The assay of SOD employs xanthine and xanthine oxidase to generate superoxide radicals which on reaction with 2-(4-iodophenyl) -5-phenyltetrazolium chloride (INT) forms a red formazan dye. The absorbance is measured at 505nm by a spectrophotometer. SOD is measured from the degree of inhibition of this reaction and expressed as units/ml. One unit is equal to the amount of sod to cause 50 percent inhibition of reduction rate. For details, my paper on serum SOD as tuberculosis diagnostics may be gone through.

For glutathione peroxidase (GPx) assay, GPx catalyses the oxidation of glutathione by cumene hydroperoxide. In presence of glutathione reductase and NADPH(H+) oxidized glutathione is immediately converted to its reduced form with concomitant oxidation of NADPH(H+) to NADP+.The decrease in absorbance at340nm is measured spectrophotometrically. The concentration of the enzyme in whole blood is expressed as Units/Litre.

For assay of catalase several assay procedures are there. Catalase breaks down the substrate hydrogen peroxide to water and nascent oxgen. By measuring volume and hence mass of nascent oxgen, catalase enzyme concentration may be expressed as per international units by breakdown of number of micromoles of hydrogen peroxide in specified time.

Thank you very much for the reply, but I do not need this explanation, but I need a working method for extraction and estimation.

I only had elaborated the principles of estimation. For detailed measures of substrate, working ingredients, period of incubation etc you may login to internet and hands on experiment in details you might get from there.

here’s a practical, researcher-level set of methods you can use to extract and measure catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH / glutathione peroxidase options). I give: (A) sample prep / extraction (for tissue or cells), (B) protein assay (for normalization), (C) detailed assay protocols (with reagent recipes, volumes, wavelengths, calculations and units), (D) troubleshooting & tips, and (E) safety/quality notes. I cite canonical methods you can cross-check. If you want these written as a lab SOP file or a formatted methods section for a paper, tell me and I’ll format it for copy-paste.

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A. Sample collection & extraction (tissue or cultured cells)

Work on ice / 4°C for all steps unless stated.

1. Materials / buffers (prepare fresh or store aliquots at −20 to −80 °C):

Phosphate buffer (50 mM, pH 7.0): e.g. for catalase extraction and assays. (Prepare from NaH2PO4/Na2HPO4; check pH.)

Potassium phosphate buffer (50–100 mM, pH 7.8): used in many SOD assays.

Homogenization buffer (general): 50 mM phosphate pH 7.0, 0.1 mM EDTA, 1 mM DTT (optional), 0.1% Triton X-100 (optional for membrane release), protease inhibitor cocktail (if required). Do not include reducing agents when measuring thiols unless required for total GSH (see below).

Cold (4°C) reagents, clean tubes, Dounce/potter homogenizer or bead-mill.

2. Tissue:

Weigh ~50–200 mg tissue, place on ice, add 5–10 volumes (w/v) of cold homogenization buffer (e.g., 100 mg tissue + 0.5–1.0 mL buffer).

Homogenize with Dounce (10–20 strokes) or mechanical homogenizer until uniform.

Centrifuge at 10,000–15,000 × g for 10–20 min at 4°C. Collect supernatant (crude enzyme extract). Keep on ice.

Aliquot and measure protein immediately or freeze aliquots at −80°C for short term. Avoid repeated freeze–thaw.

3. Cells (cultured):

Harvest cells, wash in cold PBS, resuspend in 5–10 volumes of homogenization buffer, sonicate briefly (2–3 × 5-s pulses, on ice) or pass through a 26G needle, then centrifuge at 10,000–15,000 × g for 10–20 min at 4°C.

4. Notes:

For GSH measurements, avoid oxidizing conditions. Use 5% metaphosphoric acid (MPA) or 10% trichloroacetic acid (TCA) to precipitate proteins and stabilize GSH if using colorimetric/enzymatic assays (protocol dependent). See section C for choices.

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B. Protein determination (normalize enzyme activities to mg protein)

Use Bradford or BCA assay. Quick Bradford mini-protocol:

Prepare BSA standards (0, 2, 4, 6, 8, 10 µg/µL) in same buffer as samples (diluted).

Mix 10 µL sample + 200 µL Bradford reagent in 96-well plate, incubate 5–10 min at room temp, read at 595 nm.

Determine protein (µg/µL) from standard curve. Normalize enzyme activities to mg protein (U/mg protein).

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C. Enzyme assays — step-by-step

1) Catalase (CAT) — Aebi / H2O2 decomposition method (spectrophotometric)

Principle: follow decrease in absorbance of H2O2 at 240 nm after adding sample; rate of decrease ∝ catalase activity. Many labs use the extinction coefficient 39.4 M⁻¹cm⁻¹ (Aebi and commonly cited) — confirm for your instrument/pathlength or standardize H2O2 stock.

Reagents:

50 mM phosphate buffer, pH 7.0 (ice cold).

30% H2O2 stock (prepare fresh dilutions). For assay typical working concentration: 10–30 mM H2O2 in buffer (many protocols use 10 mM final).

Quartz cuvettes (or UV-transparent microplate, correct pathlength).

Example cuvette assay (1 cm pathlength):

Prepare reaction mix in cuvette: 2.95 mL 50 mM phosphate buffer (pH 7.0) containing 10 mM H2O2. Equilibrate to 25 °C (or 30–37 °C if preferred).

Start: add 50 µL enzyme extract (diluted as needed) to 2.95 mL reaction, mix quickly, and immediately record A240 every 10 s for 1–2 minutes.

Blank: run buffer + H2O2 without enzyme. Also run sample + buffer without H2O2 (control for baseline).

Calculations:

ΔA240/min (linear portion) → convert to rate of H2O2 consumption using Beer-Lambert:

\text{[H2O2] consumed (M/min)} = \frac{\Delta A_{240}/\text{min}}{\varepsilon_{240}\ (\text{M}^{-1}\text{cm}^{-1}) \times \text{pathlength (cm)}}

multiply by 10^6 to get nmol/min, then divide by mg protein in the assay to give U/mg protein, where 1 U = 1 µmol H2O2 decomposed per minute (or some labs define as µmol/min).

Example: ΔA/min = 0.050 → rate = 0.050 / 39.4 = 0.00127 M/min = 1.27 µmol/mL/min. If 0.05 mL enzyme in 3.0 mL total, calculate µmol per minute in cuvette and normalize to mg protein present.

Units & reporting:

Report as µmol H2O2 consumed/min/mg protein (U/mg protein) or as kU/g tissue whichever your field prefers. Provide both sample volume and protein used.

Notes:

Standardize H2O2 concentration daily — H2O2 degrades over time; measure A240 of diluted stock to verify concentration.

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2) Superoxide dismutase (SOD) — NBT / photochemical or xanthine-xanthine oxidase methods

Principle (classic Beauchamp & Fridovich): superoxide anion (O2•−) reduces nitroblue tetrazolium (NBT) to formazan which absorbs at 560 nm; SOD present inhibits NBT reduction. The % inhibition is used to compute SOD units. Alternative is xanthine/xanthine oxidase→cytochrome c reduction read at 550 nm.

Reagents (photochemical riboflavin-NBT method):

50–67 mM potassium phosphate buffer pH 7.8 (some use 50 mM).

0.1 mM EDTA

0.1–0.12 mM riboflavin (fresh, light-sensitive)

0.75–1.5 mM NBT (prepare stock in buffer, protect from light)

10 mM methionine (electron donor)

L-cysteine or other modifiers not generally used

96-well plate (photochemical) assay (adaptation of classic method):

1. Prepare reaction mix (per well final volume 300 µL):

50 mM phosphate buffer pH 7.8: adjust to reach 300 µL final.

13 mM methionine (final ~13 mM) — add from stock.

0.1 mM EDTA

0.12 mM riboflavin

0.75 mM NBT

2. Add appropriate dilution of sample (e.g., 10–20 µL) to each well; include blank (no enzyme) and standard (known SOD) controls.

3. Illuminate plate under fluorescent light for 10–15 min (time optimized to produce a robust signal in blanks). Read absorbance at 560 nm (or 530–560 nm depending on plate reader).

4. Calculate % inhibition:

\% \text{inhibition} = \frac{A_{\text{blank}} - A_{\text{sample}}}{A_{\text{blank}}} \times 100

Xanthine-xanthine oxidase method:

Generates O2•− enzymatically; measure inhibition of cytochrome c reduction at 550 nm. This method provides kinetic measurement and is less light-sensitive.

Notes / caveats:

Ensure linearity — dilute extracts to be within the linear inhibition range (0–50%).

Include cyanide/KO2 controls if distinguishing Cu/Zn-SOD from Mn-SOD (KCN inhibits Cu/Zn SOD).

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3) Glutathione (GSH) — options

You probably mean reduced glutathione (GSH) and/or glutathione peroxidase (GPx). I give both common choices:

A — Total GSH (reduced GSH + GSSG) by enzymatic recycling (Tietze) (recommended for sensitivity)

Principle: DTNB (Ellman’s reagent) reacts with GSH to form TNB (measured at 412 nm) while glutathione reductase and NADPH recycle GSSG back to GSH — so TNB production is amplified and rate is proportional to total glutathione. This is highly sensitive and commonly used.

Reagents:

100 mM phosphate buffer pH 7.5 (or kit buffer)

DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) in buffer (final working conc. ~0.5–1.0 mM)

Glutathione reductase (e.g., 0.5–1 U/mL final)

NADPH stock (0.5–1 mM final)

GSH standards (prepare serial dilutions, e.g., 0–10 µM to 0–100 µM range depending on sample)

Microplate assay (96-well):

1. Prepare reaction mix per well (200 µL final): buffer + DTNB + glutathione reductase. Prewarm to 25 °C.

2. Add sample (e.g., 10–20 µL) or standards.

3. Start reaction by adding NADPH (e.g., 20 µL of 1 mM to reach final ~0.1 mM).

4. Read increase in A412 every 30–60 s for 2–5 min. Use the initial linear slope to determine GSH concentration from standard curve (rate or end-point depending on kit).

5. If you need GSSG separately, block GSH with 2-vinylpyridine or N-ethylmaleimide (NEM) and measure GSSG after derivatization per protocol, then compute GSH by difference.

Notes:

Deproteinize samples prior to assay (e.g., 5% MPA or 10% TCA), neutralize supernatant before assay (follow protocol), or use kit buffers that account for acid extracts.

B — Reduced GSH by Ellman’s DTNB direct method (less sensitive than recycling)

Principle: DTNB reacts stoichiometrically with free thiols; measure A412. Better for samples with higher GSH. For biological samples, enzymatic recycling (above) is preferred because of higher sensitivity and specificity.

C — Glutathione Peroxidase (GPx) activity (if that’s what you need)

Principle (coupled assay): GPx reduces peroxides using GSH → GSSG, glutathione reductase then reduces GSSG back to GSH using NADPH — monitor decrease in NADPH absorbance at 340 nm. This is a standard coupled assay to measure GPx activity.

Reagents:

50 mM phosphate buffer pH 7.0, 1 mM EDTA

GSH (2 mM final)

Glutathione reductase (1 U/mL)

NADPH (0.2–0.3 mM)

Substrate: tert-butyl hydroperoxide (t-BuOOH) or H2O2 (careful with final conc.; e.g., 0.25–1 mM)

Sample (diluted so decrease in A340 is linear)

Procedure:

1. In cuvette, mix buffer + GSH + GR + NADPH. Pre-warm to 25–37 °C and record baseline absorbance at 340 nm.

2. Add sample to start reaction; after stable baseline, add peroxide substrate to initiate GPx activity.

3. Follow decrease in A340 over time (linear portion). Use ΔA340/min and NADPH extinction coefficient (ε340 = 6.22 mM⁻¹cm⁻¹) to calculate nmol NADPH oxidized per min. Convert to U/mg protein (1 U = 1 µmol substrate reduced/min).

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D. Worked examples & calculation templates

1. Catalase (cuvette): ΔA240/min = 0.05; ε = 39.4 M⁻¹cm⁻¹; pathlength 1 cm.

Rate (M/min) = 0.05 / 39.4 = 0.001268 M/min = 1.268 mmol/L/min = 1.268 µmol/mL/min.

If cuvette volume 3.0 mL → µmol/min in cuvette = 1.268 µmol/mL/min × 3.0 mL = 3.804 µmol/min.

If protein in assay = 0.2 mg → activity = 3.804 µmol/min / 0.2 mg = 19.02 µmol/min/mg = 19.02 U/mg.

2. GPx: ΔA340/min = 0.02; ε340 = 6.22 mM⁻¹cm⁻¹ → rate NADPH consumption = 0.02 / 6.22 = 0.003216 mM/min = 3.216 µmol/L/min. Multiply by cuvette volume to get µmol/min and normalize to mg protein as above.

(Always check pathlength in microplates — convert absorbance to equivalent 1-cm pathlength or use pathlength correction.)

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E. Troubleshooting & tips

Dilution: Crude extracts often have high activity — run serial dilutions to find a linear range.

H2O2 standardization: Freshly prepare H2O2 working solution; measure absorbance at 240 nm to standardize concentration (H2O2 degrades). Many papers use ε = 39.4 M⁻¹cm⁻¹ (Aebi) but values between ~36–44 are reported—choose and state the coefficient you used.

Interferences: Colored extracts or turbidity can interfere (especially at 240 and 412 nm). Use appropriate blanks (sample + buffer without substrate, buffer + substrate without sample).

Protein assay compatibility: Choose a protein assay compatible with buffer components (e.g., detergent may interfere with Bradford). Use BCA when detergents present.

Temperature: Enzyme activities are temperature-dependent — perform assays at consistent temperature (25 °C or 37 °C) and state it.

Storage: Freeze aliquots at −80 °C; avoid repeated freeze–thaw cycles that reduce activities.

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F. Safety and quality

Handle H2O2 and organic peroxides with care (oxidizer). Use PPE and work in fume hood when making concentrated solutions.

Dispose of reagents (e.g., DTNB, TNB, organic peroxides) per institutional hazardous waste rules.

Include assay controls and replicate measurements (technical triplicates and biological N) for statistics.

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Key references (classic methods & reviews)

Catalase (Aebi / H2O2 decomposition): Aebi-type spectrophotometric assays; see methods and examples.

SOD (Beauchamp & Fridovich; NBT reduction; xanthine oxidase options).

Glutathione (Tietze enzymatic recycling; Ellman DTNB; GPx coupled assay).