METMYOGLOBIN ASSAY (TANNIN)

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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