I'm looking for the determination of catalase enzyme in liver homogenate using Claiborne 1985 and how I can obtain the result in U/mg protein.
Sorry! but I don't fully understand your question. Why 2 step dilution. you can prepare H2O2 like this
the Sigma datasheet it says "% (w/w)" = weight per weight, which means that the solution contains 30 g H2O2 per 100 g of solution. The density of this solution is 1.11 g/ml, so 100 g have a volume of 90.09 ml. So 1 L contains 333 g of H2O2 (30 g / 90.09 ml x 1000 ml) which is a molar concentration of 9.79 mol/L (333 g/L / 34.01 g/mol).
To prepare a 19 mM solution take ~0.2 ml of your 30% solution and fill to 100ml with water (19 mM x 1000 ml / 9.79 M).
You can try this method
The assay mixture consisted of 1.95ml phosphate buffer (0.05 M, pH 7.0), 1.0ml hydrogen peroxide, H2O2 (0.019 M) and 0.05ml liver homogenate supernatant in a final volume of 3.0ml. Change in absorbance for 3 minutes at 30 seconds interval was recorded at 240 nm. Blank was prepared using TDW instead of sample. Change in absorbance per min was calculated. Difference between absorbance of test sample from blank was divided with molar extinction coefficient of 40 M-1 cm-1 and used as a measure of the enzyme’s activity.
Specific activity of catalase is expressed as µM H2O2 decomposed/min/µg protein.
total protein is estimated using standard protocol of Lowry et al. 1951
thank you dr archana the method is :
Reagents:
1- 50 mM potassium phosphate buffer:
Solution (a) was prepared by : weighing 6.81 gm of KH2PO4 dissolved in distilled water, the volume was completed to 1000 ml.
Solution (b) was prepared by : weighing 8, 90 gm of Na2HPO4. H2O dissolved in distilled water; the volume was completed to 1000 ml.
Then solution (a) was mixed with solution(b) in a proportion of 1:1.5 (v/v), and then the PH was adjusted to 7.0.
2-Substrate: Solution (a) : volume of 0.5 ml of 30 % hydrogen peroxide was diltuted to 100 ml with 50 mM phosphate buffer PH 7.0.
(This is the approximately 0.059 M H2O2).
Then 340 µl from solution (a) was diluted in phosphate buffer to 100 ml.
Approximately 19 mM H2O2 was prepared from 30% H2O2 solution 50 mM potassium phosphate buffer.
3-1% Triton X-100: Triton X-100 was diluted with phosphate buffer with ratio of 1: 100 (modified by 1: 50); Triton X-100 was used for obtaining complete lysis of all organelles.
Procedure:
I- in 4 ml glass cuvette (with a 1.0 cm light path) 2.95 ml of 19 mM H2O2 solution were added.
2-500 µl of the sample were then added to the cuvette mixture and the ensuring decrease in absorbance at 240 nm was followed for one minute.
Calculation:
-Catalase specific activity is defined in terms of micromoles of H2O2 consumed per minute per mg protein sample.
The activity of catalase in tissue was expressed as unit/ mg protein .
Tissue catalase activity = (D A/ min x 1000 x 3)/(43.6 x 2 x mg protein)
Wereras:
43.6→ represent the molar extinction coefficient of peroxidase according to ( Lowry et al., 1951) or microbiuret methods.
3→ mean the total volume in the cuvette.
2→ mean the volume of the sample.
the problem i can not understand the 2nd step of preparation of substrate if in the first step i mix solution a with solution b how in the second step i dilute it i can not understand the method in simple way
Sorry! but I don't fully understand your question. Why 2 step dilution. you can prepare H2O2 like this
the Sigma datasheet it says "% (w/w)" = weight per weight, which means that the solution contains 30 g H2O2 per 100 g of solution. The density of this solution is 1.11 g/ml, so 100 g have a volume of 90.09 ml. So 1 L contains 333 g of H2O2 (30 g / 90.09 ml x 1000 ml) which is a molar concentration of 9.79 mol/L (333 g/L / 34.01 g/mol).
To prepare a 19 mM solution take ~0.2 ml of your 30% solution and fill to 100ml with water (19 mM x 1000 ml / 9.79 M).
I think that the best methods for assessment Catalase activity are:
1. Hadwan MH, kadhum Ali S. New spectrophotometric assay for assessments of catalase activity in biological samples. Analytical biochemistry. 2018 Feb 1;542:29-33.
https://www.ncbi.nlm.nih.gov/pubmed/29175424
2. Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC biochemistry. 2018 Dec;19(1):7.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091033/
Please go through the publications attached. I hope to find useful informations.
Best wishes
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