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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 acetyladiumat 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;">
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 enzyme solution total volume | . 0.3 0.3 0.3 0.0" 5 5.0 | . 75 smol/l 2.0 smol/l in place of buffer instead of enzymes
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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