Premier Products for Superior Life Science Research

NWLSSTM
Superoxide Dismutase
Activity Assay
Product NWK-SOD02
For Research Use Only

5131 NE 94th Avenue, Suite 201
Vancouver, WA 98662
Phone 360-449-3091 or Toll Free: 888-449-3091
Fax 360-449-3092
E-mail: nwsales@nwlifescience.com
nwksod02rev010907

Simple assay kit for quantitative measurement of superoxide
dismutatse enzyme activity in biological samples.

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Table of Contents

SectionPage

Introduction

3

Intended Use

3

Test Principle

3

General Specifications

4Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

4Warnings, Limitations, Precautions

4

Storage Instructions

4

Assay/Instrument Preparation

5

Reagent Preparation

5

Sample Handling/Preparation

5Assay Protocol

7

Data Analysis

8

Performance Details

10

References

10

Statement of Limited Warranty

11

End-User Notes

11

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Statement of Limited Warranty:
Northwest Life Science Specialties, LLC (NWLSS) makes no guarantee ofany kind, expressed or implied, that extends beyond the description of thematerial in this kit, except that they will meet our specifications at the timeof delivery. Customer's remedy and NWLSS’ sole liability is limited to, at
NWLSS’ option, refund of the purchase price, or the replacement ofmaterial not meeting our specification. By acceptance of our product,
customer assumes all liability and will indemnify and hold NWLSS harmless
for the consequence of this product’s use or misuse by the customer, its
employees, or others. Refund or replacement is conditioned of customer
notifying NWLSS within twenty-one (21) days of the receipt of product.
Failure to give notice within 21 days shall constitute a waiver by thecustomer of all claims hereunder with respect to said product.
Notes:

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Performance Details:
Stability
All unopened reagents are stable until the expiration
date stated on the package label when stored at 2-8°C.
Sensitivity:

The lower limit of detection is0.2 U SOD/mL in the reaction mixtureor,
5 U SOD/mL sample added to reaction mixture.

Linearity:

Linearity is maintained up to 60 U SOD/mL sample.

Assay Precision:

Coefficient of variation (CV)at 40 U SOD/mL sample

Recovery:Recovery of spiked SOD in RBC human seminal
plasma was calculated as 90% using this assay.

Intra12%Inter
8%References:
1. Flohe L. & Otting F., Superoxide Dismutase Assays, Methods inEnzymology 105:93-104 (1984).
2. Tarpey M.M. & Fridovich I., Methods of Detection of Vascular Reactive
Species – Nitric Oxide, Superoxide, Hydrogen Peroxide and Peroxynitrite,
Circulation Research 89:224-236 (2001).
3. Martin J.P. Jr., Dailey M. & Sugarman E., Negative and Positive Assays ofSuperoxide Dismutase Based on Hematoxylin Autoxidation, Arch. Biochem.
Biophys. 255:329-336 (1987).
4. Landmesser U., Merten R., Spiekermann S., Büttner K., Drexler H. andHornig B., Vascular Extracellular Superoxide Dismutase Activity in Patients
With Coronary Artery Disease—Relation to Endothelium-Dependent
Vasodilation, Circulation 101(19):2264-2270 (2000).

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Page 3Introduction:
Superoxide radicals are involved in many physiological and pathophysiological processes. They are produced as a by-product of respiratory electrontransport and cytochome P450 reactions. Activated neutrophils and macrophages can also produce a large amount during oxidative burst. Superoxide radical can react with NO at very fast rate to form peroxynitrite (ONOO-)
a very powerful oxidant that has been shown to damage DNA, protein andother biological molecules. Removal of superoxide is a necessary step incellular defense against these damages.
The enzyme, superoxide dismutase (SOD), rapidly decomposes superoxideanion into hydrogen peroxide and oxygen. Since its initial discovery byMcCord and Fridovich in 1969, SOD has been found to be ubiquitous in all
aerobic organisms from microbes to humans. Four types of SOD have been
identified on the basis of their metal cofactors and distribution. The copperzinc form (Cu/ZnSOD) is most common with a primary distribution in thecytoplasm of eukaryotic cells. Another copper zinc form, extra-cellular(ecSOD), has more recently been identified. This SOD is often associatedwith vascular tissues but can also be found in plasma, lymph, synovial fluid,cerebrospinal fluid (CSF) and elsewhere. The manganese form (MnSOD) isgenerally associated with the mitochondria of aerobic organisms. The ironform (FeSOD) is found predominantly in prokaryotes. The relationship between SOD and various human diseases has now been well documented
and measurement of SOD activity has become commonplace in many research model systems. One unit SOD activity is defined as the amount ofenzyme that will inhibit the rate of cytochrome c reduction by half under
specific conditions.
Intended Use:
The NWLSS™ Superoxide Dismutase Activity Assay provides a simple, rate
method for determining SOD activity in tissue homogenates, cell lysates &other biological fluids or extracts where forms of SOD might be present.
Test Principle:
The NWLSS™ NWK-SOD02 method is based on monitoring the autooxidation rate of hematoxylin as originally described by Martin J. P., Jr et al
1987, with modifications to increase robustness and reliability. Briefly, inthe presence of SOD enzyme at specific assay pH, the rate of auto-oxidation
is inhibited and the percentage of inhibition is linearly proportional to theamount of SOD present within a specific range. Sample SOD activity isdetermined by measuring ratios of auto-oxidation rates in the presence andabsence of the sample and expressed as traditional McCord Fridovich“cytochrome c” units. The basic principal of the assay is shown schematically by the following equation:
SOD
O2 + HTH2
H2O2 + HT (Abs560↑)
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General Specifications:
Format:Cuvette or 96 Well Microplate
Number of Tests: 30 Cuvette or 96 Microplate
Specificity:
Cu/Zn, Mn and Fe Superoxide Dismutase
Sensitivity:
LLD =0.3 U/mL in Reaction Mix
5 U/mL in Sample Added to Reaction Mix
Kit Contents:
Assay Buffer
Hematoxylin
Sample Dilution Buffer

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Data Analysis:
1. Determine auto-oxidation rates as illustrated in Figure 2. The rate ofreaction can be obtained by regression of the uninhibited linear portion of
the rate curves, as shown in Figure 2. The equation for this linear
regression is:
Y = aX + b,

1 X 30 mL
1 vial
1 X 30 mL

Required Materials Not Provided:
Disposable semi-micro cuvettes (1.0 mL) or transparent microplate strips.
Microcentrifuge tubes.
Plastic or glass bottles.
Pipettors, adjustable 0.0 – 1.0 mL.
Disposable pippette tips.
Required Instrumentation:
Spectrophotometer (cuvette spectrophotometer, multi-cuvette changer withtemperature control module recommended but not absolutely required) orplate reader.
Note:
The assay is sensitive to temperature. When a cuvette spectrophotometer
without a temperature module is used, the experiment must be carried out
in an environment where the temperature is constant (22 – 27°C). When
there is a noticeable change of temperature, it is necessary to run samples
and a blank (Assay Buffer) back-to-back to minimize temperature effect. For
plate readers, this requirement is less restrictive since assays are carriedout in parallel.
Warnings, Limitations, Precautions:Individual components may be harmful if swallowed, inhaled or absorbed
through the skin. Contact should be minimized through the use of gloves
and standard good laboratory practices. If contact with skin or eyes occurs, rinse the site immediately with water and consult a physicianStorage Instructions:
Upon receipt, store the reagents at 2-8ºC. Do not use components beyond
the expiration date printed on the label.

where a is the decomposition rate or (∆Abs560nm/min).
A less preferred way to obtain the rate is to select two data points in therate curve and to calculate rates according to following equation:
Rate (∆Abs560nm/min) = (Y2 – Y1)/(X2 – X1).
Calculate average rates from duplicate or triplicate assays.
2. Obtain the ratio of sample rate and blank rate using the equation
Ratios/b = Rates/Rateb
where Rates is the average auto-oxidation rate of a sample, and Rateb is theaverage autooxidation rate of the blank.
3. Calculate percentage for SOD inhibition of baseline (blank) reaction rateas:
% Inhibition = (1 - Ratios/b) * 100%
4. SOD activity measured using the classical “cytochrome c reduction”
method was found to be approximately 1.25 times measurable percent
inhibition of hematoxylin auto-oxidation as established through in-house
trials. The SOD concentration in classical McCord Fridovich “cytochrome creduction” unit activity is therefore calculated as:[SOD U/mL]s = 1.25 * (% Inhibition)
This value is representative of the SOD activity in the sample added to thereaction mixture not the final concentration in the reaction mixture.
5. The SOD concentration in the original sample (“cytochrome c reduction”U/mL) is calculated using the equation:[SOD U/mL]os = [SOD U/mL]s * (Sample Dilution Factor)

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Distinguishing types of SOD (continued):
Figure 1 illustrates theeffect of KCN on
measurable Cu/Zn SOD
activity using thehematoxylin assay
method.
Addition of 1 mM KCN isshown to block Cu/Zn
mediated inhibition ofhematoxylin autooxidation.

Data Analysis:
Typical rate curves of standards with 0 U/mL and 40 U/mL are shown
below in Fig. 2.
Figure 2: Rate curves ofhematoxylin autooxidation in the absence
and presence of
CuZnSOD. SOD units asshown represent theconcentration of enzyme
added to the reaction
mixture. The experiment
was performed at room
temperature (25°C)
without a constanttemperature cuvetteholder.
In the 40 U/mL sample shown above, there is a slight inhibition of the reaction during the initial minute of recording. This is normal and is often
observed with samples of higher SOD concentration or when interfering
substances might be present as previously described in Notes 1 & 2 in thesample preparation section.

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Assay/Instrument Preparation:
Spectrophotometer (Cuvette Assay) Setup
1. Set measurement temperature at 25°C. Otherwise, carry out theexperiment at room temperature. When there is a noticable change oftemperature, it is necessary to run sample and a blank (Assay Buffer) backto-back to minimize temperature effect.
2. Zero absorbance at 560 nm using dH2O.
Plate Reader (Microplate Assay) Setup
1. Set measurement temperature at 25°C. Otherwise, carry out experimentat room temperature.
Reagent Preparation:
Allow the kit warm up to room temperature completely before using. It isacceptable to place reagents at room temperature the night before theexperiment. However, do not reconstitute the Hematoxylin reagent until
actual day of use since the reagent in liquid form is only stable for 6 hours
at room temperature.
Assay Buffer:
Supplied Ready to Use. However, please shake or invert the bottle, open to
air and repeat four (4) more times before use to saturate with O2.
Hematoxylin Solution:
Reconstitute hematoxylin as supplied with 1.2 mL (1200 µL) of dH2O. At
room temperature, it should be used within 6 hours. If only a portion of thereconstituted hematoxylin is needed in one experiment, the excess solution
should be immediately stored at –20°C (frozen) for future use.
Sample Dilution Buffer:
Supplied Ready to Use.
Sample Handling/Preparation:
Purification & Extraction:
Some samples may need to be dialyzed, extracted, or purified throughchromatography to remove interfering substances that completely inhibit
hematoxylin auto-oxidation. These purification steps are not necessary
when inhibition time is less than 7 minutes because hematoxylin autooxidation rate is linear for the first 10 minutes. Samples typically need tobe diluted with Sample Dilution Buffer before assay. For example, seminal
plasma samples can be assayed directly after 1/10 dilution.

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Page 6To Order Call: 1-888-449-3091Sample Handling/Preparation (continued):
Sample Dilution:
The following are general dilution guidelines in order to achieve the mostsensitive linear assay range between 0 and 50 U/mL:
Tissues (10% homogenates):

1/2

Red Blood Cell lysate:(after chloroform/ethanol extraction to remove hemoglobin)

1/2

Seminal Plasma:

1/10

Blood Plasma or Serum:
Normal human plasma and serum SOD activities are typically quite low(< 5 U/mL) and are therefore below the level of detection of this assay.
However, concentrations may be elevated to detectable levels undervarious experimental or treatment conditions.
Samples from at-risk patient groups or from abnormal experimental
conditions may require a different dilution factor.
Note 1:
Linearity of the assay is lost at high SOD concentration. Therefore, when
the auto-oxidation rate (A560 nm/min) of a sample is less than 50% of theauto-oxidation rate at zero SOD concentration (blank rate), it is important
not to use the rate to calculate SOD concentration in the sample. Instead,adjust the dilution factor, make a new dilution and repeat the assay.
Note 2:The auto-oxidation of hematoxylin is a self-catalyzed reaction and the rate
is linear for about 10 minutes. This reaction can be inhibited by thiols(including protein thiols), ascorbic acid, and other antioxidants. In mostcases however, the inhibition lasts only several minutes, especially at 1/5
or higher dilution factors. In these cases, samples can be assayeddirectly by the rate assay without prior treatment. In cases where excessive inhibition is noted and/or it is not possible to dilute the sample prior
to assay, samples may be treated by dialysis, extraction and/or
chromatography to remove interfering substances before the assay.
Sample Stability
Tissues:
We recommend storage of whole tissues at <-70°C with homogenization
taking place the day of assay.
Cells:For best analyte stability, we recommend storage of whole cell pellets at<-70°C with disruption via homogenization or sonication taking place on
the day of assay.
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Assay Protocol:
Cuvette Assay
1. Add 920 µL of Assay Buffer to each cuvette for assay.
2. Add 40 µL of Assay Buffer (for blank) or 40 µL of Sample. Mix andincubate for two (2) minutes.
3. Add 40 µL Hematoxylin Reagent to start the auto-oxidation reaction.
4. Mix quickly and immediately begin recording the absorbance at 560 nm
every 10 seconds or smaller time interval for at least 5 minutes.
Note:Reaction rate should be linear for approximately 10 minutes. Undialyzed
samples may need longer recording time.
Microplate Assay
1. Record sample/standard layout.
2. To each well used for testing, add 230 µL of Assay Buffer.
3. Next add 10 µL of Assay Buffer (for blank) or 10 µL Sample. Shake tomix and incubate for 2 minutes.
4. Add 10 µL of Hematoxylin Reagent to begin reaction. Preferably, use amulti-channel pipette.
5. Mix quickly using the instrument’s shaker function and immediately
begin recording the absorbance at 560 nm every 10 seconds or smaller
time interval for at least 5 minutes.
Note:The reaction rate should be linear for approximately 10 minutes. Undialyzed
samples may need longer recording time.
Employing a Control
To minimize inter-assay variation in ongoing studies, it is recommended thata control sample is prepared, aliquoted and frozen at –20°C or below. Thiscontrol sample should be assayed each time along with other samples. Finalresults should be normalized with the control sample.
Distinguishing types of SOD
EC-SOD activity can be determined specifically after chromatography toeliminate other forms of SOD’s.
To differentiate CuZnSOD and MnSOD activity separately in samples
containing both enzymes, potassium cyanide (KCN at 1 to 2 mM) can be used
to inhibit CuZnSOD but not MnSOD. Simply add the appropriate amount ofKCN (under a hood to avoid possible HCN gas exposure) to a specific volumeof assay buffer. The activity of a sample measured using the original assay
buffer represents total SOD activity, and the result when using KCN
containing assay buffer represents only MnSOD activity.

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