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    Enzymeassays 160408151239

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    168-Shivank Dwivedi
    Enzyme assays are laboratory methods used to measure enzyme activity. There are direct and indirect assay methods. Direct methods measure changes in substrate or product concentration over time through properties like absorbance, fluorescence, or pH. Indirect methods involve further treatment of reactions to detect products. Coupled assays use additional enzymes to catalyze detectable reactions from assay products. Continuous assays allow ongoing measurement while discontinuous assays stop reactions for analysis.

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    Enzymeassays 160408151239

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    168-Shivank Dwivedi
    31 views24 pages
    Enzyme assays are laboratory methods used to measure enzyme activity. There are direct and indirect assay methods. Direct methods measure changes in substrate or product concentration over time through properties like absorbance, fluorescence, or pH. Indirect methods involve further treatment of reactions to detect products. Coupled assays use additional enzymes to catalyze detectable reactions from assay products. Continuous assays allow ongoing measurement while discontinuous assays stop reactions for analysis.

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    Enzyme assays are laboratory methods used to measure enzyme activity. There are direct and indirect assay methods. Direct methods measure changes in substrate or product concentration over time through properties like absorbance, fluorescence, or pH. Indirect methods involve further treatment of reactions to detect products. Coupled assays use additional enzymes to catalyze detectable reactions from assay products. Continuous assays allow ongoing measurement while discontinuous assays stop reactions for analysis.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    31 views24 pages

    Enzymeassays 160408151239

    Uploaded by

    168-Shivank Dwivedi
    Enzyme assays are laboratory methods used to measure enzyme activity. There are direct and indirect assay methods. Direct methods measure changes in substrate or product concentration over time through properties like absorbance, fluorescence, or pH. Indirect methods involve further treatment of reactions to detect products. Coupled assays use additional enzymes to catalyze detectable reactions from assay products. Continuous assays allow ongoing measurement while discontinuous assays stop reactions for analysis.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    of 24

    ENZYME ASSAYS

    • Laboratory method for measuring enzyme


    activity.
    • Vital for study of enzyme kinetics and enzyme
    inhibition.
    • Measurement of enzyme activity – follow
    the change in concentration of substrate or
    product – measure reaction rate.
    DIRECT CONTINUOUS ASSAYS

    • Difference in properties of substrate and product –


    measured directly.
    • Continuous observation of the progress curve – most
    preferred.
    • Change in
    – Absorbance - Fluorescence.
    . - Optical rotation.
    – pH. - Viscosity.
    – Enthalpy.
    – Volume of reaction mixture.
    ABSORBANCE
    A   log10  I 
     I0 
    ◦ I - intensity of light at a specified wavelength λ passing through a
    sample.
    ◦ I0 - intensity of the light before it enters the sample.
    ● Relation between concentration and absorbance:
    A  cI
    ◦ ɛ → extinction – proportionality constant relating absorbance
    to concentration.
    ◦ c – concentration.

    A
    c
    TURBIDIMETRY
    • Light scattering not absorbance.
    • Action of enzymes on turbid polymer solutions.
    • Difficult to standardize – difficult to reproduce
    results.
    • E.g.: bacterial lysozyme assay – on dried bacterial
    cells – measured at 450nm.
    • Unit: one unit of activity – initial rate of change in
    absorbance of 0.001 per minute when the volume in
    the cuvette is 2.6ml, pH- 6.24 at 25˚C.
    FLUORESCENCE

    • Result of electronic transition – converts


    the absorbing molecule to an excited state.
    • Fluorescent molecule emits part of
    absorbed energy as light – lower energy
    but higher wavelength.
    • More sensitive than absorbance assays.
    FLUORIMETR
    Y
    • Fluorospectrophotometer – more specific
    than spectrophotometer.
    • Disadvantage: fluorescing molecules quench
    in solution.
    • E.g: anthranilate synthase
    Chorismate + L-glutamine ↔ anthranilate +
    pyruvate
    •λ exci = 325nm, λemi = 400nm.
    RADIOMETRY
    • Requirement of labelled substrates and counting
    instruments.
    • Substrates can be labelled with 14C, 3H, 32P, 35S, 125I.
    • E.g: galactosyl transferase.
    UDP-galactose* + glucosamine GT, Mn +2
    UDP + lactosamine*
    • Stop the reaction by adding EDTA.
    ↓
    Pass through ion exchange column – separate substrate and
    products.
    ↓
    Product collected – check radioactivity by scintillation counter.
    pH stat
    ● Stationary pH.
    ● Used to monitor progress of chemical reaction
    in which protons are liberated or taken up.
    ● Achieved by measuring the amount of acid or
    base required to be added to maintain constant
    pH.
    DIRECT DISCONTINUOUS ASSAY

    ● p-nitrophenol in
    alkaline condition
    – highly
    electronegative
    .
    ● Colorless in acidic
    condition and
    yellow in alkaline
    condition.
    ● Yellow
    color
    measured
    at 405nm.
    INDIRECT ASSAYS
    • Further treatment of reaction mixture –
    produce a measurable product or
    increase sensitivity of assay procedure.
    C O N T I N U O U S ASSAYS
    • Manipulation necessary to detect product formation – allows
    continuous observation of the change.
    • Less prone to errors from sample manipulation in
    discontinuous assays
    • Reagents required for color development or measurement of
    activity included in the reaction mixture.
    • E.g.: carnitine acyl transferase.
    Acyl CoA + carnitine ↔ acyl carnitine + CoASH
    CoASH + 5,5’-dithiobis-2-nitrobenzoate → 4-nitrothiolate
    anion
    (DTNB - reagent)
    • λmax = 412nm.
    D I S C O N T I N U O U S ASSAYS
    • Also called sampling assay.
    • Stopping reaction - after a fixed time.
    • Treating the reaction mixture to separate the product for
    analysis or produce a measurable change in properties of
    substrates or product.
    • Separate product for analysis (radiochemical assay)
    – No modification made on the substrate/product → can be
    considered as a direct assay.
    • Produce change in properties of one substrate/product →
    can be measured.
    – Formation of ATP can be determined by measuring light intensity in
    the presence of luciferase.
    ATP + luciferin +O2 → oxyluciferin + PPi +CO2 + AMP + light
    Coupled assays
    • Use of one or more additional enzymes to
    catalyse a reaction of one of the products
    to yield a compound that can be directly
    detected.
    • Additional enzyme – coupled enzymes.
    E xample
    s Glucose
    • Hexokinase. ATP, Mg2+
    HEXOKINASE
    – Coupling of the
    formation of ADP, Mg2+
    glucose-6-
    Glucose 6-phosphate
    phosphate to the
    NADP+
    reduction of G6P
    NADP+ in the DEHYDROGENASE

    presence of G6P N
    dehydrogenase. A
    D
    P
    Phosphofructokinase
    Fructose 6-phosphate PHOSPHOFRUCTOKINASE Fructose 1,6-bisphosphate

    AT P ADP
    PYRUVAT E
    KINASE

    Pyruvate Phosphoenolpyruvate

    NADH + H +
    LDH

    NAD+

    Lactate
    Coupled continuous assay

    1. Aspartate amino transferase (serum glutamate


    oxaloacetate transaminase)
    •
    AST/S GOT – 30˚C, pH → 7-8 in 80mM tris.
    • Change in A340 measured.

    Aspartate + α-ketoglutarate ↔ oxaloacetate +


    glutamate Oxaloacetate + NADH + H+ ↔ malate
    + NAD+
    2. Alanine aminotransferase (serum glutamate pyruvate
    transaminase)
    • ALT/SGPT.
    Alanine + α-ketoglutarate ↔ pyruvate +
    glutamate Pyruvate + NADH + H + ↔
    lactate + NAD +
    3. Decarboxylase.
    Lysine Lysine decarboxylase cadaverine + CO2

    CO2 + PEP Wheat PEP carboxylase oxaloacetate

    Oxaloacetate + NADH + H+ MDH malate + NAD+


    V alidity of
    results

    • Reaction step should not be rate limiting.


    • Velocity of the reaction increases till coupling
    enzyme reaches the rate of the first enzyme.
    • Coupling enzyme – high Km for the
    enzyme and low Km for substrate.
    Cycling coupled
    assay
    • Alcohol dehydrogenase

    Ethanol Acetaldehyde

    NAD+ NADH + H +

    Propanediol Lactaldehyde
    Forw ard coupled
    assay
    • Malate dehydrogenase.

    Malate + NAD+ MDH Oxaloacetate + NADH + H +


    Acetyl CoA
    CITRATE SYNTHASE

    CoA

    Citrate
    Reference
    s
    • Enzyme Assays by Robert Eisenthal.
    • Photometric assays – Robert A. John.
    • Principles of enzyme assays and
    kinetic studies – Keith F. Tipton.

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