Effects of pH, Temperature and Concentration on Enzyme Properties

Niyanthesh Reddy
Dr. D Alessio
Class: DA8
Date: 10/31/2015

Abstract

This experiment is meant to understand the effects of various interactions on the

activity of enzymes in the environment (in vivo and in vitro). In this study, the
effects of pH, temperature and concentration levels on enzymes are measured and
potential data is existent. Amongst pH, the greatest activity occurred at neutral (pH
7) level and started approaching baseline level at more acidic (pH 2) and basic (pH
10) levels. With greater temperature, the number of substrates colliding with the
enzymes increased (mostly at 32 C), however after optimal level, it started dropping
due to greater number of internal bonds being damaged and the overall enzyme
ending in a denatured state. With concentration, the greater the amount of enzyme,
the greater the reaction rate due to number of substrates colliding with more active
sites. The data collected to support this study was done with a spectrophotometer
(measuring the absorbance levels) and conducted using the enzyme ALP (Alkaline
Phosphatase).
Introduction
In nature, enzymes are very important as they help decrease the activation energy,
thereby increasing the rate of a chemical reaction. Every chemical reaction occurs
using an enzyme, and without it, life would be simply impossible. Enzymes for
instance help to hydrolyze macromolecules in our food to tiny subunits which can be
used for nutrients and energy needed for the growth of the body. Enzymes are also
used in medicine, especially when diagnosing diseases.
In this experiment, one will be examining the effects of pH, temperature, and
concentration on the function of enzymes using a spectrophotometer. This is done
to understand how the role of enzymes is affected by various conditions. In this
experiment particularly, the enzyme ALP (Alkaline Phosphatase), will be used. This
enzyme hydrolyzes the phosphate groups on proteins and lipids, necessary in many
in vivo chemical reactions in our body.
Understanding the effect of pH on enzymatic function will be done using acidic,
neutral and basic solutions. Conducting this part of the experiment is important, as
one can understand what the optimal pH of an enzyme is, without making
denaturation occur (Ex. breaking internal bonds).
The effect of concentration on enzymatic activity is very important to measure,
whether increasing or decreasing substrate concentration. In scientific literature,
enzyme activity increases as the concentration of substrates increases as with more
substrates bonding to enzymes, all active sites are filled up. To a point however,
when all the active sites are filled up, enzymatic activity drops. Therefore, the
optimal concentration at which enzyme activity reaches its highest will be
necessary for speeding a reaction.
Last, in this experiment, temperature will be measured. The major reason for the
denaturation of an enzyme occurs due to temperature. In this experiment, one will

be measuring the temperature of enzyme ALP at various temperatures over a

period of time.
My null hypothesis for the pH section of the lab is that pH will not have much of an
effect of enzyme concentration as the bonds holding the enzyme together are
trong. My null hypothesis for the concentration section is that concentration will
have only a partial effect on enzymatic activity. For temperature, the null hypothesis
is that enzyme activity will not change significantly even if partial structural change
occurs with temperature.
The alternative hypothesis for the effect of pH on enzyme activity is that an
imbalance of H+ ion concentration may have some effect on enzymatic activity, and
therefore the greatest rate of reaction may be around 7 (neutral pH). The
alternative hypothesis for the effect of temperature on enzymatic activity is that
since temperature increases the frequency of collisions (kinetic energy) between
the substrates and enzyme (enzyme-substrate complex), there may be an increase
in reaction rate, even with possible denaturation. The alternative hypothesis for the
effect of concentration on enzymes is that the greater the concentration, the
greater the rate of reaction due to more substrates reacting with the active site on
the enzymes.
Materials/ Procedure
Part One- Measuring pH levels for Enzyme activity of ALP

(Wilson, Properties of Enzymes, 0)

Table 1. Mixing Instructions for pH Experiment

Tube

Relative
pH

Solution
E

0.2 M
HCl

0.1 M
Na2CO3

Distilled
water

Solution D
High conc.
enzyme

Total
Volume

1
contro
l

---

3 mL

---

---

2 mL

---

5 mL

---

---

100 L

5 mL

Acidic

3 mL

1.9
mL

Neutral

3 mL

---

---

1.9 mL

100 L

5 mL

Basic

3 mL

---

1.9 mL

---

100 L

5 mL

Add Solution B, 6.5 mL, (pNPP-para nitro phenol phosphate) and 6.5 mL distilled
water to form Solution E. Label 4 cuvettes and prepare them as to the proportions of
each solution in Table 1. Place a broad pH paper on a clean, dry surface and after
mixing, remove a drop of each solution and drop on the pH paper. For each cuvette,
record the pH of the solution (only cuvettes 2-4) in Table 2. Begin procedures 1-4
with cuvette 2, 3 and 4 and add Solution D to it (right before doing the reading).
Mix carefully, using parafilm paper to invert (cuvette) and place the cuvettes in the
spectrophotometer. Record the absorbance reading at Time 0, and henceforth every
30 seconds for five minutes. Each value is to be recorded on Data Table 2. Repeat
procedures for cuvettes 3 and 4 (steps 2-7)
Notes: Since Tube 1 is the control, the spectrophotometer should be set to blank at
405 nm. Do not add solution D to any cuvette until ready for insertion into the
spectrophotometer.
Pre-Procedure for Parts Two and Three (Wilson, Properties of Enzymes)
Mix 15 mL of Solution A and 15 mL of Solution B in a 50 mL beaker and label it
Solution F. This solution will be used when measuring the effects of concentration
and temperature on enzymatic activity in reactions (ALP enzyme). Label eight
cuvettes 1a- 4a, 1b- 4b.
In this pre-procedure, the first major step is to prepare cuvette 1 and to set the
spectrophotometer to 405 nm. Re-blank the spectrophometer by adding enzyme to
cuvette 1 and label it cuvette 1a. Prepare cuvettes 1-4a using Table 3.
Use a micropipette and add 3 mL of Solution F to each of the eight cuvettes (1a-4a,
1b-4b). Prepare the b cuvettes for part 3 (Temperature) of the experiment by
following the mixing instructions in Table 4 and by placing 1b in the refrigerator (4
C), 2b at room temperature (23 C), 3b in a water bath (32 C) and 4b in a water bath
(60 C).
Table 3- Mixing table for Cuvettes 1-4a: The Effect of Concentration on
Enzymatic Rate

Tube

Relative
Enzyme Conc.

Sol C
Low ALP
(Enzyme) conc.

Sol F (comprised of
Sol A/B
Buffer/substrate
stock solution)
3 mL

---

Sol D
High ALP
(Enzyme)
conc.
---

1 blank (no
enzyme)
1a blank plus
enzyme
2a
3a

Lowest

3 mL

100 L

---

Medium
Higher medium

3 mL
3 mL

400 L
---

--200 L

4a

Highest

3 mL

500 L

---

(Wilson, Properties of Enzymes)

Table 4- Mixing Table for Cuvette 1b-4b: The Effect of Temperature on
Enzymatic Rate

Tube

Temperature (C)

1b
2b
3b
4b

4
23
32
60

Sol F
(Buffer/substrate stock
solution)
3 mL
3 mL
3 mL
3 mL

Sol C
Low ALP (Enzyme)
conc.
100 L
100 L
100 L
100 L

(Wilson, Properties of Enzymes)

Part Two: Determining the Effect of Enzyme Concentration on Reaction

Rate (Wilson, Properties of Enzymes)
Using a micropipette (preferably 100-1000 L), add 100 L of solution C (contains
low concentration of ALP) to cuvette 1a and put immediately into
spectrophotometer after mixing thoroughly (place a parafilm and invert the cuvette
for best results). After, place the cuvette into the spectrophotometer, starting with
the first reading at 0 seconds. Read the absorbance levels every 30 seconds for 5
minutes in Table 5.
Next, using a micropipette, add 400 L of Solution C (contains low ALP
concentrations) to cuvette 2a, mix it thoroughly and insert into the
spectrophotometer. Read the absorbance every 30 seconds for 5 minutes after
insertion into the spectrophotometer.
Using a micropipette, add 200 L of Solution D (high ALP enzyme concentration) to
cuvette 3a, cover with a parafilm paper, invert the cuvette to mix, and immediately
place it into the spectrophotometer. Record the absorbance level every 30 second
for five minutes post-insertion.
Using a micropipette, add 500

L of Solution D to cuvette 4a, cover with

parafilm, invert to mix, and place it into a spectrophotometer. Read the absorbance
level at 0 seconds, and read the absorbance every 30 seconds for 5 minutes.
Note: Refer to Tables 3 and 4 for detailed mixing instructions

Part Three: Determining the Effect of Temperature on Enzyme Reaction

Rate (Wilson, Properties of Enzymes)
First, obtain cuvette 1b stored in the fridge and using a micropipette, add 100 L of
solution C (containing low ALP concentration) to cuvette 1b, inverting the mixture
for better results. Immediately place the cuvette into the spectrophotometer and
record first measurement at 0 seconds, and afterwards, every 30 seconds for 5
minutes post- insertion.
Second, use a micropipette, adding 100 L of solution C to cuvette 2b and cover the
cuvette with parafilm for mixing. Place immediately into the spectrophotometer and
record the absorbance levels every 30 minutes for a 5 minute period in Table 5.
Third, obtain cuvette 3b (in the water bath at 32C) and add 100 L of solution C (low
ALP) to the cuvette. Mix the solution in the cuvette thoroughly by inverting, and
immediately place into the spectrophotometer. Begin recording the absorbance
levels every 30 seconds for 5 minutes and record in Table 5.
Fourth, get cuvette 4b, placed in the water bath heated at 60C, and add 100 L of
Solution C (low ALP). Mix thoroughly and place in the spectrophotometer. Record the
absorbance at 30 second intervals for 5 minutes. Record in Table 5.
Note: Refer to Tables 3 and 4 for detailed mixing instructions
Results- Part 1
Effect of pH on the Enzyme Controlled Reaction
Table 1. Effect of pH on the Activity of the Enzyme ALP
time (sec)

pH 2
0
30
60
90
120
150
180
210
240
270
300

pH 7
0.038
0.036
0.035
0.034
0.033
0.033
0.033
0.033
0.032
0.032
0.032

pH 10
0.076
0.083
0.084
0.089
0.094
0.098
0.101
0.105
0.112
0.115
0.118

0.07
0.072
0.075
0.076
0.077
0.08
0.082
0.084
0.086
0.088
0.09

Figure 1: Effect of pH on the Activity of Enzyme ALP over Time

0.14
0.12
0.1

pH 2

f(x) = 0x + 0.08

Linear (pH 2)

0.08 f(x) = 0x + 0.07

Absorbance Levels 0.06

ph 7
Linear (ph 7)
pH 10

0.04
0.02
0
0

Linear (pH 10)

f(x) = - 0x + 0.04
50 100 150 200 250 300 350
Time (sec)

In Table 1, pH 2 showed a decrease in the activity of the enzyme, by the absorbance

levels going from 0.038 to 0.032. The pH 7 level showed an increase from 0.076 to
0.118 and pH10 showed an increase from 0.07 to 0.09.
Figure 1 showed a linear decrease for pH 2 data points, from the equation y=0.00002x +0.0363. The pH 7 level showed a linear increase, as indiciated by the
equation, y=0.00007+0.0701. Last, pH 10 showed the same linear increase by the
equation, y= 0.0001x+0.0768

Table 2. Effect of Different pH levels on the Rate of Enzyme Reaction

pH

Rate of Reaction
2

-0.00002

0.0001

10

0.00007

Figure 2: Effect of Different pH levels on the Rate of Enzyme Reaction over Time
0
0
0
0
Rate of Enzyme Reaction 0
0
0
0

10

0
pH level

In table 2 and figure 2, the rate of reaction was most for pH 7, as indiciated by the
rate of the reaction being y=0.0001. The rate of the reaction was then the greatest
for pH 10, at y=0.00007x, and last for pH 2, at a decreasing rate of y=-0.00002x

Results- Part 2
Effect of Concentration on Enzyme Activity
Table 3: Effect of enzyme concentration on the Activity of Enzyme ALP

Time (s)

1a (low)

2a (med)

3a (med-high)

4a (high)

0.117

0.485

0.263

0.528

30

0.126

0.512

0.317

0.645

60

0.132

0.536

0.369

0.766

90

0.140

0.558

0.423

0.878

120

0.148

0.584

0.475

0.988

150

0.154

0.610

0.549

1.098

180

0.161

0.634

0.578

1.209

210

0.168

0.660

0.630

1.314

240

0.175

0.681

0.682

1.428

270

0.182

0.704

0.729

1.525

300

0.189

0.730

0.778

1.629

Figure 3: Effect of Enzyme Concentration on Enzyme Activity over

1.800
1.600 f(x) = 0x + 0.54
1.400

1a (low)
Linear (1a (low))

1.200

2a (med)
1.000
Absorbnace Levels

Linear (2a (med))

3a (med-high)

0.800
0.600

Linear (3a (med-high))

f(x) = 0x + 0.27
f(x) = 0x + 0.49

4a (high)
Linear (4a (high))

0.400
0.200
0.000
0

f(x) = 0x + 0.12
50

100

150

200

250

300

350

Time (sec)

Table 4. Effect of Enzyme Concentration on the Rate of Reaction

Enzyme concentration

Rate of Reaction

Low

0.0002

Med

0.0008

med high

0.0017

High

0.0037

Figure 4: Effect of Different Enzyme Concentration on the Rate of Enzyme Reaction over Time
0
0
0
0
Rate of Reaction 0
0
0
0
0

1a (low)

2a (med)

3a (med-high)

4a (high)

Enzyme Concentration

In Table 3 and Figure 3, it is indicated that the enzyme concentration had the
greatest effect on the Activity of Enzyme ALP indicated by cuvette 4a (high
concentration). The absorbance level for 4a rose from 0.528 to 1.629. The
absorbance level for 3a (med-high) increased from 0.263 to 0.778. The absorbance
level for 2a (medium concentration) rose from 0.475 to 0.730. Last, 1a (low
concentration), the absorbance level rose from 0.117 to 0.189. The greatest linear
trend was for the high enzyme concentration (4a).

In Table 4 and Figure 4, all enzyme concentrations showed an increase in enzymatic

activity. The rate of the reaction, in effect to level of concentration, however was
most for 4a (high concentration) at y=0.0037x. Next highest was 3a (med-high
concentration) at y=0.017x. Third was 2a (medium concentration) at a rate of
y=0.0008x. Last was 1a, at a rate of y=0.0002x

Results-Part Three
Effect of Temperature on Enzymatic Activity in Reactions
Table 5: Effect of temperature on the rate of an enzyme controlled reaction
4oC

Time (s)
0
30
60
90
120
150
180
210
240
270
300

20oC
0.053
0.057
0.061
0.062
0.066
0.069
0.072
0.075
0.078
0.082
0.084

32oC
0.077
0.081
0.085
0.092
0.097
0.102
0.107
0.111
0.116
0.121
0.126

60oC
0.084
0.091
0.101
0.11
0.12
0.128
0.137
0.144
0.151
0.158
0.166

0.122
0.125
0.13
0.134
0.137
0.14
0.143
0.146
0.149
0.151
0.154

Figure 5: Effect of Temperature on an Enzyme Reaction over Tim

0.18
0.16 f(x) = 0x + 0.08
f(x) = 0x + 0.12
0.14
4C

0.12 f(x) = 0x + 0.08

Linear (4 C)
20 C

0.1

Linear (20 C)

Absorbance Levels

32 C
0.08 f(x) = 0x + 0.05

Linear (32 C)
60 C
Linear (60 C)

0.06
0.04
0.02
0
0

50

100

150

200

250

300

350

Time (sec)

Table 6. Effect of Temperature on the Rate of an Enzymatic Reaction

Temperature (oC)

Rate of
Reaction

0.0001

20

0.0002

32

0.0003

60

0.0001

Figure 6: Effect of Temperature on the Rate of an Enzymatic Reaction over Time

0

0
Rate of Reaction
0

20

32

60

Temperature (C)

In table 5 and Figure 5, the effect of temperature on the rate of the enzyme
controlled reaction had a big impact. The absorbance level rose from 0.053 to 0.084
for 4oC temperature. The absorbance level rose from 0.077 to 0.126 for 20 oC. At 32
o
C, the highest increase in absorbance level occurred, rising from 0.084 to 0.166.
Last, at 60 oC, the absorbance level rose from 0.122 to 0.154.
In Figure 5, the effect of temperature on enzyme activity was plugged in and for 4
o
C, y=0.0001x +0.0563. For 20oC, y=0.0002x+0.0765 was the linear trend line. For
32 oC, the trend line linear equation was 0.0003x +0.0849. Last, the trend line at 60
o
C was 0.0001x+0.1232.
In Table 6, the rate of the reaction was calculated for each Temperature, based off of
y=mx, These values were placed in a tabular form, in Graph 6. The greatest rate of
reaction, as indicated in the column graph, was at 32 oC, where y=0.0001x. The
second highest rate was at 20 oC, where the rate of reaction was y=0.0002x. The

least rate of reaction occurred at temperatures 4 oC and 60 oC both of which had

y=0.0001x rates.
Discussion
In the result sections, for pH, temperature and concentration, the results all showed
patterns and my hypothesis was statistically supported.
For pH levels, the greatest enzyme reaction occurred at 7 (neutral) in comparison to
pH 2 (acidic) and pH 10 (alkaline). The reason being that changes in pH, either too
low or too high can alter an enzymes globular or fibrous state. In other words, pH
has an effect on the fundamental basis of enzymes, amino acids. Acidic amino acids
contain a carboxyl functional group (-COOH) and basic amino acids contain amine
groups. If the state of either ionized groups, carboxyl group in acidic amino acids or
amine groups in basic amino acids alters, this can lead to the substrate not binding
to the active site on the enzyme, making it hard for the enzyme stipend more
energy to go over the Activation Energy. Therefore, pH levels impact the ionic and
hydrogen bonding which holds enzyme shape and function. In Figure 3, the data to
support this is present and is similar to the figure as presented below showing the
greatest increase in the bell-curve was at optimum pH. According to the Campbell
Biology 10th Edition textbook, enzymes have a pH at which the enzyme works best.
The most effective pH is between 6-8 (optimum), however there are some
exceptions like the enzyme pepsin which works in the stomach at pH 2.
For temperature, indicated by Tables 5 and 6 and Figures 5 and 6, the greatest
effect on the rate of enzyme activity occurred at room temperature (32 oC). At 4 oC,
the temperature does not have enough kinetic energy for the enzymes to vibrate
and break, therefore making it impossible for the substrates to fit into the active
site. At 20 C, since kinetic energy increased, more substrates were interacting with
the enzyme active site. At optimum temperature (32 degrees C), the enzyme had
the greatest activity of substrates binding to active sites, and therefore had the
greatest increase in the rate of reaction. At 60 oC however, the enzyme most
probably went into a denatured state, losing its specific shape so the active site
becomes altered and the highly energetic substrate molecules cannot fit into the
enzymes active site. Therefore, the greater kinetic energy (Temperature), the
greater the enzyme reaction rate, until after the optimum temperature has been
reached .According to the Campbell Biology 10th Edition textbook, most enzymes
have optimum temperatures between 35-40 Degress Celcius, whereas some have
optimal temperatures at 70 C.

(Reece, Campbell
Biology)

For
concentration levels,
as the concentration
increases, the rate
of the reaction
increases until all of the substrate molecules have binded to their complementary
active site on the enzyme. As indicated in Tables 3 and 4, four cuvettes with
different concentrations of ALP enzyme was tested for. The ones containing the
most enzymes had the highest reaction rate as more enzymes were able to react
with the substrates, and the ones containing the least concentration of enzymes
had the least reaction rate. This was indicated by Tables 3, through absorbance
levels in the spectrophotometer, and Table 4, by measuring the rate of reaction.
Therefore, the greater the concentration of enzyme, the greater the reaction rate as
more substrates can bond to the enzymes. This increase occurs until the active sites
of all the enzymes are full.
In pH and Temperature, buffers were used as major differences occurred. As
demonstrated in the theoretical graph (Conclusion) , the bell-curve is so drastic
between pH levels 2-10 and between temperatures 30 to 75 degrees Celsius.
Buffers were therefore used to equalize the point of interaction between enzymes
and the corresponding factors (pH and temperature).
In conclusion, pH, temperature, and concentration have a great effect on the
function and shape of enzymes. Temperature, pH and concentration, if too high or
too low can damage the internal and external structures of enzymes, thereby
causing reactions to slow or not even work, as demonstrated by the enzymesubstrate complex. Therefore, to obtain statistically more correct results, further
experimentation must be done.

Work Cited
Wilson, C., D'Alessio, N., Lavin, E., & Keith, E. (n.d.). Properties of Enzymes. Nova
Southeastern University Bio 1500, Florida.
Reece, J. (2014). Campbell biology (Tenth ed.). Boston: Benjamin Cummings :.
Michael, F. (2015). Lab #4: Enzymes. Retrieved November 2, 2015, from
http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf
Mot, R., Schrijver, A., Schoofs, G., & Parret, A. (2003). The thiocarbamate-inducible
Rhodococcus enzyme ThcF as a member of the family of / hydrolases with
haloperoxidative side activity. FEMS Microbiology Letters,197-203.
Eed, John (2013) "Factors Affecting Enzyme Activity," ESSAI: Vol. 10, Article 19.