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A simple and sensitive method for the activity staining of xanthine oxidase
J. Biochem. Biophys. Methods 36 (1998) 95–100
A simple and sensitive method for the activity staining of xanthine oxidase ¨ *, Meltem Muftuoglu, ¨ ¨ Nazmi Ozer I. Hamdi Ogus Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey Received 25 October 1997; accepted 27 November 1997
Abstract Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 mU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 mU takes about 1 min whereas staining of 0.5 mU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources. 1998 Elsevier Science B.V. All rights reserved. Keywords: Xanthine oxidase; Activity staining; Sensitivity; Stability; Nitroblue tetrazolium; Light sensitivity
1. Introduction Xanthine oxidase (XOD: EC 1.1.3.22), a complex enzyme contains 2 moles of FAD, 2 gram atoms of molybdenum, and 8 gram atoms of iron per mole of the enzyme [1,2]. *Corresponding author. Tel.: 1 90 312 311 0588; fax: 1 90 312 311 6616; e-mail: [email protected] 0165-022X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 97 )00051-1
96
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
Scheme 1.
It has been purified from several sources including milk [2–5]. Its molecular weight and isoelectric point are 275 kDa and 5.3–5.4, respectively [5,6]. Its substrate specificity is low and it has a pH optimum of 4.7 [1,5,6]. Xanthine oxidase is inhibited by heavy metals, phosphate, imidazole buffer systems and salts such as sodium and potassium chloride and inactivation due to oxidation can be reversed by sulfhydryl compounds [5]. Xanthine oxidase is commercially available and widely used for the determination of xanthine, hypoxanthine, inosine and has been used as an auxiliary enzyme for the determination of guanine and guanosine [7]. It is also used for the measurement of guanase and nucleoside–phosphorylase activity [8]. Activity staining on cellulose acetate, agarose and polyacrylamide gels are commonlyused methods for the detection of the activities of several enzymes [9,10]. Xanthine oxidase is usually used as an auxiliary enzyme in the activity staining of other enzymes such as guanase, ascorbate peroxidase and in the activity staining of several blotted enzymes [11–13]. Histochemical staining methods for xanthine oxidase are available. On the other hand, there is no direct staining method for xanthine oxidase on polyacrylamide gels [14]. The following method is obtained by modifying the staining method for ascorbate peroxidase developed by Mittler and Zilinskas [12]. The reaction mechanism is the oxidation of nitroblue tetrazolium to water-insoluble formazan by the superoxide formed from xanthine by XOD (Scheme 1).
2. Material and methods
2.1. Materials Xanthine, xanthine oxidase (grade 1), nitroblue tetrazolium, cysteine, EDTA, acrylamide, bisacrylamide, and dithiothreitol (DTT) were obtained from Sigma (USA). DEAE-Sepharose fast flow is obtained from Pharmacia, Sweden. All other chemicals were standard products of Sigma or Aldrich, USA. Xanthine oxidase from fresh cow milk, without any preservative, was partially purified using toluene extraction, ammonium sulfate fractionation and chromatography on DEAE-Sepharose (fast flow). The enzyme obtained was stored in 10 mM of Tris / HCl, pH 7.6, containing 5 mM of 2-mercaptoethanol, 2.3 M of ammonium sulfate and 0.3 per cent sodium azide, at 48C.
2.2. Activity measurements Xanthine oxidase activity was determined by a method modified from Massey et al. [15]. The conversion of xanthine to uric acid was followed by monitoring the change in
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
97
absorbance at 295 nm, using Milton-Roy 3000 spectrometer, in a 1-ml reaction mixture containing 50 mM of Tris / HCl, pH 7.6 and 0.1 mM of xanthine, at 378C. The reaction was initiated by the addition of the enzyme.
2.3. Polyacrylamide gel electrophoresis ( PAGE) Discontinuous PAGE (0.75 mm) was carried out under nondenaturing and nonreducing conditions, essentially as described by Laemmli for denaturing gels [16]. Here, sodium dodecyl sulfate was omitted. Gel dimensions and thicknesses were 100 and 0.75 mm, respectively. Six per cent separator gel, without stacking gel, was prepared. After application of the samples, a constant voltage of 100 volts was applied. After application of the samples, electrophoretic separation was carried out at room temperature. Following electrophoresis, gels are stained either for activity or protein.
2.4. XOD activity staining in polyacrylamide gels This method was obtained by a modification of the method developed for ascorbate peroxidase [12]. Activity staining on polyacrylamide gels were carried out at room temperature. The reaction mixture contained 50 mM Tris / HCl, pH 7.6, 0.50 mM xanthine, 0.25 mM nitroblue tetrazolium and 630 mM TEMED. Staining of the gels were continued till the activity band(s) appear on the gels (10 s–15 min). After the appearence of the bands, gels were washed 5–6 times with distilled water. They are preserved in cold distilled water (48C, in dark).
2.5. Protein staining of gels Staining and destaining was carried out according to Laemmli, omitting the acidfixation step [16].
2.6. Protein determination Protein concentration in samples was determined by the method of Bradford, using bovine serum albumin as standard [17].
3. Results and discussion Xanthine oxidase from fresh cow milk was partially purified by a method that involves toluene extraction, ammonium sulfate fractionation, and anion-exchange chromatography on DEAE-Sepharose (fast flow) [1–4]. This enzyme had an activity of 0.61 units / ml and a specific activity of 5.05 units / mg protein. Native gel electrophoresis was carried out on 6% polyacryamide gel using Laemmli’s method but SDS was omitted [16]. A stacking gel of 4 per cent did not give any improvement on the results obtained without stacking gel, so it was omitted. Activity staining of XOD was carried out at room temperature. The results of a typical activity staining is given in Fig. 1a. Detection of 0.02 mU of the enzyme on
98
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
Fig. 1. Activity staining of xanthine oxidase from cow milk. (a) Amount of XOD (mU per well) in lanes: 1, 0.018 (0.123); 2, 0.036 (0.246); 3, 0.072 (0.49); 4, 0.144 (0.980); 5, 0.178 (1.470); 6, 0.216 (2.450); 7, 0.505 (3.430); 8, 0.721 (4.900). Values in parenthesis are mg of protein applied per well for activity staining. (b) Protein staining: amount of protein (mg per well) in lanes: 1, 0.246; 2, 0.490; 3, 0.980; 4, 1.960; 5, 2.940; 6, 4.900; 7, 6.860; 8, 9.800.
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
99
polyacrylamide gels was possible. Staining of 0.05 mU takes about 1 min whereas staining of 0.5 mU will take less than 5 s. Although, the addition of TEMED is not essential for activity staining, it increases both the rate and the intensity of the staining (not shown) [12]. The bands observed in Fig. 1a and b show parallel staining for both activity and protein. Incubation of XOD, for 2 h at room temperature, with 10 mM of dithioerythritol decreased the intensity of the upper band(s) but even 100 mM of dithioerythrytol was unable to reduce the oxidized XOD, completely. A considerable fraction of the XOD interacted and moved together with the dye, bromophenol blue, and retained its activity (Fig. 1a). The amount of XOD activity, at the dye-front, was dependent on the contact time of the XOD with bromophenol blue, at room temperature (not shown). Similar findings, for other proteins, were also reported [18]. When the intensity of staining of the protein band(s) reaches the desired intensity, the staining (reaction) solution was discarded and the gels were transferred into distilled water. Gels must be washed at least 5 times with distilled water. Insufficient washing will result in a dark background and thick diffuse bands, due to the oxidation of the remaining NBT. Gels could be stored in the distilled water, at about 48C, after covering the petri dishes with aluminium foil, for at least a month or alternatively, after drying, in polyethylene sheets for an indefinite time. In order to be able to detect the protein bands, corresponding to activity staining, the amount of protein applied per well was doubled (Fig. 1b). This direct staining method is very important for the quality control of xanthine oxidase obtained from different sources.
References [1] Hart LJ, McGartoll MA, Chapman HR, Bray RC. The composition of milk xanthine oxidase. Biochem J 1970;116:851–64. [2] Waud WR, Brady FO, Wiley RD, Rajagopalan KV. A new purification procedure for bovine milk xanthine oxidase: Effect of proteolysis on the subunit structure. Arch Biochem Biophys 1975;169:695– 701. [3] Gilbert DAD, Bergel F. The chemistry of xanthine oxidase. Biochem J 1964;90:350–3. [4] Nelson CA, Handler P. Preparation of bovine xanthine oxidase and the subunit structures of some iron flavoproteins. J Biol Chem 1968;243:5368–73. [5] Horecker BL, Heppel LA. In: Colowick SP, Kaplan NO, editors. Methods in Enzymology, vol. 2. New York: Academic Press, 1955:482–485. ¨ K, editors. The enzymes, vol. 7. New York: Academic [6] Bray RC. In: Boyer PD, Lardy HA, Myrback Press, 1963:533. [7] Coddington A. In: Bergmeyer HU. Methoden der enzymatischen analyse. Weinheim: Verlag Chemie, 1970:1862, 1866. [8] Price VE, Otey C, Plesner P. In: Colowick SP, Kaplan NO, editors. Methods in enzymology, vol. 2. New York: Academic Press, 1955:448–453. ¨ [9] Grunbaum B. Handbook for forensic individualization of human blood and bloodstains, Gottingen, West Germany: Sartorius GmbH, 1981. [10] Harris H, Hopkinson DA. Handbook of enzyme electrophoresis in human genetics. Amsterdam: North-Holland Publishing Company, P.O. Box 211.
100
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
[11] Nishikawa Y, Fukumoto K, Watanabe F. Analysis of guanase by agarose gel electrophoresis and activity staining. Enzyme 1985;33:143–6. [12] Mittler R, Zilinskas BA. Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium. Anal Biochem 1993;212:540–6. [13] Sock J, Rohringer R. Activity staining of blotted enzymes by reaction coupling with transfer membraneimmobilized auxiliary enzymes. Anal Biochem 1988;171:310–9. [14] MacGowan S, Regan MC, Malone C, Sharkey O, Young L, Gorey TF, Wood AE. Superoxide radical and xanthine oxidoreductase activity in the human heart during cardiac operations. Ann Thorac Surg 1995;60:1289–93. [15] Massey V, Brumby PE, Komai H. Studies on milk xanthine oxidase: Some spectral and kinetic properties. J Biol Chem 1969;244:1682–91. [16] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [17] Bradford MM. A rapid and sensitive method for the quatitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. ¨ ¨ Sayek I, Ozer ¨ [18] Ozer N, Erdemli O, I. Resolution and kinetic characterization of glutathione S-transferase from human jejunal mucosa. Biochem Med Met Biol 1990;44:142–50.
A simple and sensitive method for the activity staining of xanthine oxidase ¨ *, Meltem Muftuoglu, ¨ ¨ Nazmi Ozer I. Hamdi Ogus Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey Received 25 October 1997; accepted 27 November 1997
Abstract Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 mU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 mU takes about 1 min whereas staining of 0.5 mU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources. 1998 Elsevier Science B.V. All rights reserved. Keywords: Xanthine oxidase; Activity staining; Sensitivity; Stability; Nitroblue tetrazolium; Light sensitivity
1. Introduction Xanthine oxidase (XOD: EC 1.1.3.22), a complex enzyme contains 2 moles of FAD, 2 gram atoms of molybdenum, and 8 gram atoms of iron per mole of the enzyme [1,2]. *Corresponding author. Tel.: 1 90 312 311 0588; fax: 1 90 312 311 6616; e-mail: [email protected] 0165-022X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 97 )00051-1
96
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
Scheme 1.
It has been purified from several sources including milk [2–5]. Its molecular weight and isoelectric point are 275 kDa and 5.3–5.4, respectively [5,6]. Its substrate specificity is low and it has a pH optimum of 4.7 [1,5,6]. Xanthine oxidase is inhibited by heavy metals, phosphate, imidazole buffer systems and salts such as sodium and potassium chloride and inactivation due to oxidation can be reversed by sulfhydryl compounds [5]. Xanthine oxidase is commercially available and widely used for the determination of xanthine, hypoxanthine, inosine and has been used as an auxiliary enzyme for the determination of guanine and guanosine [7]. It is also used for the measurement of guanase and nucleoside–phosphorylase activity [8]. Activity staining on cellulose acetate, agarose and polyacrylamide gels are commonlyused methods for the detection of the activities of several enzymes [9,10]. Xanthine oxidase is usually used as an auxiliary enzyme in the activity staining of other enzymes such as guanase, ascorbate peroxidase and in the activity staining of several blotted enzymes [11–13]. Histochemical staining methods for xanthine oxidase are available. On the other hand, there is no direct staining method for xanthine oxidase on polyacrylamide gels [14]. The following method is obtained by modifying the staining method for ascorbate peroxidase developed by Mittler and Zilinskas [12]. The reaction mechanism is the oxidation of nitroblue tetrazolium to water-insoluble formazan by the superoxide formed from xanthine by XOD (Scheme 1).
2. Material and methods
2.1. Materials Xanthine, xanthine oxidase (grade 1), nitroblue tetrazolium, cysteine, EDTA, acrylamide, bisacrylamide, and dithiothreitol (DTT) were obtained from Sigma (USA). DEAE-Sepharose fast flow is obtained from Pharmacia, Sweden. All other chemicals were standard products of Sigma or Aldrich, USA. Xanthine oxidase from fresh cow milk, without any preservative, was partially purified using toluene extraction, ammonium sulfate fractionation and chromatography on DEAE-Sepharose (fast flow). The enzyme obtained was stored in 10 mM of Tris / HCl, pH 7.6, containing 5 mM of 2-mercaptoethanol, 2.3 M of ammonium sulfate and 0.3 per cent sodium azide, at 48C.
2.2. Activity measurements Xanthine oxidase activity was determined by a method modified from Massey et al. [15]. The conversion of xanthine to uric acid was followed by monitoring the change in
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
97
absorbance at 295 nm, using Milton-Roy 3000 spectrometer, in a 1-ml reaction mixture containing 50 mM of Tris / HCl, pH 7.6 and 0.1 mM of xanthine, at 378C. The reaction was initiated by the addition of the enzyme.
2.3. Polyacrylamide gel electrophoresis ( PAGE) Discontinuous PAGE (0.75 mm) was carried out under nondenaturing and nonreducing conditions, essentially as described by Laemmli for denaturing gels [16]. Here, sodium dodecyl sulfate was omitted. Gel dimensions and thicknesses were 100 and 0.75 mm, respectively. Six per cent separator gel, without stacking gel, was prepared. After application of the samples, a constant voltage of 100 volts was applied. After application of the samples, electrophoretic separation was carried out at room temperature. Following electrophoresis, gels are stained either for activity or protein.
2.4. XOD activity staining in polyacrylamide gels This method was obtained by a modification of the method developed for ascorbate peroxidase [12]. Activity staining on polyacrylamide gels were carried out at room temperature. The reaction mixture contained 50 mM Tris / HCl, pH 7.6, 0.50 mM xanthine, 0.25 mM nitroblue tetrazolium and 630 mM TEMED. Staining of the gels were continued till the activity band(s) appear on the gels (10 s–15 min). After the appearence of the bands, gels were washed 5–6 times with distilled water. They are preserved in cold distilled water (48C, in dark).
2.5. Protein staining of gels Staining and destaining was carried out according to Laemmli, omitting the acidfixation step [16].
2.6. Protein determination Protein concentration in samples was determined by the method of Bradford, using bovine serum albumin as standard [17].
3. Results and discussion Xanthine oxidase from fresh cow milk was partially purified by a method that involves toluene extraction, ammonium sulfate fractionation, and anion-exchange chromatography on DEAE-Sepharose (fast flow) [1–4]. This enzyme had an activity of 0.61 units / ml and a specific activity of 5.05 units / mg protein. Native gel electrophoresis was carried out on 6% polyacryamide gel using Laemmli’s method but SDS was omitted [16]. A stacking gel of 4 per cent did not give any improvement on the results obtained without stacking gel, so it was omitted. Activity staining of XOD was carried out at room temperature. The results of a typical activity staining is given in Fig. 1a. Detection of 0.02 mU of the enzyme on
98
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
Fig. 1. Activity staining of xanthine oxidase from cow milk. (a) Amount of XOD (mU per well) in lanes: 1, 0.018 (0.123); 2, 0.036 (0.246); 3, 0.072 (0.49); 4, 0.144 (0.980); 5, 0.178 (1.470); 6, 0.216 (2.450); 7, 0.505 (3.430); 8, 0.721 (4.900). Values in parenthesis are mg of protein applied per well for activity staining. (b) Protein staining: amount of protein (mg per well) in lanes: 1, 0.246; 2, 0.490; 3, 0.980; 4, 1.960; 5, 2.940; 6, 4.900; 7, 6.860; 8, 9.800.
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
99
polyacrylamide gels was possible. Staining of 0.05 mU takes about 1 min whereas staining of 0.5 mU will take less than 5 s. Although, the addition of TEMED is not essential for activity staining, it increases both the rate and the intensity of the staining (not shown) [12]. The bands observed in Fig. 1a and b show parallel staining for both activity and protein. Incubation of XOD, for 2 h at room temperature, with 10 mM of dithioerythritol decreased the intensity of the upper band(s) but even 100 mM of dithioerythrytol was unable to reduce the oxidized XOD, completely. A considerable fraction of the XOD interacted and moved together with the dye, bromophenol blue, and retained its activity (Fig. 1a). The amount of XOD activity, at the dye-front, was dependent on the contact time of the XOD with bromophenol blue, at room temperature (not shown). Similar findings, for other proteins, were also reported [18]. When the intensity of staining of the protein band(s) reaches the desired intensity, the staining (reaction) solution was discarded and the gels were transferred into distilled water. Gels must be washed at least 5 times with distilled water. Insufficient washing will result in a dark background and thick diffuse bands, due to the oxidation of the remaining NBT. Gels could be stored in the distilled water, at about 48C, after covering the petri dishes with aluminium foil, for at least a month or alternatively, after drying, in polyethylene sheets for an indefinite time. In order to be able to detect the protein bands, corresponding to activity staining, the amount of protein applied per well was doubled (Fig. 1b). This direct staining method is very important for the quality control of xanthine oxidase obtained from different sources.
References [1] Hart LJ, McGartoll MA, Chapman HR, Bray RC. The composition of milk xanthine oxidase. Biochem J 1970;116:851–64. [2] Waud WR, Brady FO, Wiley RD, Rajagopalan KV. A new purification procedure for bovine milk xanthine oxidase: Effect of proteolysis on the subunit structure. Arch Biochem Biophys 1975;169:695– 701. [3] Gilbert DAD, Bergel F. The chemistry of xanthine oxidase. Biochem J 1964;90:350–3. [4] Nelson CA, Handler P. Preparation of bovine xanthine oxidase and the subunit structures of some iron flavoproteins. J Biol Chem 1968;243:5368–73. [5] Horecker BL, Heppel LA. In: Colowick SP, Kaplan NO, editors. Methods in Enzymology, vol. 2. New York: Academic Press, 1955:482–485. ¨ K, editors. The enzymes, vol. 7. New York: Academic [6] Bray RC. In: Boyer PD, Lardy HA, Myrback Press, 1963:533. [7] Coddington A. In: Bergmeyer HU. Methoden der enzymatischen analyse. Weinheim: Verlag Chemie, 1970:1862, 1866. [8] Price VE, Otey C, Plesner P. In: Colowick SP, Kaplan NO, editors. Methods in enzymology, vol. 2. New York: Academic Press, 1955:448–453. ¨ [9] Grunbaum B. Handbook for forensic individualization of human blood and bloodstains, Gottingen, West Germany: Sartorius GmbH, 1981. [10] Harris H, Hopkinson DA. Handbook of enzyme electrophoresis in human genetics. Amsterdam: North-Holland Publishing Company, P.O. Box 211.
100
¨ et al. / J. Biochem. Biophys. Methods 36 (1998) 95 – 100 N. Ozer
[11] Nishikawa Y, Fukumoto K, Watanabe F. Analysis of guanase by agarose gel electrophoresis and activity staining. Enzyme 1985;33:143–6. [12] Mittler R, Zilinskas BA. Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium. Anal Biochem 1993;212:540–6. [13] Sock J, Rohringer R. Activity staining of blotted enzymes by reaction coupling with transfer membraneimmobilized auxiliary enzymes. Anal Biochem 1988;171:310–9. [14] MacGowan S, Regan MC, Malone C, Sharkey O, Young L, Gorey TF, Wood AE. Superoxide radical and xanthine oxidoreductase activity in the human heart during cardiac operations. Ann Thorac Surg 1995;60:1289–93. [15] Massey V, Brumby PE, Komai H. Studies on milk xanthine oxidase: Some spectral and kinetic properties. J Biol Chem 1969;244:1682–91. [16] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [17] Bradford MM. A rapid and sensitive method for the quatitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. ¨ ¨ Sayek I, Ozer ¨ [18] Ozer N, Erdemli O, I. Resolution and kinetic characterization of glutathione S-transferase from human jejunal mucosa. Biochem Med Met Biol 1990;44:142–50.