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    Home > Biochemistry News > Biotechnology News > Determination of the activity of superoxide disambiguation enzyme (SOD).

    Determination of the activity of superoxide disambiguation enzyme (SOD).

    • Last Update: 2020-10-17
    • Source: Internet
    • Author: User
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    " (Experimental Principles)


    . SOD is an enzyme containing a metal co-base, which catalyzes the following reactions:

    02 - s.02 - s.H2 02 s.02


    because of the short life of superoxyion free-base 02 - unstable, not easy to directly determine SOD activity, but the use of
    indirect method. At present, there are 3 commonly used methods, including nitrogen blue tetum (NBT) photodecent reduction method, phthalates self-oxidation method, chemical luminescence method. This experiment mainly introduces the photodynaminic reduction method, the principle of which is: nitrogen blue tetryptonin in the presence of methionine and nucleolutin, lighting after light reduction reaction and the generation of blue A, blue acetyladium at 560nm has the maximum light absorption.
    .

    . SOD can inhibit the photoresorption reduction of NBT, and its inhibition intensity is directly related to the enzyme activity in a certain range.


    < a href="""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""> reagents:
    1.50m mol/l pH7.8 phosphoric acid buffer (PBS). Includes 0.1m mol/l < a href">"
    2.220m mol /l methionine: called 3.2824g methionine, dissolved to 100ml (now available) with a 50m mol/l pH7.8 phosphate buffer.

    3.1.25m mol/l chlorinated nitrosazole blue (NBT) solution (now available): called NBT 0.10 2g, dissolved with

    50m mol/l pH7.8 phosphoric acid buffer (PBS) dissolved to 100ml.

    .

    4.0.033m mol/lr lutein: called 2.52mg dissolved with PBS to 200ml (light-shielding).


    "Experimental steps

    < p style "text-align: left;">

    1. Enzyme preparation
    plant tissue 0.5g first add 2.5ml PBS, grinding well After the slurry, add 2.5 ml PBS mix,

    4 degrees C at 10000r/min centrifugation 15min on the liquid is crude enzyme solution. The extracted liquid is used for enzyme activity determination after proper dilution.


    2.enzyme activity assay
    take 10ml small berries7, 3 assay samples, 4 as a control,

    the table below to add the trial:


    .
    reagents
    dosage (">" ml)
    final concentration (when colored)
    50m mol. /l (PBS)
    220m mol/lMet
    1.25m mol/l NBT
    0.033m mol/l the
    enzyme solution
    total volume
    4.05.
    0.3
    0.3
    0.3
    0.0" 5
    5.0
    13m mol/l.
    75 smol/l
    2.0 smol/l
    in place of buffer instead of enzymes

    After mixing the above reagents, 1 control beech to the dark, and 3 other control berries and samples together placed in
    4000lx daylight reaction 20min (requires the tubes to be subjected to the same light, high temperature time shortened, low temperature can be appropriately extended).
    finally determines the light density of the reaction fluid at 560nm. The light density of his tubes was measured by a reference to the light-not-lighted control beech, which

    the light density of his tubes.


    3.Protein content measurement

    step 1 of the upper liquid was properly diluted with the Coomas bright blue G-250 method to determine the protein content.

    .


    "experiment results and calculations"


    SOD active unit is 50% of NBT photorescientization reduction Represented as an enzyme active unit. Pressable meter

    COUNT SOD activity:


    SOD Total Activity"-
    SOD Than Vitality","SOD Total Activity/< a href""> Protein Total Concentration
    .
    type: the total activity of SOD is U/gFW, which represents the light absorption value of ACK as a control cup (light) in U/mg protein than the unit of vitality; AE is the light absorption value of the sample; V is the total volume of the sample fluid, V1 is the sample amount at the time of determination, and W is the sample weight g.
    .

    " peroxidase (POD) activity determination

    < "text-align: left;" >

    "experimental principle


    .

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