METMYOGLOBIN ASSAY
The metmyoglobin assay is a rapid
method which provides the degree of antioxidant protection possessed by an individual species. Original descriptions
of the method can be found in:
1. Halliwell, B. (1987) FASEB J.
1, 358-364
2. Southorn, P.A. (1988) Mayo
Clin. Proc. 63, 390-408
3. Miller, N.J., Rice-Evans, C.,
Davies, M.J., Gopinathan, V., and Milner, A. (1993) Clin. Sci. 84, 407-412
Our application to tannins is
described in:
4. Hagerman, A.E.; Riedl, K.M.;
Jones, G.A.; Sovik, K.N.; Ritchard, N.T.; Hartzfeld, P.W.; Riechel, T.L. High
molecular weight plant polyphenolics (tannins) as biological antioxidants. J.
Agric. Food Chem. (1998) in press.
The reagents for the metmyoglobin
method can be purchased as a kit from Randox. It is much less expensive to
prepare them yourself as follows:
Reagents
PBS Buffer (0.005 M Phosphate,
0.145 M NaCl)
A: Dissolve 0.68 g KH2PO4 and 8.5
g NaCl in 1 L of distilled water
B: Dissolve 1.14 g K2HPO4 x H2O
and 8.5 g NaCl in 1 L of distilled water
Mix the two solutions together to
give a pH of 7.4 (approximately 770 mL of A and 2 L of B). (Final concentration
in assay is 5 mM).
Myoglobin (initial concentration
between 1 mg myoglobin/mL PBS to 5 mg/mL). The initial concentration doesn't
matter since the solution will be diluted as it runs through the column. The myoglobin
is purchased from Sigma (# M-0630).
K3Fe(CN)6 (0.24
mg K3Fe(CN)6 in 1 mL water)
2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid (0.8 mg ABTS in 1 mL PBS buffer). The ABTS is purchased form Sigma (#
1888). (Final concentration in the assay is 610 μM).
Trolox Standard (0.1564 g Trolox in 250 mL
PBS buffer). The Trolox is purchased from Aldrich (# 23,881-3). If the solid
does not dissolve, sonicate gently. (Final concentration in the assay is 2.5
mM).
Tannin Sample (1 mg tannin/mL of water). Make
sure the actual concentration used is recorded in the laboratory notebook for
the calculations. Prepare 1:10 dilutions.
Hydrogen Peroxide Dilute the 30 % w/v stock
solution as follows:
#1 1 part (H2O2) plus 9
parts water
#2 1 part (#1) plus 9 parts water
#3 1 part (#2) plus 9 parts water
#4 1.7 parts (#3) plus 8.3 parts PBS buffer
(Final concentration in the assay is 250 μM).
Sephadex G-10-120 column (Height of 20 cm and
Diameter of 1 cm). Equilibrate the column with the PBS buffer before using.
This column can be prepared and reused indefinately, stored at room temperature.
Metmtoglobin. Mix equal volumes of myoglobin
and K3Fe(CN)6 solutions. Run sample through column and
collect the second fraction where the brown color starts to come off the
column. The first fraction is just buffer and the third fraction is yellow
containing the K3Fe(CN)6. Read the Abs @ 490 nm of the second
fraction. Adjust the solution with buffer to give an absorbance reading of
0.147 so that the final concentration of metmyoglobin in the assay will be 6.1 μM).
The equations used to calculate the amounts of the various forms of the
myoglobin are found in Reference #3.
Procedure
Set up an appropriate number of 0.65 mL
microfuge tubes to run each sample in duplicate or triplicate.
Add the 20 μL of the sample (water, standard,
or tannin), 250 μL of metmyoglobin and 250 μL of ABTS to the tubes. Vortex the
tubes.
Using a microcuvette (1 mL), blank the
spectrophotometer at 600 nm with water.
Read the absorbance of each sample and record
as Abs1.
Add 100 μL of the hydrogen peroxide substrate
to the tubes. After exactly 3 minutes, read the absorbance and record as Abs2.
(Hint: Add the substrate to four tubes at 30 second intervals. This provides
enough time to read each sample at exactly three minutes).
Subtract Abs1 from Abs2 for each sample. The
typical change in absorbance for the control (water as sample) is 0.296.
Calculations
The amount of putative antioxidant required
to supress absorbance of the ABTS radical cation by 50% is compared to the
amount of Trolox required for 50% supression in order to compare potency of
various antioxidants. A Trolox standard curve is run with each set of samples
because there is substantial day-today variability in the assay.
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