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    BMM PRACTICAL 1 Report PRC1 BMM Group Q (Senior Lepas)

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    SARAH SAFIAH TAJUL ARIFFIN
    The document describes 4 experiments on the enzyme alkaline phosphatase: 1) The first experiment measured the enzyme's activity over time and found increasing activity from 0 to 7 minutes. 2) The second experiment determined the Michaelis-Menten constant (Km) by measuring activity at different substrate concentrations, finding a Km value of 119 mM. 3) Temperature stability was examined next, with maximum activity at 37°C, indicating this is the optimal temperature. 4) Finally, pH effects were studied from pH 8 to 12, with highest activity at pH 10, identifying this as the optimum pH.

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    BMM PRACTICAL 1 Report PRC1 BMM Group Q (Senior Lepas)

    Uploaded by

    SARAH SAFIAH TAJUL ARIFFIN
    17 views8 pages
    The document describes 4 experiments on the enzyme alkaline phosphatase: 1) The first experiment measured the enzyme's activity over time and found increasing activity from 0 to 7 minutes. 2) The second experiment determined the Michaelis-Menten constant (Km) by measuring activity at different substrate concentrations, finding a Km value of 119 mM. 3) Temperature stability was examined next, with maximum activity at 37°C, indicating this is the optimal temperature. 4) Finally, pH effects were studied from pH 8 to 12, with highest activity at pH 10, identifying this as the optimum pH.

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    221103 BMM PRACTICAL 1 Report PRC1 BMM Group Q (Senior lepas)

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    The document describes 4 experiments on the enzyme alkaline phosphatase: 1) The first experiment measured the enzyme's activity over time and found increasing activity from 0 to 7 minutes. 2) The second experiment determined the Michaelis-Menten constant (Km) by measuring activity at different substrate concentrations, finding a Km value of 119 mM. 3) Temperature stability was examined next, with maximum activity at 37°C, indicating this is the optimal temperature. 4) Finally, pH effects were studied from pH 8 to 12, with highest activity at pH 10, identifying this as the optimum pH.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    17 views8 pages

    BMM PRACTICAL 1 Report PRC1 BMM Group Q (Senior Lepas)

    Uploaded by

    SARAH SAFIAH TAJUL ARIFFIN
    The document describes 4 experiments on the enzyme alkaline phosphatase: 1) The first experiment measured the enzyme's activity over time and found increasing activity from 0 to 7 minutes. 2) The second experiment determined the Michaelis-Menten constant (Km) by measuring activity at different substrate concentrations, finding a Km value of 119 mM. 3) Temperature stability was examined next, with maximum activity at 37°C, indicating this is the optimal temperature. 4) Finally, pH effects were studied from pH 8 to 12, with highest activity at pH 10, identifying this as the optimum pH.

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    © All Rights Reserved
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    of 8

    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.

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