Metmyoglobin Method 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: #11 part(H2O2)plus 9 parts water #2 1 part (#1)plus 9 parts water #31 part (#)plus 9 parts water #4 1.7 parts(#3)plus 8.3 parts PBS buffer (Final concentration in the assay is 250 uM) 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 uM).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 uL of the sample(water,standard,or tannin).250 uL of metmyoglobin and 250 uLof 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 Absl Add 100 uL 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 Absl from Abs2 for each sample.The typical change in absorbance for the control(water as sample)is 0.296
Metmyoglobin Method 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
Metmyoglobin Method 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-to day variability in the assay. Return to Tannin Chemistry Home Page
Metmyoglobin Method 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. © Ann E. Hagerman 1998, 2002. This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author. Return to Tannin Chemistry Home Page
Chemical preparation of ABTS radical cation Described by Re et al.(Re R,Pellegrini N,Proteggente A,Pannala A,Yang M,Rice-Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Rad Biol Med1999:26(9-10):1231-7). ABTS is prepared in the desired buffer at 3.84 mg/mL(7.01 mM).That solution (15 mL)is mixed with i mL of KS,Os(10.6 mg/mL,39.2 mM)prepared in the same buffer.The mixture is incubated at room tem perature in the dark for 16 hou rs,and is then diluted with buffer to obtain the working solution of radical cation.This method has fewer side reactions and is much The max of the ABTS radical cation is 734 nm,and there is a linear relationship between radical cation concentration and absorbance through at least an absorbance of 2.0.The extinction coefficient E is 12867 Mcm and is independent of pH at pH values 3-7.4
Chemical preparation of ABTS radical cation Described by Re et al.( Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Rad Biol Med 1999; 26(9-10):1231-7). ABTS is prepared in the desired buffer at 3.84 mg/mL (7.01 mM). That solution (15 mL) is mixed with 1 mL of K2S2O8 (10.6 mg/mL, 39.2 mM) prepared in the same buffer. The mixture is incubated at room temperature in the dark for 16 hours, and is then diluted with buffer to obtain the working solution of radical cation. This method has fewer side reactions and is much The λmax of the ABTS radical cation is 734 nm, and there is a linear relationship between radical cation concentration and absorbance through at least an absorbance of 2.0. The extinction coefficient Ε is 12867 M-1 cm -1 and is independent of pH at pH values 3-7.4
ic aion ecof in complexes As described in Riedl,K.M.;Hagerman,A.E.Tannin-protein complexes as radical scavengers and radical sinks.Journal of Agricultural and Food Chemistry 2001 49,4917-4923. Quenching:Protein and procyanidin are mixed in 1.5 mL cuvettes by combining 450 uL 734 nm.The decolorization reaction was initia ted by adding and immediately mixing by inversion and p condions.thea thbe actos aboutpectrophotometer.Under these acing the mixture in th Capacity:To maintain a large excess of radical,163 nmoles(150 uL of 1.09 mM ABTS")is added to 900 uL of solution containing 0.203 nmoles(I ug)of PC at the desired pH.The starting A7s is about 2.0.The absorbances of both tannin-free control,and of PC-containing samples,are monitored during the reaction to account for the spontaneous decolorization of corresponds to the diffe mple.The extinction coefficient of the ABTScan be used to calculate the number of moles of radical scavenged per mole of PC
ABTS radical cation quenching by tannin-protein complexes ABTS radical cation decolorization capacity As described in Riedl, K.M.; Hagerman, A.E. Tannin-protein complexes as radical scavengers and radical sinks. Journal of Agricultural and Food Chemistry 2001 49, 4917-4923. Quenching: Protein and procyanidin are mixed in 1.5 mL cuvettes by combining 450 µL protein solution (0-180 µg/mL) with 450 µL of PC solution (4-8 µg/mL). The solution was inverted to mix, and incubated for 10 min at room temp before zeroing the spectrophotometer at 734 nm. The decolorization reaction was initiated by adding 100 µL of 65 µM ABTS+•, and immediately mixing by inversion and placing the mixture in the spectrophotometer. Under these conditions, the A734 at the beginning of the reaction was about 0.7. Capacity: To maintain a large excess of radical, 163 nmoles (150 µL of 1.09 mM ABTS+•) is added to 900 µL of solution containing 0.203 nmoles (1 µg) of PC at the desired pH. The starting A734 is about 2.0. The absorbances of both tannin-free control, and of PC-containing samples, are monitored during the reaction to account for the spontaneous decolorization of ABTS+•, which increases as pH is increased. The amount of ABTS+• scavenged at any time corresponds to the difference in absorbance between the control and the PC sample. The extinction coefficient of the ABTS+• can be used to calculate the number of moles of radical scavenged per mole of PC
Protein Precipitation PROTEIN PRECIPITATION BY TANNINS Although the ability to precipitate protein is the defining characteristic of tannins,the detailed chemistry of the interaction is still only partly understood.It is now clear that both the type of interaction and the strength of interaction are dictated by both the chmeistry of the tannin and the chemistry of the protein In addition,the interaction is influenced by reaction conditions including temperature,pH,solvent compostion,and tannin:protein ratio Review articles which summarize the current knowledge of tannin-protein interactions have been published by Hagerman;Haslam;and Butler.Recent comparisons of condensed to hydrolyzable tannins are useful (Hagerman,A.E.;Rice,M.E.;Ritchard,N.T.J.Agric.Food Chem.1998,in press). Numerous methods for determining tannins which take advantage of the interaction between tannin and protein have been devised.The radial diffusion method is convenient;the protein-precipitable phenolics method is robust and gives excellent results with many types of tannins;the radiochemical method is very sensitive but requires specialized equipment;a similar but less sensitive method can be done with blue dye-labeled protein.Qualitative assessments of binding can be made with electrophoretic methods A method for assessing phlorotannin-protein interactions has also been described. Return to Tannin Chemistry Home Page
Protein Precipitation PROTEIN PRECIPITATION BY TANNINS Although the ability to precipitate protein is the defining characteristic of tannins, the detailed chemistry of the interaction is still only partly understood. It is now clear that both the type of interaction and the strength of interaction are dictated by both the chmeistry of the tannin and the chemistry of the protein. In addition, the interaction is influenced by reaction conditions including temperature, pH, solvent compostion, and tannin:protein ratio. Review articles which summarize the current knowledge of tannin-protein interactions have been published by Hagerman; Haslam; and Butler. Recent comparisons of condensed to hydrolyzable tannins are useful (Hagerman, A.E.; Rice, M.E.; Ritchard, N.T. J. Agric. Food Chem. 1998, in press). Numerous methods for determining tannins which take advantage of the interaction between tannin and protein have been devised. The radial diffusion method is convenient; the protein-precipitable phenolics method is robust and gives excellent results with many types of tannins; the radiochemical method is very sensitive but requires specialized equipment; a similar but less sensitive method can be done with blue dye-labeled protein. Qualitative assessments of binding can be made with electrophoretic methods. A method for assessing phlorotannin-protein interactions has also been described. © Ann E. Hagerman 1998, 2002. This material may be copied for use within a single laboratory but cannot be copied for distribution or publication without permission of the author. Return to Tannin Chemistry Home Page