Introduction

There has been an increasing interest in studying the action of metal on enzyme activity and in using their biochemical changes in the aquatic biota as an index of metal toxicity (Lan et al. 1995). In this context, copper, an essential metal ion, has been intensively studied as a potential toxic compound (Gill et al. 1992, Guasch et al. 2002). This element is widely used in agriculture to control fungal diseases and its increasing concentration in the environment has been attributed to mining operations, sewage sludge disposal and industrial discharges (WHO 1998). Copper sulphate has also been used in the control of natural phytoplanktonic communities (Larsen et al. 2003; Le Jeune et al. 2006). The increase of availability and potential toxicity of copper in water may have detrimental effects on aquatic organisms including algae, an important component of primary production and thus the entire aquatic food chain. Algae are able to bioconcentrate and to metabolize several aquatic pollutants like insecticides and polycyclic aromatic hydrocarbons (Warshawsky et al. 1995; Jonsson et al. 2001).

Pseudokirchneriella subcapitata (formely Selenastrum capricornutum), an unicellular Chlorophyceae (green) algae present in the freshwater aquatic and terrestrial compartments (Keddy et al. 1995), has been widely used in studies of pollutants effects (Jonsson et al. 1998; Okamura et al. 2002) and is recommended by regulatory national (Gherardi-Goldstein et al. 1990; Jonsson and Maia 1999) and international (OECD 1984) agencies as a test organism.

Acid phosphatase (EC 3.1.3.2) plays important roles in the metabolism such as decomposing organic phosphates into free phosphates and organic compounds. Several functions have been attributed to algae acid phosphatases, such as autophagic digestive processes, hydrolysis of phospholipid materials (Cooper et al. 1974), fertilization (breakdown of plasmalemma and absorption of flagella) (Braten 1975), availability of inorganic phosphate from the extracellular medium (Sommer and Blum 1965), recycling of inorganic phosphate for its reassimilation (Theodorou et al. 1991), endomembrane recycling (Domozych 1989) and spore differentiation (Tsekos and Schnepf 1991). In the freshwater algae S. tenue, the enzyme was found in the cell wall, plasmalemma and vacuole (Michetti et al. 2006). The induction of acid phosphatase activity in unicellular green algae at low N and P concentrations has been observed by Kruskopf and Du Plessis (2004).

It has been reported that copper or other metal ion are needed for the catalytic action of different acid phosphatases. Vincent and Averill (1990) have observed that the oxidized form of some mammalian acid phosphatases exhibited a iron–phosphorus interaction indicating that phosphate, and presumably the phosphate esters, bound to one of both irons present in the protein. Reilly et al. (1999) demonstrated that the addition of copper restored nearly all the enzymatic activity of an acid phosphatase from Haemophilus influenzae. The acid phosphatases from the microorganisms Mycoplasma fermentans, Pseudomonas aeruginosa and Yarrowia lipolytica are enhanced in the presence of copper (Domenich et al. 1992; Noda et al. 1994; Ito et al. 2007). This last effect was also observed by Bounias et al. (1996) in the kinetic studies for alkaline and acid phosphatases present in a gut crude extract obtained from honeybees .

The activities of algal enzymes like esterase (Franklin et al. 2001), β-D-galactosidase (Peterson and Stauber 1996), nitrate redutase, urease, ATPase (Rai and Rai 1997) and antioxidants enzymes (Li et al. 2006) were affected in presence of the metal.

Previously, we reported the activator effect of copper on the activity of acid phosphatase from P. subcapitata. Also, we demonstrated that the metal behaved as a slight antagonist for the inhibitor effect of mercury on the enzyme activity (Jonsson and Aoyama 2007). In the present work, we studied the effect of copper ions on the enzyme activity from the algae. We analyzed the stability and determined dose effects and kinetic parameters in order to understand the magnitude and mechanism of metal ion–phosphatase interaction.

Materials and methods

Materials

p-Nitrophenylphosphate (pNPP) was obtained from Sigma Chemical Co. Stock solutions of CuSO45H2O (Merck) were prepared in Milli-Q water or in liquid medium (OECD 1984). All the other reagents were AR grade.

Organisms and growth conditions

Unicellular green algae Pseudokirchneriella subcapitata was maintained and subcultured in an inorganic liquid medium prepared as recommended by OECD (OECD 1984). Cultures were grown in 250 ml flasks sealed with cotton bungs and containing 200 ml of sterilized medium. The flasks were incubated in a controlled temperature chamber (20 ± 2°C) under a continuous white fluorescent light of 3,000–4,000 lux and manually shaken twice a day. Every 40–60 days, a new stock culture maintained at 4°C (in dark) was prepared by inoculating approximately 5 × 104 cells ml−1 (Jonsson and Aoyama 2007).

Harvesting and preparation of extracts

All centrifugation procedures were carried out at 4°C. Exponential phase organisms were harvested by centrifugation at 4,000 r.p.m. for 5 min in a Beckman J2-21 refrigerated centrifuge (rotor SER# 7644, JA-20) and washed twice with 0.1 M sodium acetate buffer, pH 5.0. The algae pellet was suspended in 0.1 M sodium acetate buffer (1:4 w/v) and the cell suspension was submitted to the cell disruption procedure for phosphatase extraction as described previously (Jonsson and Aoyama 2007): The sample was frozen at −20°C, thawed at room temperature and submitted to a probe sonication at 0°C (ice bath) for 50 s followed by 20 s interval (1 cycle) with an amplitude of 70 (Vibra Cell, Sonics Materials Inc., 45 mm tipped probe). This procedure was repetead twice. The resultant cell disrupted suspension was centrifuged at 10,000 r.p.m. for 20 min and the supernatant fluid (extract) was used for acid phosphatase activity determination.

Assay of phosphatase activity

Acid phosphatase activity was routinely assayed at least in duplicate by incubating the enzyme with pNPP as substrate and measuring the p-nitrophenol (pNP) produced as previously described (Prazeres et al. 2004). The enzyme activity was determined in a final volume of 1 ml containing 0.1 M sodium acetate buffer (pH 5.0) and 10 mM substrate. After incubation for 40 min at 37°C, the reaction was terminated by the addition of 1 ml of 1 M NaOH. The pNP released was measured at 405 nm in a UNICAM 8625 UV/VIS spectrophotometer. For the initial velocity (V) determination, the amount of pNP produced was calculated using a molar extintion coefficient of 18,300 M−1cm−1 (Chaimovich and Nome 1970). Units (U) of enzymatic activity are defined as μmoles of pNP released per min.

Effect of Cu2+concentration

The enzyme activity was determined in the absence (control) or in the presence of several copper concentrations in a range of 0.1–200 μM Cu2+. Lineweaver–Burk plot was used to determine the Cu2+-enzyme dissociation constant by plotting the velocity change (delta V) versus the reciprocal of copper concentration (1/[Cu2+]), as described by Dixon and Webb (1979).

Enzyme stability

The enzyme activity was determined after preincubation at 37°C for 0, 20, 40 and 60 min in the absence or in the presence of 0.2 mM Cu2+.

Apparent Michaelis constant (Km) and maximum velocity (Vmax) determination

The enzyme was preincubated in the absence or in the presence of 0.2 mM Cu2+, and after that the enzyme activity was determined, using pNPP as substrate, in a concentration range of 0.1–10.0 mM. The apparent Km and Vmax values were calculated from Lineweaver–Burk plot.

Activation energy determination

The enzyme activity was determined after preincubation at different temperatures (27, 32, 37, 42 and 47°C) in the absence or in the presence of 0.2 mM Cu2+. The activation energy values were determined from the slopes of the Arrhenius plots (log V versus 1/T (Kelvin)), according to Dixon and Webb (1979).

Data analysis

The results were analyzed by a Simples Regression program with a Statgraphics® Plus Version 2 software package.

Results

In vitro effect of copper on P. subcapitata acid phosphatase activity

A remarkable increase in pNPP dephosphorylation was observed in the presence of Cu2+ up to a concentration of 0.05 mM, when this metal was previously incubated with the enzyme (Fig. 1). The dissociation constant (KdCu2+), for the complex Cu2+-enzyme was calculated from the double reciprocal plot to be 22.04 μM (Fig. 1, inset). In contrast to the results described in the Fig. 1, no significant enzyme activity changes were observed in the presence of Cu2+ without preincubation (results not shown).

Fig. 1
figure 1

Effect of copper concentration on acid phosphatase activity. The preincubation of the enzyme and Cu2+(at different concentrations) was performed for 20 min at 37°C. At the end of this period, the reaction was initiated by the addition of pNPP 10 mM as substrate, as described in Methods. The activity in the absence of Cu2+ was considered as 100% (control) and the other values were determined as percentages of this value. Determination of dissociation constant for the interaction Cu2+-acid phosphatase (inset). Bars represent the standard deviations of the averages of two replicates

Full size image

Stability of acid phosphatase in the presence of copper

The enzyme was stable for at least 60 min at 37°C when preincubated in the presence of 0.2 mM Cu2+. In contrast, a loss of acid phosphatase activity of about 60% was observed when the enzyme was preincubated in the absence of the metal ion (Fig. 2).

Fig. 2
figure 2

Preincubation time course of acid phosphatase activity. The enzyme activity was determined at the indicated times by preincubation at 37°C in the absence (●) or in the presence (■) of 0.2 mM Cu2+. Bars represent the standard deviations of the averages of two replicates

Full size image

Effect of Cu2+on the acid phosphatase kinetic parameters

The values of apparent Km and Vmax were determined from the double reciprocal plot of Lineweaver–Burk, and the activation energy from the Arrhenius plot (not shown). These results are shown in the Table 1. In the presence of 0.2 mM Cu2+, the apparent Km value decreased from 1.21 to 0.37 mM and the activation energy from 26.8 to 13.6 kJ mol−1; no significant difference was observed for the Vmax values (0.55 and 0.75 mU ml−1).

Table 1 Effect of Cu2+on the kinetic parameters for P. subcapitata acid phosphatasea
Full size table

Discussion

The metallic ion Cu2+ has been generally used in enzymatic systems in order to inactivate the reactions by binding to essential sulfhydryl groups located at or near the active site. In the case of acid phosphatases, we have previously reported that the enzyme purified from bovine kidney (Granjeiro et al. 1997) and from castor bean seed (Granjeiro et al. 1999) were inhibited 97 and 93% in the presence of 10 mM and 1 mM Cu2+, respectively.

In the present work we demonstrated that Cu2+ when preincubated with P. subcapitata acid phosphatase activated the pNPP-directed reaction. Lineweaver–Burk plot was used to determine the dissociation constant value for Cu2+ binding to the enzyme (KdCu2+), as described by Dixon and Webb (1979) (Fig. 1, inset). Youngs et al. (2000) have also applied this method to determine the dissociation constant for the Cd-Mn-peroxidase complex. The low magnitude of the KdCu2+ value (22.04 μM) obtained for the complex Cu2+-acid phosphatase indicated a strong binding capacity of this metal to the enzyme.

Even after preincubation of Cu2+, and enzyme, no activation effect was observed for the acid phosphatase obtained from the algae Ochromonas danica (Patni and Aaronson 1974). However, others in vitro studies showed that acid phosphatases from crude extracts of Rizopus delemar (Tsekova and Galabova 2003) and honeybee guts (Bounias et al. 1996) were activated by Cu2+.

In order to explain the reasons for the activation of the P. subcapitata acid phosphatase by Cu2+, we performed three different experiments: enzyme stability study, determination of apparent Km, and determination of activation energy, in the absence and in the presence of the metal.

The presence of Cu2+promoted a protection of the P. subcapitata acid phosphatase from thermal inactivation at 37°C for at least 1 h (Fig. 2), which supports an enzyme–Cu2+ interaction. Protection of enzymes by metal ions in stability studies was also described for fungal laccase by Cu2+ (Baldrian and Gabriel 2002), fungal peroxidase by Cd2+and Mn2+ (Youngs et al. 2000), and E. coli alkaline phosphatase by Cu2+and other metals (Trotman and Greenwood 1971).

The kinetic parameter apparent Km for pNPP decreased (3.3 fold) in the presence of Cu2+ (Table 1), suggesting an enhancement on the affinity enzyme-substrate by copper.

Bounias et al. (1996) also described a decrease of the Km value for honeybee gut acid phosphatase contained in the crude extract in the presence of copper. The apparent Km value of 0.37 mM, obtained in the presence of Cu2+is similar to those reported for other algae species when the enzyme was assayed in a non-preincubated system and in the absence of the metal (Bennum and Blum 1966; Patni and Aaronson 1974). The decrease of the activation energy value from 26.8 to 13.6 kJ mol−1 in the presence of Cu2+ (Table 1), also contributed to the activating effect of Cu2+on the P. subcapitata acid phosphatase activity.

Summarizing, different factors contributed to the increase in the acid phosphatase activity in the presence of Cu2+: protection of the enzyme against thermal inactivation, increase in the enzyme-substrate affinity, and reduction of the activation energy. The high sensitivity of the enzyme crude extract to copper may be useful as a tool to detect this metal in natural waters and other environmental samples.

We hope that the results presented in this work can improve the understanding of the basic events of the impact of copper at biochemical levels in primary producer organisms. Moreover, they could provide a sound basis for comparison with enzymatic processes affected by other pollutants.