2.1 Chemicals and Materials

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

Purified clover leaf seedlings containing amine oxidase were prepared as reported by Emmanuel Delhaize and John Webb [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 (NH₄)₂SO₄ 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 [(NH₄)₂SO₄fraction]. Step 3: The dialysed fraction wasthen precipitated with precooled (- 20°C) acetone (Me₂CO, 50 mL /100 mL enzyme solution) with stirring and centrifuged at 15 000 g for 20 min.Further Me₂CO 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 (Me₂COfraction). Step 4: The Me₂CO 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% (NH₄)₂SO₄saturation, 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% (NH₄)₂SO₄, 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

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]. 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

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].

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].

where K_a1 and K_a2 are the dissociation constant of protonated groups that control the dependence of kinetic parameters on pH, and y₀ is the pH independent value of the parameter y (k_cat or k_cat/K_m) [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]:

where Z_a and Z_b are the charges of the species involved in the formation of activated complex and k₀ 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.

3.1 Substrate Specificity

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) were used as the enzymatic substrates. The steady-state kinetic parameters for the oxidation reaction catalyzed by TPAO are summarized in 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^-1mg^-1, which is approximately 14 folds as that of tyramine (18 μM min^-1mg^-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 theV_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.

Fig. 1.Full-screen View

Molecular structures of different amines used as substrates of TPAO.

3.2 pH Dependence of Catalytic Parameters

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), 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 pK_a values which can be calculated by fitting the data to Eq-3. The pK_avalues for different amine-containing substrates in the TPAO catalyzing reactions are summarized in 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.

Fig. 2.Full-screen View

Effect of pH on log kcat for TPAO catalyzing different amine-containing substrates. The plots of kcat against pH for different amine-containing substrates are represented by putrescine (□), cadaverine (■), spermidine (△), spermine (▲), tyramine (○), and phenylethylamine (●), respectively. And the solid lines were obtained by a non-linear least squares fit of the plot data to Eq-3. Experiments were carried out at 37 °C with a pH ranging from 4.0 to 9.5.

Generally, the variability of k_cat/K_m values should be not much sensitive to the changes of pH. Fig. 3 shows the plot ofk_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. Moreover, the corresponding pK_a values also show low degrees of variability (Table 2) compared with those derived from Fig. 2, in that free TPAO hasa high ionization potentialand the free amine-containing substrates are easily protonated [23].

Fig. 3.Full-screen View

Effect of pH on k_cat/K_m for TPAO catalyzing different amine-containing substrates. The plots of k_cat/K_m against pH for different amine-containing substrates are represented by putrescine (□), cadaverine (■), spermidine (△), spermine (▲), tyramine (○), and phenylethylamine (●), respectively. And the solid lines were obtained by a non-linear least squares fitting. Experiments were carried out at 37 °C with a pH ranging from 4.0 to 9.5.

3.3 Dependence of V_max and K_m on Ionic Strength

The effect of ionic strength (I) on the kinetic parameters ofV_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) 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.

Fig. 4.Full-screen View

Effect of ionic strength on logK_m of TPAO catalyzing different amine-containing substrates. The plots of logK_m against ionic strength (I^1/2) for different amine-containing substrates are represented by putrescine (□), cadaverine (■), spermidine (△), spermine (▲), tyramine (○), and phenylethylamine (●), respectively. And the solid lines were obtained by a non-linear least squares fitting to Eq-4. The correlational coefficient of 0.99 was determined for all substrates. Experiments were carried out at 37 °C, pH 7.0 and with an ionic strength ranging from 5 – 120 mM by using NaCl solution.

The dependence of all kinetic parameterson ionic strength, the slopes (2CZ_aZ_b)which can be determined from the plots of logK_m against I^1/2, have beensummarized in Table 3. Among these data, the slopes derived from V_max/K_m fitting curves are believed to be able to reflect the interaction between substrates and enzymes. Here from Table 3, it can be clearly concluded that the ionic interaction appears with the same order as V_max/K_mfor different amine-containing substrates, indicating that 2CZ_aZ_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, 25].

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.