BIOCHEMISTRY AND MOLECULAR MEDICINE

GENERAL MODULE 1

PRACTICAL 1:
ENZYME ACTIVITY

PREPARED BY:

GROUP Q
STUDENT’S NAME STUDENT ID
ADELIA BINTI MOHD YUSOF 2021458822
AHMAD DARIMI BIN MD ARIFFIN 2021887676
AHMAD NABIL MUSTAQIM BIN MOHD RAHIMI 2021829606
ALYA ADRIANA BINTI SAREH AZMI 2021840028
AMAL IKHLAS BINTI SHARAF 2021823636
LUQMAN HAKIM BIN AZEMAN (LEADER) +60137278170 2021817414
MASYITAH BINTI OMAR 2021494758
NIK BISARAH BINTI NIK PAUZI 2021817244
NUR ZAHIRAH BINTI RADIN ZULKARNAIN 2021494538
NURUL SYAHIRAH BINTI AIMISAHRIMAN 2021485884
SHAHRIL HAFIZEE BIN SHAFIE 2021485878
 CONTENTS
TITLE PAGE/S
Contents i
1.0 Experiment 1: Serum Alkaline Phosphatase (ALP) Estimation 1
I. Result of The Experiment
II. Calculation of Enzyme Activity
III. Graph of Velocity versus Time
2.0 Experiment 2: Km Estimation for Serum Alkaline Phosphatase 2–3
I. Result of The Experiment
II. Calculation on Enzyme Activity
III. Graph of 1/V versus 1/[S]
3.0 Experiment 3: Temperature Stability of Serum Alkaline Phosphates 4
I. Result of The Experiment
II. Enzyme Activity Calculation
III. Graph of Enzyme Activity versus Temperature
4.0 Experiment 4: Estimation of the Optimum pH for Alkaline Phosphatase 5–6
I. Calculation of Enzyme Activity
II. Graph of Enzyme Activity versus pH

i
1.0 EXPERIMENT 1: SERUM ALKALINE PHOSPHATASE (ALP) ESTIMATION
I. Result of The Experiment:
Time Point (min) 0 2 3 4 5 6 7
OD 0.120 0.144 0.158 0.175 0.195 0.208 0.210
Δ OD 0 0.024 0.038 0.055 0.075 0.088 0.090
Enzyme Activity, V
0 65.808 104.196 150.810 205.650 241.296 246.78
(U)

II. Calculation of Enzyme Activity:

Min 0 = 0 × 2742 = 0
Min 2 = 0.024 × 2742 = 65.808
Min 3 = 0.038 × 2742 = 104.196
Min 4 = 0.055 × 2742 = 150.810
Min 5 = 0.075 × 2742 = 205.650
Min 6 = 0.088 × 2742 = 241.296
Min 7 = 0.090 × 2742 = 246.278
III. Graph of Velocity versus Time:

1
2.0 EXPERIMENT 2: KM ESTIMATION FOR SERUM ALKALINE PHOSPHATASE
I. Result of The Experiment:
Volume Reagent A (S) 0.5 0.75 1.0 1.5 2.0
OD (0 minute) 0.112 0.114 0.11 0.112 0.116
OD (3 minutes) 0.184 0.210 0.249 0.328 0.38
Δ OD 0.072 0.096 0.139 0.216 0.264
Enzyme Activity, (V) 65.808 87.744 127.046 197.424 241.296
1/V 0.0151 0.0114 0.0079 0.0051 0.0041
Substrate Concentration, 3.137 4.706 6.275 9.412 12.549
[S]
1/[S] 0.319 0.212 0.159 0.106 0.080

II. Calculation of Enzyme Activity:

0.072
𝐹𝑜𝑟 0.5𝑚𝑙 = × 2742 = 65.808
3
0.096
𝐹𝑜𝑟 0.75𝑚𝑙 = × 2742 = 87.744
3
0.139
𝐹𝑜𝑟 1.0𝑚𝑙 = × 2742 = 127.046
3
0.216
𝐹𝑜𝑟 1.5𝑚𝑙 = × 2742 = 197.424
3
0.264
𝐹𝑜𝑟 2.0𝑚𝑙 = × 2742 = 241.296
3

2
III. Graph of 1/V versus 1/[S]:

1/V versus 1/[S] y = 0.0476x + 0.0004

0.02

0.015
Rate of reaction, 1/V (U^-1)

0.01

0.005

0
-0.100 0.000 0.100 0.200 0.300 0.400

-0.005
Substrate Concentration, 1/[S] (mM^-1)

1
y-intercept = 𝑉𝑚 = 0.0004

𝑉𝑚 = 2500 𝑈
−1
x-intercept = 𝐾𝑚 = −8.4034 × 10−3

𝐾𝑚 = 119 𝑚𝑀

3
3.0 EXPERIMENT 3: TEMPERATURE STABILITY OF SERUM ALKALINE PHOSPHATASES
I. Result of The Experiment
Temperature (°C) 5 25 37 50
OD (0 minute) 0.113 0.111 0.114 0.112
OD (5 minute) 0.157 0.179 0.209 0.167
Δ Abs 0.044 0.068 0.095 0.055
Enzyme Activity, V
24.1296 37.2912 52.098 30.162
(U)

II. Enzyme Activity (V) Calculation:

0.044
𝐹𝑜𝑟 5℃ = × 2742 = 24.1296
5
0.068
𝐹𝑜𝑟 25℃ = × 2742 = 37.2912
5
0.095
𝐹𝑜𝑟 37℃ = × 2742 = 52.098
5
0.055
𝐹𝑜𝑟 50℃ = × 2742 = 30.162
5
III. Graph of Enzyme Activity versus Temperature:

Graph of Enzyme Activity versus Temperature

60
52.098
50
Enzyme Activity ,V (U)

40 37.2912

30.162
30
24.1296

20

10

0
5 25 37 50
Temperature (°C)

Therefore, the optimum temperature for enzyme activity is 37℃

4
4.0 EXPERIMENT 4: ESTIMATION OF THE OPTIMUM PH FOR ALKALINE PHOSPHATASES
I. Calculation of Enzyme Activity:
pH 8 9 10 11 12
OD (0 minutes) 0.112 0.114 0.11 0.112 0.116
OD (15 minutes) 0.19 0.214 0.237 0.235 0.175
Delta OD 0.078 0.100 0.127 0.123 0.059
Enzyme Activity, V 14.26 18.28 23.22 22.48 10.79
(U)

𝐹𝑜𝑟 𝑝𝐻 = 8, 𝛿𝑂𝐷 = 0.19 − 0.112 = 0.078

0.078
𝑇ℎ𝑢𝑠, 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 2742 = 14.26 𝑈
15
𝐹𝑜𝑟 𝑝𝐻 = 9, 𝛿𝑂𝐷 = 0.214 − 0.114 = 0.100
0.1
𝑇ℎ𝑢𝑠, 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 2742 = 18.28 𝑈
15
𝐹𝑜𝑟 𝑝𝐻 = 10, 𝛿𝑂𝐷 = 0.237 − 0.11 = 0.127
0.127
𝑇ℎ𝑢𝑠, 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 2742 = 23.22 𝑈
15
𝐹𝑜𝑟 𝑝𝐻 = 11, 𝛿𝑂𝐷 = 0.235 − 0.112 = 0.123
0.123
𝑇ℎ𝑢𝑠, 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 2742 = 22.48 𝑈
15
𝐹𝑜𝑟 𝑝𝐻 = 12, 𝛿𝑂𝐷 = 0.175 − 0.116 = 0.059
0.059
𝑇ℎ𝑢𝑠, 𝑒𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = × 2742 = 10.79 𝑈
15

5
II. Graph of Enzyme Activity versus pH:

Therefore, the optimum pH value for enzyme activity is 10.