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Influence of pH and Concentration to enzyme Activity
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I TITLE OF EXPERIMENT :
Effect of pH and concentration enzyme by enzyme activity
II DATE OF EXPERIMENT :
November 24th, 2015
III END OF EXPERIMENT :
November 24th, 2015
IV PURPOSE :
To showing that pH and concentration of enzyme effected enzyme activity
V BASIC THEORY
“ nzym ”
With the exception of a small group of catalytic RNA molecules, all enzymes
are proteins. Their catalytic activity depends on the integrity of their native
protein conformation. If an enzyme is denatured or dissociated into its subunits,
catalytic activity is usually lost. If an enzyme is broken down into its component
amino acids, its catalytic activity is always destroyed. Thus the primary,
secondary, tertiary, and quaternary structures of protein enzymes are essential to
their catalytic activity.
Enzymes, like other proteins, have molecular weights ranging from about
12,000 to more than 1 million. Some enzymes require no chemical groups for
activity other than their amino acid residues. Others require an additional
chemical component called a cofactor—either one or more inorganic ions, such as
Fe2, Mg2, Mn2, or Zn2, or a complex organic or metalloid-organic molecule called a
coenzyme. Some enzymes require both a coenzyme and one or more metal ions for
activity. A coenzyme or metal ion that is very tightly or even covalently bound to
the enzyme protein is called a prosthetic group. A complete, catalytically active
enzyme together with its bound coenzyme and/or metal ions is called a
holoenzyme. The protein part of such an enzyme is called the apoenzyme or
apoprotein.
Coenzymes act as transient carriers of specific functional groups. Most are
derived from vitamins, organic nutrients required in small amounts in the diet. We
consider coenzymes in more detail as we encounter them in the metabolic
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pathways discussed in Part II. Finally, some enzyme proteins are modified
covalently by phosphorylation, glycosylation, and other processes. Many of these
alterations are involved in the regulation of enzyme activity.
HOW ENZYMES WORK
The enzymatic catalysis of reactions is essential to living systems. Under
biologically relevant conditions, un-catalyzed reactions tend to be slow—most
biological molecules are quite stable in the neutral-pH, mild temperature, aqueous
environment inside cells. Furthermore, many common reactions in biochemistry
entail chemical events that are unfavorable or unlikely in the cellular environment,
such as the transient formation of unstable charged intermediates or the collision
of two or more molecules in the precise orientation required for reaction.
Reactions required to digest food, send nerve signals, or contract a muscle simply
do not occur at a useful rate without catalysis.
An enzyme circumvents these problems by providing a specific environment
within which a given reaction can occur more rapidly. The distinguishing feature
of an enzyme-catalyzed reaction is that it takes place within the confines of a
pocket on the enzyme called the active site (Fig. 6–1). The molecule that is bound
in the active site and acted upon by the enzyme is called the substrate. The surface
of the active site is lined with amino acid residues with substituent groups that
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bind the substrate and catalyze its chemical transformation. Often, the active site
encloses a substrate, sequestering it completely from solution.
The enzyme substrate complex, whose existence was first proposed byCharles-Adolphe Wurtz in 1880, is central to the action of enzymes. It is also the
starting point for mathematical treatments that define the kinetic behavior of
enzyme-catalyzed reactions and for theoretical descriptions of enzyme
mechanisms.
ENZYME ACTIVITY DEPENDS ON PH
Enzymes have an optimum pH (or pH range) at which their activity is
maximal, at higher or lower pH, activity decreases. This is not surprising. Amino
acid side chains in the active site may act as weak acids and bases with critical
functions that depend on their maintaining a certain state of ionization, and
elsewhere in the protein ionized side chains may play an essential role in the
interactions that maintain protein structure. Removing a proton from a His
residue, for example, might eliminate an ionic interaction essential for stabilizing
the active conformation of the enzyme. A less common cause of pH sensitivity is
titration of a group on the substrate.
The pH range over which an enzyme undergoes changes in activity can
provide a clue to the type of amino acid residue involved. A change in activity near
pH 7.0, for example, often reflects titration of a His residue. The effects of pH must
be interpreted with some caution, however. In the closely packed environment of
a protein, the pKa of amino acid side chains can be significantly altered. For
example, a nearby positive charge can lower the pKa of a Lys residue, and a nearby
negative charge can increase it. Such effects sometimes result in a pKa that is
shifted by several pH units from its value in the free amino acid. In the enzyme
acetoacetate decarboxylase, for example, one Lys residue has a pKa of 6.6
(compared with 10.5 in free lysine) due to electrostatic effects of nearby positive
charges. Every enzyme has an optimum pH (or pH range) at which it has maximal
activity.
EXAMPLES OF ENZYMATIC REACTIONS
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Thus far we have focused on the general principles of catalysis and on
introducing some of the kinetic parameters used to describe enzyme action. We
now turn to several examples of specific enzyme reaction mechanisms. An
understanding of the complete mechanism of action of a purified enzyme requires
identification of all substrates, cofactors, products, and regulators.
Moreover, it requires a knowledge of (1) the temporal sequence in which
enzyme-bound reaction intermediates form, (2) the structure of each intermediate
and each transition state, (3) the rates of interconversion between intermediates,
(4) the structural relationship of the enzyme to each intermediate, and (5) the
energy contributed by all reacting and interacting groups to intermediatecomplexes and transition states. As yet, there is probably no enzyme for which we
have an understanding that meets all these requirements. Many decades of
research, however, have produced mechanistic information about hundreds of
enzymes, and in some cases this information is highly detailed.
We present here the mechanisms for four enzymes: chymotrypsin,
hexokinase, enolase, and lysozyme. These examples are not intended to cover all
possible classes of enzyme chemistry. They are chosen in part because they are
among the best understood enzymes, and in part because they clearly illustrate
some general principles outlined in this chapter. The discussion concentrates on
selected principles, along with some key experiments that have helped to bring
these principles into focus. We use the chymotrypsin example to review some of
the conventions used to depict enzyme mechanisms. Much mechanistic detail and
experimental evidence is necessarily omitted; no one book could completely
document the rich experimental history of these enzymes. Also absent from these
discussions is the special contribution of coenzymes to the catalytic activity of
many enzymes. The function of coenzymes is chemically varied, and we describe
each as it is encountered in Part II.
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VI TOOLS AND MATERIALS
A Tools
- Steam Bath
-
Beaker Glass
B Materials
- Saliva solution
- Starch solution 0.4 mg/mL
- Iodine solution
VII FLOW CHART
1.
Influence of pH to enzyme activity
saliva
- diluted 100x with distillate
Enzyme solution
1 mL starch with pH (1,3,5,7 and 9)
- pour into test tube- Let it for about 2 minutes
+ 10 drops enzyme solution
- Mixed it well and wait for 3minute
+ 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
1 mL starch solution
- pour into test tube B
(Blank)- Let it for about 2 minutes+ 10 drops distillate water
- Mixed it well and wait for 3
minute+ 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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2. Influence of concentration to enzyme activity
saliva
- diluted 100, 200, 300, 400,
500 x with distillate water
Enzyme solution
1 mL starch solution
- pour into test tube- Let it for about 2 minutes+ 10 drops enzyme solution
concentration 100, 200, 300,400, 500- Mixed it well and wait for 3
minute
- heated in 60 ˚C
+ 1 mL iodine solution+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
1 mL starch solution
- pour into test tube- Let it for about 2 minutes+ 10 drops distillated water
- Mixed it well and wait for 3minute- heated in 60 ˚C + 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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VIII RESULT OF EXPERIMENT
Procedure of the ExperimentsObservation data
Reaction/Assumption ConclusionBefore After
1. Effect of pH towards enzyme activity - Starch
solution:
turbid solution
-
Distillatewater:
colorless
solution
- Iodine: yellow
solution
- Enzyme:
turbid solution
blank solution
- Starch + I2
= blackish
blue-
+ distillate
water =
blackish
blue
Test tube I U
- Starch pH 1
+ enzyme +
iodine =
blue (+++)
solution
- + distillate
water =blue (+++)
solution
Tube II U
- Starch pH 3
+ enzyme +
pH affects to the
enzyme activation
and enzyme work
effectively at pH 7
+
+ I2 ↛
+ I2 ↛
saliva
- diluted 100x with distillate
Enzyme solution
1 mL starch with pH (1,3,5,7 and 9)
- pour into test tube
- Let it for about 2 minutes
+ 10 drops enzyme solution
- Mixed it well and wait for 3
minute
+ 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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turbid
solution
- + iodine =
blue (++)
solution
- + distillate
water =
blue (++)
solution
Tube III U
- Starch pH 5
+ enzyme =
turbid
solution
- + iodine +
distillate
water =
blue (+)
solution
Tube IV U
- Starch pH 7
+ enzyme =
turbid
solution
-
+ iodine =
∆ A1 = 0.887
∆ A3 = 0.863
∆ A5 = 0.825
∆ A7 = 0.066
∆ A9 = 0.117
- Enzyme activation affected by pH. pH
optimum of amylase is 7 (Mohammad
Sadikin, 2002)
1 mL starch solution
- pour into test tube B
(Blank)
- Let it for about 2 minutes
+ 10 drops distillate water
- Mixed it well and wait for 3minute
+ 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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light brown
- + distillate
water =
colorless
Tube V U
- Starch pH 9
+ enzyme =
turbid
solution
- + iodine +
distillate
water =
light blue
2. influence of concentration to enzyme activity - starch solution
= turbid
solution
- iodine solution
= yellow
solution
- distillate
water =
colorless
- enzyme
solution =
turbid solution
Blank test tube
-
starch +
water =
colorless
-
+ I2 = deep
blue
Test tube
- Starch +
enzyme
(dilute
100x) =
colorless +
Concentration of
substrate can affect to
the enzyme
activation.
Enzyme work
effectively in
concentration 100x
diluted
+
saliva
- diluted 100, 200, 300, 400,
500 x with distillate water
Enzyme solution
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I2 =
colorless
solution
-
Starch +
enzyme
(dilute
200x) =
colorless +
I2 = light
blue
- Starch +
enzyme
(dilute
300x) =
colorless +
I2 = blue
(++)
- Starch +
enzyme
(dilute
400x) =
colorless +
I2 = blue
(+++)
-
Starch +
∆ A100 = 0.030
∆ A200 = 0.487
∆ A300 = 0.682
∆ A400 = 0.745
∆ A500 = 0.545
Greater concentration of enzyme cause
enzyme work properly (Hafiz Soewoto, 2000)
+ I2 ↛
+ I2 ↛
1 mL starch solution
- pour into test tube
- Let it for about 2 minutes
+ 10 drops distillated water- Mixed it well and wait for 3
minute
- heated in 60 ˚C + 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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enzyme
(dilute
500x) =
colorless +
I2 = blue
(+)
1 mL starch solution
- pour into test tube
- Let it for about 2 minutes
+ 10 drops enzyme solutionconcentration 100, 200, 300,
400, 500
- Mixed it well and wait for 3
minute
- heated in 60 ̊ C + 1 mL iodine solution
+ 10 mL distillate water
Result the solution
- read the absorbance
Result the absorbance
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IX EXPLANATION
In this experiment, we do the experiment about factor which effected
enzyme amylase activity in saliva where breaking starch solution. Factor which
effected enzyme activity there’s a concentration of enzyme, pH, temperature, and
substrate concentration.
Effect of pH towards enzyme activity
First experiment, with the aim to knowing effected of pH to enzyme activity.
We used tube B for blank solution and tube U for diluted saliva. In tube B, added
with 1 mL starch solution 1% turbid solution, which the function of starch is as a
substrate. Then, let it 2 minutes to make starch work correctly and starch can
degradation perfectly. Next add with 10 drops distillate water colorless solution,
where the function of distillate as a changes of enzyme in blank solution. Then,
mixed well and wait about 3 minute for the solution mixed correctly. Add 1 mL
iodine solution yellow solution, where the function is for indicator of amylum and
for formed complex solution in starch solution. Then add again with 10 mL
distillated water colorless solution. Distillated water is for the solution not too
concentrated. After that read the absorbance in UV-Vis spectrophotometer.
In 5 tubes U for diluted saliva, we add 100x dilute saliva in each tube 10
drops. Then we added with starch solution pH 1, starch solution pH 3, starch
solution pH 5, starch solution pH 7, and starch solution pH 9, where the function of
starch is for substrate too. Then let it for 2 minute, to make the starch mix
correctly. Then add 10 drops enzyme solution, with function for knowing
enzymatic reaction from enzyme amylase and break amylum become glucose.
Then, wait for 3 minute to make the saliva broke and changes become glucose
correctly. After that, added with 1 mL iodine yellow solution with the function as
indicator color change from saliva which specific for tested existence of amylum
and for formed complex solution in starch solution. Then, added with 10 mL
distillate water to make the saliva not too concentrated. After that, read the
absorbance in UV-Vis spectrophotometer. The principle of absorbance is from
starch solution is colorless solution, so for measuring absorbance using
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spectrophotometer, starch solution must change become complex solution for
giving color in solution and can measured the absorbance.
After we tested absorbance in UV-Vis spectrophotometer, we get the data
and the color of solution like:
pH Abs
blanko 0.937
1 0.887
3 0.863
5 0.825
7 0.066
9 0.117
pH 1: Blue (+++) solution
pH 3: Blue (++) solution
pH 5: Blue (+) solution
pH 7: Colorless solution
pH 9: Light blue solution
From this experiment, we can conclude that value of pH affects to the
enzyme activation. And enzyme work effectively at pH 7, because in pH 7 is pH
optimum. So the enzyme correctly reacted with starch very fast, so the color
become colorless.
Influence of concentration to enzyme activity
Second experiments, we used saliva which has been diluted with 100x, 200x,
300x, 400x, and 500x. Next, we prepare test tube for blank solution and 5 tubes for
saliva solution.
+
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First, to make blank solution, we used 1 mL starch turbid solution where the
function of starch as substrate. Then let it for 2 minutes to make starch can
degradation perfectly. After that, add 10 drops distillate water colorless solution,
where the function as changes of saliva in blank solution. Mixed it well and wait
for 3 minute, for the solution mixed correctly. Then, heated solution in 60 ˚C in 1
minute, where the function of heated is for make the reaction work faster. Then
added with iodine yellow solution, where the function of iodine is as indicator for
determining existence of amylum. After that, add with 10mL distillate water
colorless solution, to make the solution not too concentrated. And read the
absorbance in UV-Vis spectrophotometer, and we get the absorbance.
For 5 tubes saliva solution which has been diluted in 100x, 200x, 300x, 400x,
and 500x. First tube, we add with starch turbid solution where the function of
starch as substrate. Then, let it for 2 minute is for the starch degradation perfectly.
After that, add with 10 drops enzyme turbid solution 100-500x diluted in each
tube and keep it until 3 minute. It will make the starch solution happened
hydrolysis partial. Starch solution can be hydrolysis by enzyme amylase in saliva
so become glucose. After that, heated the saliva in 60 ˚C 1 minute to made the
reaction work faster. Then, added with 1mL iodine yellow solution with function
as indicator to determining existence of amylum. Then added with 10mL distillate
water colorless solution is to make the solution not too concentrated. After that
read the absorbance in UV-Vis spectrophotometry. The principle of absorbance is
from starch solution is colorless solution, so for measuring absorbance using
spectrophotometer, starch solution must change become complex solution for
giving color in solution and can measured the absorbance. And we get for the
result:
Conc Abs
blanko 0.953
100x 0.03
200x 0.487
300x 0.682
400x 0.745
500x 0.545
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Conc. 500x: Blue (+) solution
Conc. 400x: Blue (+++) solution
Conc. 300x: Blue (++) solution
Conc. 200x: Light blue solution
Conc. 100x: Colorless solution
From this experiment, we can conclude that concentration of substrate can
affected to the enzyme activation and enzyme work affectively in concentration
100x dilute. Concentration affected of this enzyme is product formed, where
bigger concentration of enzyme as much as product which produce so we can
conclude that rate reaction inversely proportional with enzyme concentration.
X CONCLUSION
Based on our experiment we can conclude that:
1. pH affect to the enzyme activation and enzyme work affectively at pH optimum
is pH 7
2. Concentration of substrate can affected to the enzyme activation. Enzyme work
affectively in concentration 100x diluted
XI QUESTION AND ANSWERS
QUESTION
1. Create curve which showed the relationship between reaction rate
enzymatic (V= ∆A/minute) with pH
+
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2. Create curve which showed the relationship between reaction rate
enzymatic (V= ∆A/minute) with concentration/ diluted enzym
ANSWERS
1.
2.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 3 5 7 9
B
- U
pH
Absorbance vs pH
B - U
y = -0.1288x + 0.8416
R² = 0.52460
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.80.9
1
100x 200x 300x 400x 500x
a b s o r b a n c e
concentration
absorbance vs concentration
absorbance
Linear (absorbance)
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REFFERENCESS
Anonym. 2015. Enzyme. (online). (https://en.wikipedia.org/wiki/Enzyme accesses at
November 30th , 2015)
Bresnick, S., 2004, Intisari Kimia Organik , Hipokrates, Jakarta.
Gritter, A., 1991, Biokimia 1 , PT. Gramedia, Jakarta.
Lehninger, A.L., Dasar-Dasar Biokimia , Penerbit Erlangga, Jakarta
https://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzyme
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ATTACHMENT
Experiment 1 : Influence of pH to enzyme activity
1 ml starch solution poured
tube B(blanco)
Added 10 drops aquadest Wait for 3 minutes and added
4 drops I2 solution
Result after added I2 Added 8 ml aquadest Saliva/enzyme
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Put into disstilate tube Added aquadest until the line Enzyme dilute 100 x
1 ml starch in pH 9 1 ml starch in pH 1 1 ml starch in pH 3
1 ml starch in pH 5 1 ml starch in pH 7 Let it for 2 minutes
Added 10 drops of enzyme in Wait for 3 minutes
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each pH
Added 4 drops I2 solution in each pH Added 8 ml aquadest in each
pH
result
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Experiment 2 : Influence of concentration to enzyme activity
Enzyme in conc 100 Enzyme in conc 200 Enzyme in conc 300
Enzyme in conc 400 Enzyme in conc 500 1ml starch solution poured in
tube B(blanco conc)
1ml starch solution poured 1ml starch solution poured 1ml starch solution poured
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into tubes labelled 500 into tubes labelled 400 into tubes labelled 300
1ml starch solution poured
into tubes labelled 200
1ml starch solution poured
into tubes labelled 100
Added 10 drops aquadest to
tube B
Added 10 drops enzyme conc
500 to tube labelled 500
Added 10 drops enzyme conc
400 to tube labelled 400
Added 10 drops enzyme conc
300 to tube labelled 300
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Added 10 drops enzyme conc
200 to tube labelled 200
Added 10 drops enzyme conc
100 to tube labelled 100
Result
Heated in 60OC Added 1 ml I2 in Tube B Added 1 ml I2 in Tube labelled
500
Added 1 ml I2 in Tube labelled
400
Added 1 ml I2 in Tube labelled
300
Added 1 ml I2 in Tube labelled
200
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Added 1 ml I2 in Tube labelled
100
Added 8 ml aquadest to tube
B
Added 8 ml aquadest to tube
labelled 500
Added 8 ml aquadest to tube
labelled 400
Added 8 ml aquadest to tube
labelled 300
Added 8 ml aquadest to tube
labelled 200
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Added 8 ml aquadest to tube
labelled 100
Result
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ph WL B - U
blanko 0.937
1 0.887 0.05
3 0.863 0.074
5 0.825 0.112
7 0.066 0.871
9 0.117 0.82
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 3 5 7 9
B
- U
pH
Absorbance vs pH
B - U
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Conc. WL absorbance
blanko 0.953
100x 0.03 0.923
200x 0.487 0.466
300x 0.682 0.271
400x 0.745 0.208
500x 0.545 0.408
y = -0.1288x + 0.8416
R² = 0.52460
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
100x 200x 300x 400x 500x
a b s o r b a n c e
concentration
absorbance vs concentration
absorbance
Linear (absorbance)