A rapid and sensitive assay method for measuring amine oxidase based on hydrogen peroxide titanium complex formation

Plant Science 157 (2000) 157 163 www.elsevier.com/locate/plantsci A rapid and sensitive assay method for measuring amine oxidase based on hydrogen peroxide titanium complex formation Sudipa Nag, Kalpana Saha, Monojit A. Choudhuri * Department of Botany, Uni ersity of Burdwan, Burdwan 713 104, India Received 23 September 1999; received in revised form 8 January 2000; accepted 12 April 2000 Abstract Hydrogenperoxide (H 2 O 2 ) is an end product of diamine and polyamine oxidation by their respective oxidase enzymes. A new sensitive assay method is based on a H 2 O 2 titanium (Ti) complex formation as an indicator of H 2 O 2 production due to polyamine oxidation. The orange yellow coloured H 2 O 2 Ti complex was measured at 410 nm in a Shimadzu spectrophotometer. The assay conditions for maximum diamine oxidase (DAO) and polyamine oxidase (PAO) as standardized here using the hypocotyl tissues of Vigna catjang Endl. cv Pusa Barsati consisted of ph 7.4 (40 mm potassium phosphate buffer), 3 mm substrate (putrescine or spermine), 37 C incubation temperature and 30 min incubation time in the presence of catechol (10 2 M) used as an inhibitor of both peroxidase and catalase activity. The method described here was significantly more sensitive than the starch iodide method [T.A. Smith, Biochem. Biophys. Res. Commun. 41 (1970) 1452 1456], which could be improved further if measured under the same assay conditions as described for the H 2 O 2 Ti method. Sensitivity of the present method was tested by assaying DAO/PAO activity in auxin treated hypocotyls of Vigna and comparing it with the starch iodide method in two other plant samples. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diamine oxidase; Indole-3-butyric acid; Polyamine; Polyamine oxidase; Vigna catjang Endl.; Vigna radiata L. cv 105; Brassica alba Hook cv B9 1. Introduction Diamine oxidase (DAO) and polyamine oxidase (PAO) cause oxidation of diamines and polyamines, respectively, where hydrogenperoxide (H 2 O 2 ) is an end product [1,2]. Activity of these enzymes are reportedly measured either by estimating the residual substrate [3] or by estimating the end product H 2 O 2 or pyrrolline derivatives in an indirect way [4 7]. One such indirect method involves the development of a starch iodide complex due to the oxidation of I to I 0 by the liberated H 2 O 2 [5]. Another method consists of measurement of the rate of peroxidative oxidation Abbre iations: H 2 O 2, hydrogenperoxide; DAO, diamine oxidase; PAO, polyamine oxidase; Ti, titanium; CAT, catalase; POX, peroxidase; IBA, indole-3-butyric acid. * Corresponding author. Tel./fax: +91-342-56260. E-mail address: dsabot@dte.vsnl.net.in (M.A. Choudhuri). of guaiacol by H 2 O 2 in presence of peroxidase enzyme [6], or of the evolved O 2 from the breakdown of H 2 O 2 in presence of catalase enzyme using an oxygen electrode [7]. A direct measurement of H 2 O 2 produced during DAO/PAO activity seems to be a more sensitive method for measuring their activities in plant systems. Titanium ions strongly react with H 2 O 2 forming a brilliant orange yellow coloured complex, which is used for measuring H 2 O 2 in plant tissues [8]. Thus, in the present communication, determination of DAO/ PAO activity in terms of H 2 O 2 measurement was standardized and established to be more sensitive than some other methods. A valid enzyme assay is substantiated by (1) the rate of product formation which must be linear with respect to the reaction time of the assay; and (2) the quantity of product formed during a fixed reaction time that should increase proportionately 0168-9452/00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-9452(00)00281-8

158 S. Nag et al. / Plant Science 157 (2000) 157 163 Table 1 Effect of inhibitor catechol (10 2 M) on authentic catalase (CAT) and peroxidase (POX) activity (n=9) Treatment CAT catechol CAT+catechol POX catechol POX+catechol Enzyme activity (EU min 1 mg 1 protein) S.E. 0.26 0.002 0.0 0.0 8.22 0.001 0.072 0.001 15R, USA). The clear supernatant fraction containing 170 g protein ml 1, was used as enzyme source during assays. To test the role of catechol as an inhibitor of CAT and POX enzymes, the activity of standard CAT (Sigma, USA) was performed as described by Snell and Snell [10] with some modifications [11] and POX (Sigma, USA) activity was measured according to the method of Kar and Mishra [12] in the presence and absence of catechol (10 2 M). 2.3.2. Substrate concentration To standardize the optimum substrate concentration, the reaction was initiated by adding 0.5 ml of reaction mixture containing 0.5, 1, 2, 3, 4, 5 and 6 mm putrescine and previously standardized aswith the amount of enzyme supplied to the reaction. 2. Materials and methods 2.1. Plant materials Vigna catjang Endl. cv Pusa Barsati seeds were grown in sand in a controlled growth room with a 16 h photoperiod at 222 mol m 2 s 1 intensity (400 700 nm) for 7 days. Hypocotyls of 7-day-old seedlings were taken for assaying the diamine oxidase (DAO) and polyamine oxidase (PAO) activity. 2.2. Extraction and estimation of DAO/PAO Enzymes were extracted following the method of Rinaldi et al. [7]. Hypocotyl tissue (1 g) was homogenized in a prechilled mortar using 1.5 ml of 50 mm potassium phosphate buffer (ph 7.0) containing catechol (10 2 M), used as an inhibitor of catalase (CAT) and peroxidase (POX) [9]. The homogenate was centrifuged at 10 000 rpm for 15 min at 4 C in a Beckman centrifuge (Model GS- 2.3. Standardization of conditions for maximum enzyme acti ity using putrescine as substrate 2.3.1. Assay ph To standardize the optimum ph of assay buffer, the reaction was started by the addition of 0.5 ml of reaction mixture containing 2 mm substrate (putrescine) and 40 mm potassium phosphate buffer separately at ph 7.0, 7.2, 7.4 and 7.5 with 0.3 ml of enzyme extract at 30 C for 30 min. The ph of the actual reaction was determined after mixing enzyme extraction with reaction mixture. After incubation, the reaction was terminated by adding 0.1 ml of 15% (W/V) titanium sulphate (TiSO 4 ) in 23% H 2 SO 4. The incubation mixture was then centrifuged at 10 000 rpm for 10 min and absorbance of the orange yellow coloured solution was taken at 410 nm in a Shimadzu UV-vis spectrophotometer. In controls, TiSO 4 was added prior to the addition of enzyme solution. Table 2 Determination of actual ph of reaction mixture for maximum diamine oxidase (DAO) a PH of the assay buffer PH of the extraction buffer ph of the actual reaction mixture H 2 O 2 production ( A/30 min) S.E. 7.0 7.0 7.0 0.166 0.002 7.5 7.0 7.2 0.39 0.001 8.0 7.0 7.4 0.556 0.003 8.3 7.0 7.5 0.406 0.002 a Activity at incubation temperature 30 C and time 30 min and substrate concentration 2 mm (n=9).

S. Nag et al. / Plant Science 157 (2000) 157 163 159 Fig. 1. (a) Determination of substrate concentration for maximum diamine oxidase (DAO) activity at incubation temperature 30 C, time 30 min and at standardized ph 7.4. (b) Determination of enzyme volume at standardized substrate concentration (3 mm putrescine), and ph 7.4 and at incubation temperature 30 C and time 30 min for maximum DAO activity. (c) Determination of incubation time at standardized substrate concentration (3 mm putrescine), ph 7.4 enzyme volume 0.3 ml and at incubation temperature 30 C for maximum DAO activity. (d) Determination of incubation temperature for maximum DAO activity at standardized substrate concentration (3 mm putrescine), ph 7.4, enzyme volume 0.3 ml and incubation time 30 min. DAO activity in terms of A/30 min. Bars indicate standard error (n=9). say buffer (40 mm potassium phosphate buffer at ph 7.4) with 0.3 ml enzyme extract at 30 C for 30 min. The reaction was terminated by adding 0.1 ml of 15% TiSO 4 in 23% H 2 SO 4. After centrifugation of the mixture, absorbance of the H 2 O 2 titanium complex was measured at 410 nm. 2.3.3. Enzyme concentration To standardize the optimum enzyme concentration, 0.5, 0.75, 1.00, 1.50 and 2.0 g tissue was separately extracted using 1.5 ml of 50 mm potassium phosphate buffer (ph 7.0) with catechol (10 2 M) and then the reaction was initiated by adding 0.3 ml of enzyme extract separately from each extraction with 0.5 ml reaction mixture containing 3 mm putrescine (standardized) and 40 mm potassium phosphate buffer (ph 7.4) (standardized) at 30 C for 30 min. The reaction was terminated by adding 0.1 ml of 15% TiSO 4 in 23% H 2 SO 4. After centrifugation of the mixture, absorbance of the H 2 O 2 titanium complex was measured at 410 nm. To standardize the optimum enzyme volume 1.0 g tissue (standardized) was extracted using 1.5 ml of 50 mm potassium phosphate buffer (ph 7.0) with catechol (10 2 M) and then the reaction was initiated by adding 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml of enzyme extract separately with 0.5 ml reaction mixture containing 3 mm putrescine (standardized) and 40 mm potassium phosphate buffer at ph 7.4 (standardized) at 30 C for 30 min. The reaction was terminated by adding 0.1 ml of 15% Table 3 Estimation of protein contant and hydrogen peroxide production for maximum diamine oxidase (DAO) activity (n=9) Tissue (g tissue/1.5 ml buffer) Content of protein ( g/ml) H 2 O 2 Production ( A/30 min) S.E. 0.50 80 0.22 0.002 0.75 116.3 0.44 0.001 1.00 170.3 0.6 0.003 1.50 2.00 237.3 333.3 0.69 0.001 0.72 0.003

160 S. Nag et al. / Plant Science 157 (2000) 157 163 Fig. 2. (A) Diamine oxidase (DAO) activity (mm H 2 O 2 produced/30 min) in presence and absence of catechol. Starch iodide method. (B) Starch iodide method done under assay conditions of H 2 O 2 Ti method. (C) H 2 O 2 Ti method. Bars indidicate standard error (n=9). TiSO 4 in 23% H 2 SO 4. After centrifugation of the mixture, absorbance of the H 2 O 2 titanium complex was measured at 410 nm. 2.3.4. Incubation time By keeping all other conditions as standardized above fixed, the incubation time for maximum enzyme activity was standardized by varying the time from 10 to 60 min at intervals of 10 min at 30 C temperature and the optimum incubation time was determined. The enzyme activity was calculated as AXT v /tx, where A is the absorbance of sample after incubation minus absorbance at zero time control, T v is the total volume of the filtrate, t is the time (min) of incubation with substrate, and is the volume of filtrate actually taken for incubation [13]. 2.3.5. Temperature To standardize the optimum temperature for incubation, the reaction mixture, consisting of 0.3 ml enzyme extract (standardized), 0.5 ml reaction mixture containing 3 mm putrescine (standardized) and 40 mm potassium phosphate buffer at ph 7.4 (standardized) was separately incubated at 20, 25, 30, 37, 40, and 45 C for 30 min (standardized). After incubation, 0.1 ml of 15% TiSO 4 reagent was added and after centrifugation at 10 000 rpm for 10 min, absorbance of the H 2 O 2 titanium complex was read at 410 nm. Table 4 Effect of IBA (25 M) treatment (48 72 h) on diamine oxidase (DAO) and polyamine oxidase (PAO) a Treatment DAO S.E. PAO S.E. Control 0.79 0.001 0.770 0.002 IBA (48 h) 0.89 0.003 0.81 0.003 IBA (72 h) 1.36 0.004 1.29 0.003 a Activity in terms of A/30 min. (n=9).

S. Nag et al. / Plant Science 157 (2000) 157 163 161 Table 5 Comparison of the H 2 O 2 Ti method and the traditional starch iodide method on other plant samples a Plant samples Starch idodide method H 2 O 2 Ti method DAO PAO DAO PAO S.E. S.E. S.E. S.E. V.catjang 0.7 0.02 0.6 0.03 2.6 0.02 V. radiata 0.5 0.01 0.4 0.01 2.0 0.02 B. alba 0.6 0.01 0.5 0.02 2.0 0.01 2.4 0.01 1.8 0.01 1.9 0.01 a DAO and PAO activities in terms of mm H 2 O 2 produced /30 min. 2.4. Estimation of protein content Amount of protein was determined by the method of Bradford [14] with bovine serum albumin as the standard. 2.5. Comparison with starch idodide method of Smith [5] To examine the sensitivity of the present method based on H 2 O 2 titanium complex formation due to diamine oxidase activity, it was compared with the starch iodide method at the assay conditions reported by Smith [5]. DAO activity was measured employing the starch iodide method under two assay conditions. In one, the conditions as described by Smith [5] were strictly followed, while in the other, the conditions as described above for H 2 O 2 titanium method was followed. Thus in the starch iodide method, the assay mixture contained 0.3 ml of crude enzyme (previously extracted with 100 mm potassium phosphate buffer, ph 6.5) and 0.5 ml reaction mixture consisting of 1.3% soluble starch, 20 mm potassium iodide and 10 mm putrescine in 1 mm potassium phosphate buffer (ph 5.8) for a period of 30 min at 37 C [5]. The enzyme activity was also measured following the starch iodide formation method under the assay conditions reported here in presence and absence of catechol (10 2 M). After incubation, the absorbance was measured at 550 nm. Enzyme activities in the above mentioned methods were expressed in terms of mm H 2 O 2 produced/30 min as determined from separate standard curves. 2.6. DAO/PAO acti ities in IBA treated seedlings Changes in DAO and PAO activities were measured using H 2 O 2 Ti method in the hypocotyl region after treating the cuttings of Vigna with IBA (indole-3-butyric acid, 25 M) for 48 and 72 h. Where PAO activity was measured, putrescine was replaced by spermine. 2.7. Comparison of the H 2 O 2 Ti and starch iodide method on a range of plant samples To justify the significance of the H 2 O 2 Ti method and to make the method valid, DAO and PAO activities were measured in two other plants viz, V. radiata L. cv 105 and B. alba Hook. cv B9. All the experiments were repeated three times with at least three replicates each time with reproducible results. 3. Results and discussion 3.1. Effect of an orthodiphenol inhibitor catechol (10 2 M) It is evident from Table 1 that the standard CAT and POX showed approximately 0.0 and 0.87% activity respectively in presence of the inhibitor (catechol, 10 2 M). Homogenates of hypocotyl tissues were, therefore, treated with catechol, an orthodiphenol compound, to inhibit the POX and CAT activity which were responsible for breakdown of enzymatically generated H 2 O 2. 3.2. Optimum conditions for DAO acti ity The optimum activity of DAO was standardized under H 2 O 2 Ti method at varying ph (7.0, 7.2, 7.4 and 7.5) (Table 2). The optimum concentration of the substrate putrescine (0.5, 1, 2, 3, 4, 5 and 6 mm) was then standardized (Fig. 1a). Concentration of the enzyme (0.5, 0.75, 1.0, 1.50 and 2.0 g tissue/1.5 ml phosphate buffer, ph 7.0) was determined (Table 3). Next the enzyme volume (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml) was standardized and shown in Fig. 1b. This was followed by determination of incubation time (10, 20, 30, 40, 50 and 60 min) which was depicted in Fig. 1c, and temperature (20, 25, 30, 37, 40 and 45 C) was shown in Fig. 1d. It was observed that the activity of DAO

162 S. Nag et al. / Plant Science 157 (2000) 157 163 (extraction buffer ph 7.0) was optimum under the assay conditions of ph 7.4, 3 mm putrescine, 1.0 g of tissue in 1.5 ml phosphate buffer equivalent to 170 g protein ml 1, 0.3 ml enzyme volume, 30 min incubation time and 37 C temperature. It is evident from Fig. 1a that 3 mm putrescine is probably saturating since further increases in substrate concentration ( 3 mm) led to very small changes in activity of the enzyme. It is evident from Fig. 1b that the quantity of the product formed during the fixed reaction time (30 min) increased proportionately with the amount of enzyme supplied to the reaction up to 0.3 ml enzyme volume and beyond that volume it did not show any linearity. Fig. 1c shows that the rate of product formation was linear with increasing reaction time up to 30 min. Further increase in incubation time did not increase the product formation linearly. It may be due to limitation of the substrate. Enzyme activity was linear up to 30 min incubation time. Temperature affects the rate of enzyme catalysed reaction. A rising temperature (Fig. 1d) increased the rate of reaction up to a certain limit (20 37 C) above which the enzyme activity was inhibited. However, higher temperature ( 37 C) might lead to the denaturation of the enzyme which caused a decrease in catalytic activity. 3.3. Sensiti ity of the present method To examine the sensitivity of the present method for measuring amine oxidase activity, DAO was measured by the present H 2 O 2 Ti method as standardized above (Fig. 2) and the activity was compared with the starch iodide method under the assay conditions described by Smith [5], as well as under the assay conditions standardized for H 2 O 2 Ti method reported here. It was observed that DAO activity significantly increased (10 times) in terms of mm H 2 O 2 produced/30 min if measured under the present H 2 O 2 Ti method over the starch iodide method. Furthermore, there was a significant improvement in DAO activity (about 2.5 times) over the method standardized by Smith [5], if measured by starch iodide method under the assay conditions reported for H 2 O 2 Ti method instead of those reported by Smith. These results clearly reveal greater sensitivity of the H 2 O 2 Ti method than that of the starch iodide method. 3.4. Effect of IBA (25 M) on DAO/PAO Table 4 shows that activity of both DAO and PAO measured by the standardized H 2 O 2 Ti method in the hypocotyls of IBA (25 M)-treated seedlings greatly increased over untreated controls in both treatment hours. 3.5. DAO/PAO in other plants Table 5 shows that the H 2 O 2 Ti method is more sensitive and more reproducible than the starch iodide method. Results show that activities of both DAO and PAO with the H 2 O 2 Ti method were more sensitive than with the starch iodide method in the three separate plant samples. It has been also known that DAO activities are greater than PAO in all the plants studied with either method. Results of the present enzyme kinetics with DAO/PAO confirm the general criteria of enzyme kinetics and hence the H 2 O 2 Ti method is valid. Acknowledgements The authors are grateful to Dr D. Sengupta, Professor of Biochemistry, University of Calcutta for his valuable advice and suggestions for interpreting the results in proper perspective. References [1] A.W. Galston, Polyamines as modulators of plant development, Bioscience 33 (1983) 382 388. [2] T.A. Smith, Polyamines, Annu. Rev. Plant Physiol. 36 (1985) 117 143. [3] B.I. Naik, R.G. Goswami, S.K. Srivastava, Rapid and sensitive colorimetric assay of amine oxidase, Annu. Biochem. 111 (1981) 146 148. [4] B. Holmsted, L. Larsson, R. Tham, Further studies on spectrophotometric method for the of amine oxidase activity, Biochim. Biophys. Acta 48 (1961) 182 186. [5] T.A. Smith, Polyamine oxidase in higher plants, Biochem. Biophys. Res. Commun. 41 (1970) 1452 1456. [6] T.A. Smith, Polyamine oxidation by enzymes from Hordeum ulgare and Pisum sati um seedlings, Phytochemistry 13 (1974) 1075 1081. [7] A. Rinaldi, G. Floris, A. Finazzi-Agro, Purification and properties of diamine oxidase from Euphorbia latex, Eur. J. Biochem. 127 (1982) 417 422. [8] W.M. MacNevin, P.P. Uron, Separation of hydrogen peroxide from organic hydroperoxides, Anal. Chem. 25 (1953) 1760 1761.

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