incubate the sample from 30 to 60 minutes. Shake at 150rpm.

After 30 minutes, glucose concentration was determined by using DNS method.

The absorbance obtained for each test tube was recorded and repeat incubation for 60 min.


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2.1.4 Effect of substrate concentration on the activity of amylase enzyme

2.1.5 Effect of inhibitor

The starch solution was prepared at concentration 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 and 3.0 &(w/v) as the substrate. The substrate was placed into 0.5ml of various concentrations into the

test tubes.

The reaction mixture was incubated for 30 min at 30C with shaking at 150 rpm.

The amylase activities were determine by using DNS method, and then plot the graph of

substrate concentration against amylase activity.

2 ml of amylase solution, 2 ml of 1 % starch and 10 drops of 0.1 M Pb(NO3)2 was added

into a dry test tube

Mix and incubate in water bath for 30 min. After 30 min, determine glucose concentration

using DNS method at 540 nm. Record the absorbance obtains.


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3.0Result

3.1 Concentration of Glucose for Standard Glucose

Table 1: The absorbance and concentration of glucose for standard glucose

Concentration of glucose (mg/L) Absorbance

0.000 0.132

200.0 0.145

400.0 0.194

600.0 0.208

800.0 0.270

1000 0.373

Figure 1: Absorbance against the concentration of glucose for standard glucose

y = 0.0002x + 0.1065

R = 0.905

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 200 400 600 800 1000 1200

Absorbance

Concentration of glucose standard

Graph 1: Absorbance Vs Concentration of Glucose Standard


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3.2 Determination of Time and Enzyme Concentration

Table 2: The absorbance and enzyme activities at different duration

Figure 2: The enzyme activities against time

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 20 40 60 80 100 120 140

EnzymeActivities

Time

Graph 2: Enzyme Activities Vs Time

Time (minutes) Absorbance Enzyme activities

30 0.620 0.0856

60 0.646 0.0450

90 0.701 0.0330

120 0.691 0.0244


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3.3 Effect of Temperature on the Amylase Activity

Table 3:The absorbance and enzyme activities for 30 minutes at different temperature

Temperature Absorbance Enzyme activities

Ice water bath 0.836 0.1216

30 0.833 0.1211

40 0.816 0.1183

50 0.840 0.1223

100 0.797 0.1151

Figure 3:Enzymes activities against temperature for 30 minutes

0.114

0.115

0.116

0.117

0.118

0.119

0.12

0.121

0.122

0.123

0 20 40 60 80 100 120

EnzymeActivities

Temperature

Graph 3: Enzymes Activities Vs Temp 30 min


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3.4 Effect of Temperature on the Amylase Activity

Table 4:The absorbance and enzyme activities for 60 minutes at different temperature

Temperature Absorbance Enzyme Activities

Ice water bath 0.754 0.0540

30 0.687 0.0484

40 0.687 0.0484

50 0.708 0.0501

100 0.734 0.0523

Figure 4:Enzymes activities against temperature for 60 minutes

0.047

0.048

0.049

0.05

0.051

0.052

0.053

0.054

0.055

0 20 40 60 80 100 120

EnymeActivities

Temperature

Graph 4: Enzyme Activities Vs Temp 60 min


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3.5 Effect of pH on the Amylase Activity

Table 5:The absorbance and enzyme activities for 30 minutes at difference pH

pH Absorbance Enzyme Activities

0 0.301 0.0324

6 0.191 0.0141

7 0.158 0.0086

8 0.158 0.0086

9 0.182 0.0126

Figure 5:The enzyme activities against pH for 30 minutes

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0 1 2 3 4 5 6 7 8 9 10

EnzymeActiviyies

pH

Graph 5: Enzyme Activities Vs pH for 30 min


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3.6 Effect of pH on the Amylase Activity

Table 6:The absorbance and enzyme activities for 60 minutes at difference pH

pH Absorbance Enzyme Activities

0 0.396 0.0241

6 0.308 0.0168

7 0.233 0.0105

8 0.251 0.0120

9 0.197 0.0075

Figure 6:The enzyme activities against pH for 60 minutes

0

0.005

0.01

0.015

0.02

0.025

0.03

0 1 2 3 4 5 6 7 8 9 10

EnzymeActivities

pH

Graph 6: Enzyme Activities Vs pH for 60 min


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3.7 Effect of Substrate Concentration on the Activity of Amylase Enzyme

Table 7:The absorbance and concentration of glucose at difference concentration of substrate

Concentration of substrate

(%)

Absorbance Enzyme Activities

0.25 0.214 0.0179

0.5 0.326 0.0366

0.75 0.219 0.0188

1.0 0.262 0.02591.5 0.238 0.0219

Figure 7:The enzyme activities against concentration of substrate

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

EnzymeActivities

Concentration of substrate (%)

Graph 7: Enzyme Activities Vs Concentration of Substrate


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3.8 Effect of Inhibitor

Table 8:The absorbance and enzyme activities at 30 minutes

Test tube Absorbance Enzyme Activities

1 0.824 0.1196

2 0.836 0.1216

3 0.833 0.1211

4 0.816 0.1183

5 0.840 0.1223

Figure 8:The enzyme activities against inhibitor

0.118

0.1185

0.119

0.1195

0.12

0.1205

0.121

0.1215

0.122

0.1225

0.81 0.815 0.82 0.825 0.83 0.835 0.84 0.845

EnzymeActivities

Inhibitor

Graph 8: Enzymes Activities Vs Inhibitor


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4.0Discussion

Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all

catalysts, speed up reactions without being used up themselves. This occurs by lowering the

activation energy of a reaction. All biochemical reactions are catalyzed by enzymes. Most

enzymes have optimum activity at a neutral pH and at body temperature. Enzymes are also very

specific, it only act on one substrate or one class of related substrate molecules. Typically, only

one kind of substrate will fit into the active site.

This experiment was conducted to investigate the factors that may affect enzyme activity.

Enzyme activity means the ability of an enzyme that catalyzes a specific chemical reaction under

certain conditions. Temperature, pH, concentration of substrate and inhibitor were tested. There

are several factors that can affect this enzyme activity. The glucose standard graph was plotted as

shown in figure 1.

Based on figure 1, the linear equation will get y=0.0002x + 0.1065. Theoretically

according effect of concentration, enzyme activities calculated with concentration of glucose and

time. During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an

enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a high

concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes place,

and then the product is released. Based on figure 2 shows that the enzymatic activities are

decrease with the duration of sample incubated with shaking at 150 rpm is an increase. The

longer an enzyme is incubated with its substrate, the greater the amount of product that will be

formed.

Enzyme catalyst 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. This is why

at 90 minutes to 120 minutes; the product formed was getting low.


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Other factor that may affect the enzymatic activity is temperature. As the temperature

rises, reacting molecules have more kinetic energy. This increases the chances of a successful

collision and so the rate increases. Theoretical, all reactions are faster at a higher temperature.

However, enzyme-catalyzed reactions become slower or stop if the temperature becomes too

high, because enzymes become denatured at high temperatures. Therefore, enzymes have an

optimum temperature that corresponds to maximum activity. (At higher or lower temperatures,

the activity of the enzyme is lower.) The optimum temperature is usually around body

temperature (37C).

As shown in Figure 3, the enzyme activities decreasing with high temperature, and then

abruptly declines with further increase of temperature. The result a show was not followed the

theory. This might be because of some errors. For example, the sample prepared was not been

pipette properly because the pump was not functioning. Therefore the amount that supposed to

be ready was incorrect. Most animal enzymes rapidly become denatured at temperatures above

40C. This is why the graph was slightly decreased at temperature 40C. This experiment was

repeated again for 60 minutes. The data obtains can be shown in figure 3. The enzyme activities

product for 30 minutes was much higher than at 60 minutes. Supposedly at 60 minutes, the

enzyme activities produced was more because the longer the time for incubation, the greater the

amount of product formed. Improper preparing the sample could be contributed to this

undesirable result.

Changes in pH also may affect the enzymatic activity. Since enzymes are proteins, they

are very sensitive to changes in pH. Each enzyme has its own optimum range for pH where it

will be most active. Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. It is

present in the saliva of humans and some other mammals, where it begins the chemical process

of digestion. In this case, amylase optimum pH is at 6. Referring to figure 6, it was show not

bell-shaped. The result a show was not followed the theory.


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The amount of enzyme activities produced was higher at pH 0 where amylase most

active. As the pH increase, the enzyme activities produced was decreased. Compared to figure 5,

which the incubation duration was 30 minutes, the optimum pH was at pH 0. Besides that, the

enzyme activities produced at pH 7.0 was the lowest. The enzymes activities produced for 30

minutes of incubation process was more than for 60 minutes incubation. This result was not

followed the theory since the longer the time for incubation, the greater the amount of product

formed. This condition happened again because improper in preparing the sample to be

investigated.

Figure 7 shows the enzyme activities against the concentration of substrate. According to

Michaelis-Menten kinetics, enzyme-substrate reactions are actually comprised of two elementary

reactions. The first is the when the substrate forms a complex with the enzyme and then in the

second, the complex decomposes to product and enzyme. As the substrate concentration is

increased, each enzyme is able to locate and react with more substrate molecules and the

observed enzyme activity increases. When this maximum velocity had been reached, all the

available enzyme has been converted to ES, the enzyme substrate complex. This point is

designated as Vmax. However the data obtained was fluctuated. This result is away from the

theory. Therefore the Vmaxcannot be identified.

0.1 M Pb(NO3)2 is an inhibitor. The function of inhibitor is to reduce the rate of enzyme

activity, usually by binding with the enzyme and interfering with the formation of the enzyme-

substrate complex. There are three common types of enzyme inhibition; competitive, non-

competitive and substrate inhibition. Competitive inhibition occurs when the substrate and a

substance resembling the substrate are both added to the enzyme. Non-competitive inhibitors are

considered to be substances which when added to the enzyme alter the enzyme in a way that it

cannot accept the substrate. In this experiment 0.1 M Pb(NO3)2 caused the reaction rate to bedecreased. The figure 8 shows that as the incubation time increased the enzyme activities

produced will be increased too.


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5.0Conclusion

Enzyme activity means the ability of an enzyme that catalyses a specific chemical reaction

under certain conditions. There are several factors that can affect enzyme activity, from the

experiment that was conducted temperature, pH, concentration of substrate and inhibitors were

tested to determine the factors affecting enzyme activities. Enzyme-catalyzed reactions become

slower or stop if the temperature becomes too high, because enzymes are denatured at high

temperatures. As for the pH, the result shows the longer the time for incubation, the greater the

amount of product formed, but this was against the theory due to error during conducting the

experiment. According to the Michaelis-Menten kinetics, the Vmaxcannot be identified because

the data obtained was fluctuated. By using an inhibitor the incubation time increased the enzyme

activities produced will be increased too. The precaution is to avoid from making parallax error

for most of the measured content for an accurate final results.


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6.0References

1)

Raymond Chang, Chemistry 10thEd, McGraw Hill

2) Nelson, J., Chemistry: The Central Science, 3rd

Ed, Prentice Hall ( 1985).

3) Eed, John (2013) "Factors Affecting Enzyme Activity,"ESSAI: Vol. 10, Article

19.Available at:http://dc.cod.edu/essai/vol10/iss1/19.

4) 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.

5) Petersen, Chris E., and Barbara J. Anderson. Investigation in the Biology 1151

Laboratory. Champaign, IL: Stipes L.L.C., 2005.

exp 3 biochemistry enzyme activity

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