Characterization of the copper-containing amine oxidase from trifolium pratense seedlings

Characterization of the copper-containing amine oxidase from trifolium pratense seedlings 1 corresp first-author Corresponding Authors. E-mail address: abel518@hotmail.com; Corresponding Authors. E-mail address: abel518@hotmail.com; abel518@hotmail.com 2 3 3 3 3 corresp fengzhang1978@hotmail.com. fengzhang1978@hotmail.com. fengzhang1978@hotmail.com College of Life Science and Technology, Inner Mongolia Normal University, Hohhot 010022, China; College of Life Science and Technology, Inner Mongolia Normal University, Hohhot 010022, China; Sugar beet Physiological Institute, Inner Mongolia Agricultural University, Hohhot 010019, China; Sugar beet Physiological Institute, Inner Mongolia Agricultural University, Hohhot 010019, China; Agricultrual Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China. Agricultrual Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China. We studied the kinetic characterizations of the trifolium pratense seedlings copper-containing amine oxidase (TPAO) by using various amine-containing substrates. The catalyzing rate for all of amine-containing substrates can be ordered as: diamines > polyamines > aromatic monoamines, and it shows an apparent trend in each category of substrates such as: the longer the carbon chain, the lower the V max is, so does the V max / K m values but is opposite for the K m value of TPAO. The distinct differences between the kinetic parameters for different amine-containing substrates indicated that the rate-determining step of the catalytic reaction strongly depends on the substrate's chemical structure. It is concluded that both pH and ionic strength can affect the catalytic activity of TPAO via influencing the coulomb interaction-mediated enzyme-substrate docking processes, which can be attributed to the potential of charged groups from both substrates and the activity sites of TPAO by the regulation of ionic strength. Amineoxidase Trifolium pretense Kinetic characterization pH dependence;Ionic strength. 1 INTRODUCTION 1 Amine oxidases (AOs) catalyze the oxidative deamination of polyamines which are ubiquitous polycationic compounds involved in many significant events of the cell cycles [ 1 1 ]. The amine oxidases can be classified into two categories: copper-containing amine oxidases (CuAOs; EC1.4.3.6) and flavin-containing polyamine oxidases (PAOs; EC1.5.3.11). CuAOs are a ubiquitous and novel group of quinoenzymes that catalyze the oxidation of aromatic and aliphatic primary amines to be corresponding aldehydes with concomitant reduction of molecular oxygen to hydrogen peroxide [ 2 2 ], which can be concluded as following reaction formula (in which R represents aromatic or aliphatic moieties): <math display="block" id="M1"><mrow><msub><mrow><mtext>R-CH</mtext></mrow><mtext>2</mtext></msub><msub><mrow><mtext>-NH</mtext></mrow><mtext>2</mtext></msub><msub><mrow><mtext>+ H</mtext></mrow><mtext>2</mtext></msub><msub><mrow><mtext>O + O</mtext></mrow><mn>2</mn></msub><mo>→</mo><msub><mrow><mtext> R-CHO + NH</mtext></mrow><mtext>3</mtext></msub><msub><mrow><mtext>+ H</mtext></mrow><mtext>2</mtext></msub><msub><mtext>O</mtext><mtext>2</mtext></msub></mrow></math> Diamines like putrescine and cadaverine can be oxidized by mono-amine orpoly-amineAOs with a low catalytic activity, however, they have been considered preferred substratesfor the characterization of CuAOs in plants [ 3 3 ]. CuAOs can be highly purified and characterized from a variety of microorganisms including fungi and bacteria [ 4 4 ]. In plants, CuAOs have been detected in many species and particularly have higher levels when observing dicotyledons [ 5 5 ]. Some of them such as Lens esculenta [ 3 3 ], Pisum sativum [ 6 6 ], Triticum aestivum [ 7 7 ], Eurphorbia characias [ 8 8 ], and Vigna radiate [ 9 9 ] have been highly purified and characterized. At this point in time the kinetic mechanism of CuAOs in plants is not well understood, although it has been extensively studied [ 10 10 - 14 14 10-14 ]. The biological role of AOs has been reported mainly on the production of polyamine catabolism in hydrogen peroxide. Several extensive reviews have been recently published[ 1 1 , 15 15 - 17 17 15-17 ]. In this article, we have thoroughly studied the kinetic characteristics of trifolium pratense seedlings amine oxidase (TPAO) with using 8 different kinds of amine-containing substrates under different pH and ionic strengths. We have intensivelydiscussed the catalyzingmechanism of TPAO for different amine derivatives and the effects of both pH and ionic strength. 2 EXPERIMENTAL SECTION 1 2.1 Chemicals and Materials 2 All chemicals were of the analytical grade and used without further purification. All of amine substrates were purchased from Sigma-Aldrich (Biodee, Beijing, China). N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylamine was a gift from Dojin Laboratories (Kumamoto, Japan). Horseradish-peroxidase was purchased from Wako Pure Chemical (Osaka, Japan). Red clover ( trifolium pratense L . ) seeds were sterilized with 0.1% benzalkonium chloride, thoroughly soaked with tap water for eight hours, and grown on moist filter paper (Whatman No. 1) for ten days at 25 °C in total darkness. After treatments, the etiolated seedlings were harvested, frozen in liquid nitrogen, and stored at -80 °C until use. The HPLC data was collected with a Hitachi Chromaster system. Spectrophotometric measurements were measured with a Hitachi UV-2800 instrument (Hitachi Co., Tokyo, Japan). 2.2 Enzyme Purification and Copper Determination 2 Purified clover leaf seedlings containing amine oxidase were prepared as reported by Emmanuel Delhaize and John Webb [ 18 18 ]. Briefly, a six step purification procedure has beenadopted in the present investigation for purification of TPAO from 72 hr germinated clover leaf seedlings. All the operations werecarried out at 4°C.Step 1: Clover leaf seedlings were homogenized in sodium phosphate buffer(10 mM, pH 7.5). The homogenate was filtered through cheesecloth and centrifuged at 10000 g for 20 min. Step 2: The crudeextract was subjected to (NH 4 ) 2 SO 4 fractionation and theprotein precipitating at 35-65% saturation was dispersed in sodium phosphate buffer (10 mM, pH 7.5) and dialysed against the same buffer for24 h [(NH 4 ) 2 SO 4 fraction]. Step 3: The dialysed fraction wasthen precipitated with precooled (- 20°C) acetone (Me 2 CO, 50 mL /100 mL enzyme solution) with stirring and centrifuged at 15 000 g for 20 min.Further Me 2 CO at -20°C was added (150mL / 100 mL enzyme solution) in the same manner and the precipitation recovered bycentrifugationat 20000 g for 30 min was dissolved in sodium phosphate buffer (10 mM, pH7.5), dialyzed and centrifuged at 5000 g for 5min (Me 2 COfraction). Step 4: The Me 2 CO fraction was applied to a DEAE-cellulose column (25 cm2.2 cm) previously equilibrated with sodium phosphate buffer (0.1 M, pH 7.5) and the column was washed with200 mL of the same buffer. The enzyme fractions were eluted fromthe column with 0.1 M sodium phosphate buffer containing 0.2 M NaCl. Theactive fractions were pooled, brought to 70% (NH 4 ) 2 SO 4 saturation, centrifuged at 10000 g for 20 min, the precipitation dissolved in10 mM sodium phosphate buffer and dialysed. Step 5: The dialysed enzymefraction (10 mL) was passed through the DEAE-Sephadex (A-25)column (33 cm1.8 cm) equilibrated with sodium phosphate buffer (0.1 M,pH 7.5) and inactive protein was washed from the column with sodium phosphate buffer containing 0.1 M NaCl. The enzyme was then elutedfrom the column with 0.3 M NaCl in sodium phosphate buffer (0.1 M, pH 7.5),precipitated with 70% (NH 4 ) 2 SO 4 , centrifuged at 10000g for20 min, dissolved in 10 mM sodium phosphate buffer and dialysed overnight.Step 6: The column (4 cm2 cm) of hydroxylapatite was equilibrated with sodium phosphate buffer (0.1 M, pH 7.5). The enzyme fractionobtained in step 5 was passed through the column and the columnwas washed with the same buffer and then the enzyme was elutedwith sodium phosphate buffer (0.3 M, pH 7.5). Copper contents were determined by atomic absorption using an IL 951 atomic absorption spectrometer (Instrumentation Laboratory, Wilmington, DE). The spectral line was chosen 324.7 nm for copper. 2.3 Measurements of Enzyme Activity 2 The hydrogen peroxide-producing amine oxidase activity was continuously monitored by spectrophotometric detection in a reaction mixture with using the modified quinoneimine dye method that, in a previous report, we discussed using the purification of a novel FAD-dependent amine oxidase from oat seedlings [ 19 19 ]. The mixture for a standard assay contained 50 mM potassium phosphate buffer (pH 7.0),0.5 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylamine, 0.5 mM 4-aminoantipyrine, 3.3 purpurogallin units horseradish-peroxidase, and enzyme solution in a total volume of 3.0 mL. The reaction was initiated with the addition of amine. The mixture was incubated at 37 °C and the enzyme activity was determined by the absorbance at 555 nm. Enzyme stability, as a function of pH, was determined in air-saturated solutions at 37 °C in the assay mixture including each of the following buffers: 50 mM acetate buffer, 100 mM K-phosphate buffer, 50 mM Tris-HCl buffer, or 50 mM glycine-NaOH buffer. The pH was adjusted to the desired range (4.0 to 9.5) with the addition of NaOH or HCl. Enzyme activity measurements, as a function of ionic strength, were carried out in air-saturated solutions at 37 °C in the presence of 0.1 mM EDTA. The concentrated NaCl solutions were mixed with different amines as substrates. Controlled experiments without NaCl were performed throughout the entire experiment. 2.4 Analysis of Kinetic Parameters 2 K m and V max values, were obtained by fitting the kinetic data to the Michaelis-Menten equation (Eq-2) by non-linear regression analysis with using Microcal Origin 4.1 software, and assuming a TPAO molecular weight of 150 kDa [ 18 18 ]. <math display="block" id="M2"><mrow><mi>v</mi><mo>=</mo><msub><mi>V</mi><mrow><mtext>max</mtext></mrow></msub><mrow><mo>[</mo><mtext>S</mtext><mo>]</mo></mrow><mo>/</mo><mrow><mo>(</mo><mrow><msub><mi>K</mi><mtext>m</mtext></msub><mo>+</mo><mtext> </mtext><mrow><mo>[</mo><mtext>S</mtext><mo>]</mo></mrow></mrow><mo>)</mo></mrow></mrow></math> The data reported accounts for the average of three experiments by using at least six amine concentrations each. The standard deviation was about ten percent of the measured value. Kinetic parameters k cat and k cat / K m values were graphically plotted against the proton concentration and fitted to an equation (Eq-3) that determined measurements of enzyme stability as a function of pH [ 20 20 ]. <math display="block" id="M3"><mrow><mi>y</mi><mo>=</mo><msub><mi>y</mi><mn>0</mn></msub><mo>/</mo><mrow><mo>(</mo><mrow><mn>1</mn><mo>+</mo><mrow><mo>[</mo><mrow><msup><mi>H</mi><mo>+</mo></msup></mrow><mo>]</mo></mrow><mo>/</mo><msub><mi>K</mi><mrow><mtext>a1</mtext></mrow></msub><mo>+</mo><msub><mi>K</mi><mrow><mtext>a2</mtext></mrow></msub><mo>/</mo><mrow><mo>[</mo><mrow><msup><mrow><mtext> H</mtext></mrow><mo>+</mo></msup></mrow><mo>]</mo></mrow></mrow><mo>)</mo></mrow><mo>,</mo></mrow></math> where K a1 and K a2 are the dissociation constant of protonated groups that control the dependence of kinetic parameters on pH, and y 0 is the pH independent value of the parameter y ( k cat or k cat / K m ) [ 21 21 ]. The kinetic data, V max or V max / K m , on ionic strength ( I ) at pH 7.0 at 37 °C, was analyzed according to the Debye Huckel equation (Eq-4) [ 22 22 ]: <math display="block" id="M4"><mrow><mtext>Log </mtext><msub><mi>K</mi><mtext>m</mtext></msub><mo>=</mo><mtext> log </mtext><msub><mi>k</mi><mtext>0</mtext></msub><mo>+</mo><mtext> </mtext><mn>2</mn><mi>C</mi><msub><mi>Z</mi><mtext>a</mtext></msub><msub><mi>Z</mi><mtext>b</mtext></msub><msup><mi>I</mi><mrow><mn>1</mn><mo>/</mo><mn>2</mn></mrow></msup><mo> </mo><mo>,</mo></mrow></math> where Z a and Z b are the charges of the species involved in the formation of activated complex and k 0 is the kinetic rate constant, K m , at I = 0 and the constant C is 0.518 M 1/2 for a reaction occurring in water at 37 °C. 3 RESULTS 1 3.1 Substrate Specificity 2 The atomic absorption measurements have proved the purified TPAO was one of typical copper containing AOs (data not shown). In order to characterize the substrate specificity of TPAO, different amine derivatives ( Fig. 1 Fig. 1 ) were used as the enzymatic substrates. The steady-state kinetic parameters for the oxidation reaction catalyzed by TPAO are summarized in Table 1 Table 1 . The V max values of the analyzed substrates show distinct differences. The highest value of V max in putrescine is 251 μM min -1 mg -1 , which is approximately 14 folds as that of tyramine (18 μM min -1 mg -1 ). All of amine-containing substrates can be ordered as: diamines > polyamines > aromatic monoamines, and in each category of substrates, it shows an apparent trend as: the longer the carbon chain, the lower the V max is, which is also same as the V max / K m values but is opposite for the K m value of TPAO, in that the highest K m value of aromatic monoamines shows approximately 2 orders of magnitude higher than that of diamines. The distinct differences between the kinetic parameters for different amine-containing substrates indicated that the catalyzing activity of TPAO depends on the molecular structure of itssubstrates. Both terminals of the spermidine molecule contain an amino-butyl end, so its structure is rather similar to putrescine. Other groups like phenyl and hydroxyl groups also show different influences on the catalytic efficiency of TPAO, for example the V max / K m value for tyramine is much larger than both phenylethylamine and benzylamine. All of the kinetic measurements are taken in standard assay mixture as described in the materials and methods at pH 7.0 and 37 ℃. All the sets of measurements are run in triplicate * n.d. means the sample is not detectable due to the too low readout 1 1 Substrate 1 1 V max (μM min -1 mg -1 ) 1 1 K m (μM) 1 1 V max / K m (min -1 mg -1 ) 1 1 Putrescine 1 1 252 1 1 38 1 1 6.632 1 1 Cadaverine 1 1 197 1 1 89 1 1 2.213 1 1 1,8-Diaminooctane 1 1 122 1 1 196 1 1 0.622 1 1 Spermidine 1 1 98 1 1 348 1 1 0.282 1 1 Spermine 1 1 63 1 1 698 1 1 0.090 1 1 Benzylamine 1 1 46 1 1 2380 1 1 0.019 1 1 Phenylethylamine 1 1 18 1 1 3750 1 1 0.005 1 1 Tyramine 1 1 41 1 1 1920 1 1 0.021 1 1 Propylamine 1 1 n.d. * 1 1 n.d. 1 1 n.d. 1 1 Hexylamine 1 1 n.d. 1 1 n.d. 1 1 n.d. 3.2 pH Dependence of Catalytic Parameters 2 To deepen the understanding the pH effect on the catalytic activity of TPAO, a pH ranging from 4.0 to 9.5 was chosen to this end. An asymmetrical bell-shaped curve obtained by plotting k cat against pH ( Fig. 2 Fig. 2 ), from which the k cat values for diamines are highest compared to polyamines and aromatic monoamines. The maximum peaks of the curves for putrescine and cadaverine are found around neutral pH, however, others are centered at alkaline region. As we know, the pH effecton the catalyzing activity is caused by protonation/deprotonation of the corresponding protonated groups, and it can be well described as the appartent p K a values which can be calculated by fitting the data to Eq-3. The p K a values for different amine-containing substrates in the TPAO catalyzing reactions are summarized in Table 2 Table 2 , from which we can figure out that the pK a values also show an increasing trend as the sequence for K m values except for tyramine, which could be attributed tothe interfere of the additional hydroxyl group. These results indicate that charged state of substrates might affect the catalyzing activity of TPAO by controlling the enzyme-substrate binding affinities upon changing pH of reaction solution. 1 1 Substrate 2 1 k cat 2 1 k cat / K m 1 1   1 1 pK a1 1 1 pK a2 1 1 pK a3 1 1 pK a4 1 1 Putrescine 1 1 6.22 ± 0.11 1 1 8.04 ± 0.15 1 1 6.75 ± 0.15 1 1 8.13 ± 0.10 1 1 Cadaverine 1 1 6.53 ± 0.12 1 1 8.66 ± 0.13 1 1 7.15 ± 0.09 1 1 8.95 ± 0.12 1 1 1,8 diaminooctane 1 1 6.82 ± 0.12 1 1 8.94 ± 0.11 1 1 7.73 ± 0.14 1 1 8.93 ± 0.12 1 1 Spermidine 1 1 6.68 ± 0.10 1 1 8.59 ± 0.13 1 1 7.26 ± 0.12 1 1 8.86 ± 0.10 1 1 Spermine 1 1 6.93 ± 0.10 1 1 8.90 ± 0.09 1 1 7.54 ± 0.14 1 1 9.01 ± 0.16 1 1 Benzylamine 1 1 7.50 ± 0.13 1 1 9.19 ± 0.17 1 1 7.61 ± 0.15 1 1 9.34 ± 0.10 1 1 Phenylethylamine 1 1 7.62 ± 0.13 1 1 9.28 ± 0.11 1 1 7.63 ± 0.16 1 1 9.30 ± 0.13 1 1 Tyramine 1 1 6.41 ± 0.12 1 1 8.39 ± 0.14 1 1 7.02 ± 0.10 1 1 8.54 ± 0.15 Generally, the variability of k cat / K m values should be not much sensitive to the changes of pH. Fig. 3 Fig. 3 shows the plot of k cat / K m values against different pH, which are much smoother, more parabolic and parallel to each other compared with the curves in Fig. 1 Fig. 1 . Moreover, the corresponding pK a values also show low degrees of variability ( Table 2 Table 2 ) compared with those derived from Fig. 2 Fig. 2 , in that free TPAO hasa high ionization potentialand the free amine-containing substrates are easily protonated [ 23 23 ]. 3.3 Dependence of V max and K m on Ionic Strength 2 The effect of ionic strength ( I ) on the kinetic parameters of V max, K m and V max / K m were studied by using a Lineweaver-Burk equation (Eq-4) with the same amine-containing substrates. All of the measurements were carried out with the I ranging from 10 mM to 120 mM by adjusting the assay solutions withconcentrated solutions of NaCl. Linear fitting curves with a correlational coefficient of 0.99 were obtained, from which the K m ( Fig. 4 Fig. 4 ) and V max / K m (data not shown) values appear strongly dependent on ionic strength, in contrast, the V max values (data not shown) of the tested substrates are almost independent. The dependence of all kinetic parameterson ionic strength, the slopes (2 CZ a Z b )which can be determined from the plots of log K m against I 1/2 , have beensummarized in Table 3 Table 3 . Among these data, the slopes derived from V ma x /K m fitting curves are believed to be able to reflect the interaction between substrates and enzymes. Here from Table 3 Table 3 , it can be clearly concluded that the ionic interaction appears with the same order as V max /K m for different amine-containing substrates, indicating that 2 CZ a Z b values depend on the relative spatial distribution of the charges present in the active site of enzymes and of those amine-containing substrates [ 24 24 , 25 25 ]. 1 1 Substrate 3 1 2 CZ a Z b 1 1   1 1 V max 1 1 K m 1 1 V max / K m 1 1 Putrescine 1 1 −0.8 ± 0.3 1 1 +6.0 ± 0.5 1 1 −5.7 ± 0.1 1 1 Cadaverine 1 1 −0.6 ± 0.5 1 1 +6.2 ± 0.3 1 1 −5.5 ± 0.6 1 1 1,8 diaminooctane 1 1 −0.7 ± 0.2 1 1 +5.5 ± 0.1 1 1 −5.2 ± 0.3 1 1 Spermidine 1 1 −0.4 ± 0.6 1 1 +5.1 ± 0.5 1 1 −4.1 ± 0.2 1 1 Spermine 1 1 −0.5 ± 0.7 1 1 +4.6 ± 0.4 1 1 −3.8 ± 0.5 1 1 Benzylamine 1 1 +0.2 ± 0.2 1 1 +1.8 ± 0.4 1 1 −1.9 ± 0.2 1 1 Phenylethylamine 1 1 +0.1 ± 0.3 1 1 +1.9 ± 0.7 1 1 −1.8 ± 0.5 1 1 Tyramine 1 1 +0.2 ± 0.1 1 1 +2.4 ± 0.2 1 1 −2.2 ± 0.1 Amine oxidase activities were spectrophotometrically determined at 555 nm as described by Zhang et al. in 2012.Ionic strength was determined by using NaCl with the concentrations ranging from10 mM to120 mM. 4 DISCUSSION 1 AOs exist widely in different organisms and their organs; they are involved in biogenic amine metabolisms. Based on their different structural and biological functions, AOs have a physiological role in organisms. In order to understand the characterization of AOs, one must take into account that past researches were always conducted in vitro assays. In our previous publication [ 19 19 ], we demonstrated that an oat AO, which has exclusive substrate specificity to phenylethylamine and benzylamine, surprisingly does not have detectable activity when compared with other amines. Currently, we have carried out TPAO activity measurements under the same experimental physicochemical conditions and used different amine derivatives as substrates. During this procedure the results indicate diamines (putrescine and cadaverine) have the highest affinitiesto TPAO compared withpolyamines (spermidine and spermine) and aromatic amines (phenylethylamine, benzylamine and tyramine). This behavior of TPAO appears common to AOs from other species like pea and lentil seedling [ 21 21 ], and soybean seedling [ 3 3 ]. TPAO was found to be a copper-containing diamine oxidase by Emmanuel and John [ 18 18 ]. A slight difference of substrate specificity of TPAO was reported in a contemporary article with respect to pea seedling amine oxidase [ 26 26 ] and bovine serum amine oxidase [ 27 27 ]. The activity of TPAO depends strongly on the carbon chain length and the structure of amines which are peaking at putrescine (C4) among diamines, spermidine (C4 + C3) among polyamines, and tyramine (phenyl + C2) among aromatic amines, all of which are measured to obtain both K m and V max values.The results indicate that the kinetic catalyzing parameters of TPAO are relatively dependent on both chemical state of the activity sites of TPAO and the structures of its substrates. Furthermore, the higher V max value of spermidine compared with that of spermine could result from the fact that amino-butyl group has much higher activity than amino-propyl group to TPAO, so does the situation of putrescine and cadaverine. In order to understand the catalytic mechanism of TPAO, it should be better to resolve the active site of this enzyme by using spectroscopic and x-ray diffraction techniques. The dependence of TPAO activity on pH is different with different amine-containing substrates. The plotsof k cat values versus pH display the classical bell-shaped curves ( Fig. 2 Fig. 2 ). The reason we selected this pH range is because that the enzyme activity is very poor beyond this range. Fig. 2 Fig. 2 suggests that the protonating and deprotonating processes are involved in recognition behaviors between substrates and enzyme activity sites. As to all of the highest values of k cat from pH 6.5 to 8.0, the maximum difference was found between putrescine and phenylethylamine, which can be attributed to the polarity of substrates since phenylethylamine contains amuch more nonpolar groups (phenyl-) compared with those aliphatic amines. A hypothesis that the proton interacts with both the enzyme-substrate and enzyme-product adducts due to the increased possibility of a hydrophobic site, is consistent with the findings reported in previous articles [ 21 21 , 28 28 ]. From the analysis of the kinetic parameters versus I 1/2 , it shows that both K m and V max / K m are significantly dependent on ionic strength. The large variabilityof both K m and V max / K m indicates that electrostatic/coulomb/ionic interactionplays an important role during the substrate-enzyme docking process, in which all of the charged groups from both amine-containing substrates and the enzyme activity sites can be regulated by ionic strengths, which has already been discovered in the studies on rat mitochondrion AO [ 25 25 , 29 29 ].The ionic strength’s influence actuallycan result from the possibility of formation on stable interaction between two negative charges of the active site with two positive charges of diamines. There are three and four nitrogen atoms for spermidine and spermine, respectively, however there are only two positive charges on both ends of the molecular structures that could interact with two negatively charged residues in the enzyme activity site. 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DOI: 10.1042/BJ20021055 http://doi.org/10.1042/BJ20021055 Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates 10.1007/s41365-016-0066-x Supported by the Department of Education of Inner Mongolia and Inner Mongolia Natural Science Foundation of China for Basic Research (No. NJZY13054), the National Natural Science Foundation of China (Nos. 31360214, 21171086 and 81160213), the Inner Mongolia Autonomous Region Science and Technology Department (No. 211-202077), the Inner Mongolia Grassland Talent (No. 108-108038), the Natural Science Foundation of Inner Mongolia Autonomous Region of China (Nos. 2013MS1121 and 2015MS0806) and the Inner Mongolia Agricultural University (Nos. 109-108040, 211-109003 and 211-206038) . 2015-08-23 2015-10-26 2015-11-02 2016-05-12 2016-06-20 2021-04-11 © China Science Publishing & Media Ltd. (Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2016 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-016-0066-x http://dx.doi.org/10.1007/s41365-016-0066-x . 2016 Nuclear Science and Techniques 03 27