ANALYTICAL
BIOCHEMISTRY
Staining
72,428~432
for Lipoxygenase BENITO 0. DELUMEN
Campbell
Institute
For Food Camden,
(1976)
Activity AND
in Electrophoretic
STANLEY
Gels
J. KAZENIAC
Research, Basic Research New Jersey 08101
Department,
Received September 22, 1975; accepted December 2, 1975 A staining procedure for lipoxygenase in electrophoretic gels based on the oxidation of a dye, 3,3’-dimethoxybenzidine hydrochloride, by hydroperoxides is described. The specificity of the stain to the dye was established by enzyme inhibition, by use of nonsubstrates, by enzyme heat-inactivation and by proportionality of the staining intensity to the amount of enzyme as observed visually. Cyanide is included with the substrate to inhibit heme-catalyzed oxidation. The gels which had little or no background staining have been stored for 18 months at 4°C without any color deterioration. As little as 220 units of soybean lipoxygenase and 5 ~1 of a 30% tomato homogenate gave detectable staining.
The detection of lipoxygenase activity in electrophoretic gels is becoming increasingly important as isoenzymes have been found present in different plant tissues (1,2). Grossman et al. (3) used Koch’s thiocyanate test for peroxides (4) to localize lipoxygenase activity in cellulose acetate gels, while Guss et al. (5) employed acidic potassium iodide on polyacrylamide gels impregnated with starch. With both methods, we experienced background staining which made detection of low activity samples difficult. Furthermore, the color development continued upon storage so that a permanent record was not possible unless photographs were taken immediately. Gardner et al. (6) reported a high percentage of failure in stain development with Guss’ procedure due possibly to a high threshold requirement for hydroperoxide to release I2 in the gel. We report in this study the use of a dye, 3,3’-dimethoxybenzidine hydrochloride (DBH) to visualize hydroperoxide bands in polyacrylamide gels with practically no background staining. The method is relatively sensitive, and the gels had been stored for 18 months at 4°C without color deterioration. MATERIALS
AND METHODS
Gel ElectrophoresiA
Electrophoresis on polyacrylamide gels was performed essentially according to the method of Davis (7) except that no stacking gel was used. Samples (5 to 20 ~1) were added directly on top of the gel and mixed with 5 ,!A of tracking dye (bromphenol blue, 0.5% in H20) and 10 ~1 of 40% sucrose added previously. Electrophoresis was carried out at pH 8.8 428 Copyright 0 1976 by Academic Pras. Inc. All rightc of reproduction in any form reserved.
STAINING
FOR LIPOXYGENASEACTIVITY
429
(0.38 M, Tris-HCI) on 7% polyacrylamide with acurrent of 2.5 to 3 mA per tube for 3 hr at 4°C. Protein bands were stained with Coomassie brilliant blue. Lipoxygenase
Staining
Immediately after electrophoresis, the gels were rinsed with distilled water and placed in 1.6 x 15 cm tubes containing buffered linoleic acid (2 mM) and inhibitors when necessary. Reaction was carried out at room temperature for 30 min. The gels were rinsed with distilled water, transferred to test tubes containing 0.1% DBH (Eastman Kodak Co.), and stained overnight at room temperature. The dye was washed off and the gels were stored in water. Lipoxygenase
Sources
Crystalline soybean lipoxygenase (Sigma Chemical Co.) containing 21.600 units/mg solids was used. Homogenates (30-50%) of tomatoes were prepared by blending fruits in a Waring Blender for 60 set at 4°C. The tomato homogenates were stored in liquid nitrogen until ready to use. Substrate
Preparation
Linoleic acid (Grade III, 99% pure, Sigma Chem. Co.) was prepared by dispersing 0.62 ml of acid in 50 ml of water containing two drops of Triton X-100 with a Branson sonifier for 5 min at 0°C. It was diluted with water to give a stock solution of 20 mM. The working solution was prepared by dilution of the stock to 2 mM either with pH 8.6,O. 1 M Tris-HCl or pH 5.8,O. 1 M citrate-phosphate buffer. Immediately before using, the substrate was oxygenated for 20-30 min at room temperature. RESULTS
AND
DISCUSSION
Gels that contained 20 pg (432 units), 40 pg (864 units) and 80 pg (1728 units) of soybean lipoxygenase exhibited increasingly darker reddish brown stains as observed visually while the inactivated control (lOO’C, 10 min) showed no band at all (Fig. 1). Soybean lipoxygenase containing 220 units is detectable with this technique, compared to 500 units reported by Guss (5). Five microliters of tomato homogenate showed sufficient staining. Staining of tomato was more effective when the fatty acid was incubated at pH 8.8, possibly due to greater solubility of the substrate, although we found that tomato lipoxygenase had a pH optimum around 5.8. No staining was evident when the gels were incubated at pH 8.8 followed by stepwise incubation at pH 5.8 according to the procedure of Gardner (6). Soybean lipoxygenase has been shown to have at least two isoenzymes
430
DE LUMEN
AND KAZENIAC
FIG. 1. Effect of soybean lipoxygenase concentration on intensity of staining. (1) Coomasie blue staining for protein, (2) 80 fig, (3) 40 pg, (4) 20 pg. and (5) heat inactivated control. The dark bands at the bottom ofgels 2 through 5 suggest oxidation of the substrate and of the dye by ammonium persulfate.
(3,8). At a concentration of 10d3 M, nordihydroguaiaretic acid (NDGA) prevented staining of both bands, whereas only the second band stained weakly at a concentration of lop4 M. No bands were evident when linoelaidic and oleic acids were used as substrates. These fatty acids do not have the cis, cis-1, 4-pentadiene system for which lipoxygenase is specific. Sodium cyanide ( low3 M), an inhibitor of heme-catalyzed peroxidation, did not inhibit staining in both soybean lipoxygenase and tomato homogenates. No experiments were done using heme proteins to produce hydroperoxides. Maier and Tappel (9) reported that heme-catalyzed oxidation of linoleic acid required an induction period of 24 hr at less than 7 mM
STAINING
FOR LIPOXYGENASE
ACTIVITY
FIG. 2. Isoenzyme patterns of tomato (2) and corncob (3). The protein pattern of tomato is shown in (I). The dark band in bottom of gel 3 suggests oxidation of the substrate and of the dye by ammonium persulfate.
linoleate concentration. Ah substrate concentrations used in these experiments were 2 mM. Lipoxygenase is a far more powerful catalyst than heme proteins (lo), and should be the predominant catalyst of lipid oxidation in plant tissues where heme proteins are relatively low in concentration. In spite of these observations, sodium cyanide (10e3 M) is included routinely with the substrate to inhibit any contribution of heme catalysts to the formation of hydroperoxides. As an application of this technique, we have been studying the fipoxygenase isoenzymes of tomato and other fruits. Isoenzyme patterns of tomato and corncob are shown in Fig. 2. Tomato and corncob hpoxygenases have at least four isoenzymes with very different distributions as compared with the two isoenzymes of soybean.
432
DE LUMEN
AND KAZENIAC
The best solvent for the dye was water, the solution having a pH of 3.5. Some precipitation was observed in aqueous DBH when the gel was introduced into the solution with pH increasing to about 8.6. No staining was observed with dilute acid solvents while dilute base precipitated out the dye. The gels had a clear background and they had been stored at 4°C for as long as 18 months without color deterioration. Storage of the gels in 7.5% acetic acid removed the stain. The sharpness of the stained bands may have been affected because a stacking gel was not used or because some diffusion of protein and hydroperoxide may have occurred during the 30 min reaction period at room temperature of the enzyme with linoleic acid. ACKNOWLEDGMENT We are grateful to Dr. Yu Bang Lee for his help in the electrophoresis during the initial stages of these studies.
experiments
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Pinsky, A., Grossman, S., and Trop, M. (1971)J. Food Sci. 36, 571. Hale, S., Richardson, T., Vonelbe, J. H., and Hagedom. D. J. (1969) Lipids 4, 209. Grossman, S., Pinsky, A., and Goldweitz, Z. (1971)Ann/. Biochem. 44, 642. Koch, R. B., Stem, B., and Ferrari, C. G. (1958)Arch. Biochem. Biophys. 78, 165. GUSS, P. L., Richardson, T., and Stahmann, M. A. (1967) Cereal Chem. 44, 607. Gardner, H. W., Christianson, D. D., and Kleiman, R. (1973) Lipids 8, 271. Davis, B. J. (1964)Ann. N. Y. Acad. Sci. 121, 404. Christopher, J., and Axelrod, B. (1971) Biochem. Biophys. Res. Commun. 44, 731. Maier, V. P., and Tappel, A. L. (1959) J. Amer. Oil Chem. Sot. 36, 8. Eriksson, C. (1975) J. Agric. Food Chem. 23, 126.
BIOCHEMISTRY
Staining
72,428~432
for Lipoxygenase BENITO 0. DELUMEN
Campbell
Institute
For Food Camden,
(1976)
Activity AND
in Electrophoretic
STANLEY
Gels
J. KAZENIAC
Research, Basic Research New Jersey 08101
Department,
Received September 22, 1975; accepted December 2, 1975 A staining procedure for lipoxygenase in electrophoretic gels based on the oxidation of a dye, 3,3’-dimethoxybenzidine hydrochloride, by hydroperoxides is described. The specificity of the stain to the dye was established by enzyme inhibition, by use of nonsubstrates, by enzyme heat-inactivation and by proportionality of the staining intensity to the amount of enzyme as observed visually. Cyanide is included with the substrate to inhibit heme-catalyzed oxidation. The gels which had little or no background staining have been stored for 18 months at 4°C without any color deterioration. As little as 220 units of soybean lipoxygenase and 5 ~1 of a 30% tomato homogenate gave detectable staining.
The detection of lipoxygenase activity in electrophoretic gels is becoming increasingly important as isoenzymes have been found present in different plant tissues (1,2). Grossman et al. (3) used Koch’s thiocyanate test for peroxides (4) to localize lipoxygenase activity in cellulose acetate gels, while Guss et al. (5) employed acidic potassium iodide on polyacrylamide gels impregnated with starch. With both methods, we experienced background staining which made detection of low activity samples difficult. Furthermore, the color development continued upon storage so that a permanent record was not possible unless photographs were taken immediately. Gardner et al. (6) reported a high percentage of failure in stain development with Guss’ procedure due possibly to a high threshold requirement for hydroperoxide to release I2 in the gel. We report in this study the use of a dye, 3,3’-dimethoxybenzidine hydrochloride (DBH) to visualize hydroperoxide bands in polyacrylamide gels with practically no background staining. The method is relatively sensitive, and the gels had been stored for 18 months at 4°C without color deterioration. MATERIALS
AND METHODS
Gel ElectrophoresiA
Electrophoresis on polyacrylamide gels was performed essentially according to the method of Davis (7) except that no stacking gel was used. Samples (5 to 20 ~1) were added directly on top of the gel and mixed with 5 ,!A of tracking dye (bromphenol blue, 0.5% in H20) and 10 ~1 of 40% sucrose added previously. Electrophoresis was carried out at pH 8.8 428 Copyright 0 1976 by Academic Pras. Inc. All rightc of reproduction in any form reserved.
STAINING
FOR LIPOXYGENASEACTIVITY
429
(0.38 M, Tris-HCI) on 7% polyacrylamide with acurrent of 2.5 to 3 mA per tube for 3 hr at 4°C. Protein bands were stained with Coomassie brilliant blue. Lipoxygenase
Staining
Immediately after electrophoresis, the gels were rinsed with distilled water and placed in 1.6 x 15 cm tubes containing buffered linoleic acid (2 mM) and inhibitors when necessary. Reaction was carried out at room temperature for 30 min. The gels were rinsed with distilled water, transferred to test tubes containing 0.1% DBH (Eastman Kodak Co.), and stained overnight at room temperature. The dye was washed off and the gels were stored in water. Lipoxygenase
Sources
Crystalline soybean lipoxygenase (Sigma Chemical Co.) containing 21.600 units/mg solids was used. Homogenates (30-50%) of tomatoes were prepared by blending fruits in a Waring Blender for 60 set at 4°C. The tomato homogenates were stored in liquid nitrogen until ready to use. Substrate
Preparation
Linoleic acid (Grade III, 99% pure, Sigma Chem. Co.) was prepared by dispersing 0.62 ml of acid in 50 ml of water containing two drops of Triton X-100 with a Branson sonifier for 5 min at 0°C. It was diluted with water to give a stock solution of 20 mM. The working solution was prepared by dilution of the stock to 2 mM either with pH 8.6,O. 1 M Tris-HCl or pH 5.8,O. 1 M citrate-phosphate buffer. Immediately before using, the substrate was oxygenated for 20-30 min at room temperature. RESULTS
AND
DISCUSSION
Gels that contained 20 pg (432 units), 40 pg (864 units) and 80 pg (1728 units) of soybean lipoxygenase exhibited increasingly darker reddish brown stains as observed visually while the inactivated control (lOO’C, 10 min) showed no band at all (Fig. 1). Soybean lipoxygenase containing 220 units is detectable with this technique, compared to 500 units reported by Guss (5). Five microliters of tomato homogenate showed sufficient staining. Staining of tomato was more effective when the fatty acid was incubated at pH 8.8, possibly due to greater solubility of the substrate, although we found that tomato lipoxygenase had a pH optimum around 5.8. No staining was evident when the gels were incubated at pH 8.8 followed by stepwise incubation at pH 5.8 according to the procedure of Gardner (6). Soybean lipoxygenase has been shown to have at least two isoenzymes
430
DE LUMEN
AND KAZENIAC
FIG. 1. Effect of soybean lipoxygenase concentration on intensity of staining. (1) Coomasie blue staining for protein, (2) 80 fig, (3) 40 pg, (4) 20 pg. and (5) heat inactivated control. The dark bands at the bottom ofgels 2 through 5 suggest oxidation of the substrate and of the dye by ammonium persulfate.
(3,8). At a concentration of 10d3 M, nordihydroguaiaretic acid (NDGA) prevented staining of both bands, whereas only the second band stained weakly at a concentration of lop4 M. No bands were evident when linoelaidic and oleic acids were used as substrates. These fatty acids do not have the cis, cis-1, 4-pentadiene system for which lipoxygenase is specific. Sodium cyanide ( low3 M), an inhibitor of heme-catalyzed peroxidation, did not inhibit staining in both soybean lipoxygenase and tomato homogenates. No experiments were done using heme proteins to produce hydroperoxides. Maier and Tappel (9) reported that heme-catalyzed oxidation of linoleic acid required an induction period of 24 hr at less than 7 mM
STAINING
FOR LIPOXYGENASE
ACTIVITY
FIG. 2. Isoenzyme patterns of tomato (2) and corncob (3). The protein pattern of tomato is shown in (I). The dark band in bottom of gel 3 suggests oxidation of the substrate and of the dye by ammonium persulfate.
linoleate concentration. Ah substrate concentrations used in these experiments were 2 mM. Lipoxygenase is a far more powerful catalyst than heme proteins (lo), and should be the predominant catalyst of lipid oxidation in plant tissues where heme proteins are relatively low in concentration. In spite of these observations, sodium cyanide (10e3 M) is included routinely with the substrate to inhibit any contribution of heme catalysts to the formation of hydroperoxides. As an application of this technique, we have been studying the fipoxygenase isoenzymes of tomato and other fruits. Isoenzyme patterns of tomato and corncob are shown in Fig. 2. Tomato and corncob hpoxygenases have at least four isoenzymes with very different distributions as compared with the two isoenzymes of soybean.
432
DE LUMEN
AND KAZENIAC
The best solvent for the dye was water, the solution having a pH of 3.5. Some precipitation was observed in aqueous DBH when the gel was introduced into the solution with pH increasing to about 8.6. No staining was observed with dilute acid solvents while dilute base precipitated out the dye. The gels had a clear background and they had been stored at 4°C for as long as 18 months without color deterioration. Storage of the gels in 7.5% acetic acid removed the stain. The sharpness of the stained bands may have been affected because a stacking gel was not used or because some diffusion of protein and hydroperoxide may have occurred during the 30 min reaction period at room temperature of the enzyme with linoleic acid. ACKNOWLEDGMENT We are grateful to Dr. Yu Bang Lee for his help in the electrophoresis during the initial stages of these studies.
experiments
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Pinsky, A., Grossman, S., and Trop, M. (1971)J. Food Sci. 36, 571. Hale, S., Richardson, T., Vonelbe, J. H., and Hagedom. D. J. (1969) Lipids 4, 209. Grossman, S., Pinsky, A., and Goldweitz, Z. (1971)Ann/. Biochem. 44, 642. Koch, R. B., Stem, B., and Ferrari, C. G. (1958)Arch. Biochem. Biophys. 78, 165. GUSS, P. L., Richardson, T., and Stahmann, M. A. (1967) Cereal Chem. 44, 607. Gardner, H. W., Christianson, D. D., and Kleiman, R. (1973) Lipids 8, 271. Davis, B. J. (1964)Ann. N. Y. Acad. Sci. 121, 404. Christopher, J., and Axelrod, B. (1971) Biochem. Biophys. Res. Commun. 44, 731. Maier, V. P., and Tappel, A. L. (1959) J. Amer. Oil Chem. Sot. 36, 8. Eriksson, C. (1975) J. Agric. Food Chem. 23, 126.
