Báo cáo khoa học: Kinetic properties of catecholoxidase activity of tarantula hemocyanin pot

Kinetic properties of catecholoxidase activity of tarantula hemocyanin Elmar Jaenicke and Heinz Decker Institut fu ¨ r Molekulare Biophysik, Johannes Gutenberg Universita ¨ t, Mainz, Germany Hemocyanins are oxygen transport proteins in the hemolymph of many arthropod and mollusk species. Although the overall molecular architecture of arthro- pod and mollusk hemocyanins seems to be unrelated, and to represent two different protein folds, their active site and immediate surroundings are very similar [1–4]. Both types of hemocyanin bind dioxygen in l:g 2 -g 2 coordination at type 3 copper centers [5,6]. Type 3 copper centers cannot only reversibly bind oxygen, as in hemocyanins, but can also activate and metabolize oxygen in enzymes such as tyrosinase (EC 1.14.18.1) and catecholoxidase (EC 1.10.3.1). Tyrosi- nase catalyzes the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphe- nols to o-quinones, whereas catecholoxidase only cata- lyzes the latter reaction [7–9]. The common name phenoloxidase is used for both tyrosinases and cat- echoloxidase of arthropod origin. Both enzymes are found in almost all organisms, and serve diverse func- tions, such as pigmentation, arthropod cuticle scleroti- zation after molting, wound healing and the innate immune response, where they kill pathogens by either encapsulating them with melanin or exposing them to highly reactive o-quinones [10,11]. The production of o-quinones by phenoloxidase is the first step in mela- nin biosynthesis [7,12,13]. The structure of phenol- oxidases involves two different types of molecular architecture. Arthropod phenoloxidases are very simi- lar to arthropod hemocyanins in terms of sequence, whereas phenoloxidases found in all other phyla seem to be more closely related to the protein fold of mol- lusk hemocyanin [3,14,15]. Keywords Eurypelma; hemocyanin; micelle; phenoloxidase; SDS Correspondence E. Jaenicke, Institut fu ¨ r Molekulare Biophysik, Johannes Gutenberg Universita ¨ t, Jakob Welder Weg 26, 55128 Mainz, Germany Fax: +49 6131 3923557 Tel: +49 6131 3923570 E-mail: elmar.jaenicke@uni-mainz.de (Received 2 January 2008, revised 22 January 2008, accepted 25 January 2008) doi:10.1111/j.1742-4658.2008.06311.x Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmenta- tion and sclerotization in arthropods. Many hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a pheno- loxidase has not been identified in the hemolymph, and thus hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for hemocyanin from several cheli- cerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The acti- vated state of Eurypelma hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated hemocyanin. For dopamine, a K M value of 1.45 ± 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated hemocyanin. This indicates that the activated hemocyanin behaves as a normal phenoloxidase. Abbreviations k cat , turnover number; K I , inhibition constant; K M , Michaelis–Menten constant; L-DOPA, L-dihydroxyphenylalanine; NADA, N-acetyldopamine; V max , maximum velocity. 1518 FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS Hemocyanins as oxygen carriers normally do not exhibit phenoloxidase activity. However, some are also latent phenoloxidases, as phenoloxidase activity can be induced by at least three different methods: firstly, by proteolytic cleavage of the N-terminus, resembling the activation of phenoloxidase by the phenoloxidase cas- cade [16,17]; secondly, by complex formation with either antimicrobial peptides or clotting enzyme in the horse- shoe crab [18,19]; thirdly, by interaction with amphi- philic compounds, such as SDS, which has recently been shown to induce a conformational change in hemocya- nin resulting in the activation of phenoloxidase activity [20]. This anionic synthetic compound is widely used to activate latent enzymatic activities, not only in pheno- loxidase, but also, for example, in the proteasome [21– 31]. In the proteasome, a reversible conformational change is induced in the presence of SDS, which mimics activation by fatty acids – a mode of activation that has also been demonstrated in phenoloxidases [26,27,32]. Most arthropods possess a phenoloxidase in their hemolymph, hemocytes or cuticle [11]. However, in some chelicerates, such as scorpions, tarantulas and horseshoe crabs, no phenoloxidase has been found to date, even though hemocytes from a tarantula were specifically examined for the presence of immune- related gene transcripts [33]. Because of their close relationship and physiological similarity with other arthropods, it seems unlikely that these animals totally lack a phenoloxidase, which is needed for important functions, such as cuticle sclerotization after molting and the immune response. Thus, it is proposed that, in these animals, hemocyanin substitutes for phenoloxi- dase, fulfilling its physiological function [2,3]. The acti- vation of hemocyanin by either antimicrobial peptides or clotting enzyme, as observed in the horseshoe crab Tachypleus tridentatus, supports this idea [18,19]. Although, in the last years, phenoloxidase activity has been reported for various hemocyanins, a thor- ough kinetic characterization of its activity is still lack- ing as, especially in crustaceans, the activity is very weak and transient, making characterization very diffi- cult [2]. In contrast, hemocyanin of the chelicerate Eurypelma californicum forms a stable activated state and its enzymatic properties are reported for the first time in this article. Results Activation of hemocyanin In the first experiment, the activation kinetics, i.e. the time dependence of the development of phenoloxidase activity after the addition of SDS, were studied (Fig. 1). After the addition of SDS, the catecholoxi- dase activity of Eurypelma hemocyanin developed gradually over a period of 5 min until a constant activ- ity was reached (Fig. 1). As an equilibrium condition (i.e. constant activity) is a prerequisite for the measure- ment of enzyme kinetics, the conditions with constant activity were established. This was accomplished by preincubating hemocyanin with 5 mm SDS for 5 min before adding the substrate. Using this condition, which was employed for all further experiments, the reaction proceeded at a constant rate for at least 5 min more (Fig. 1). Dependence of activation on SDS concentration SDS has a complex behavior when dissolved in aque- ous medium, as it exists as a monomer at low concen- trations and forms micelles at concentrations higher than the critical micellar concentration. To determine how the SDS concentration influences the catecholoxi- dase activity of hemocyanin, the catecholoxidase activ- ity was measured at different SDS concentrations and, furthermore, under conditions with different critical micellar concentrations (Fig. 2). The critical micellar concentration of SDS in sodium phosphate buffer depends almost exclusively on the sodium concentra- tion; therefore, sodium phosphate buffers with differ- ent concentrations were used to set different critical micellar concentrations [34]. Critical micellar concen- trations of 1.3 ± 0.1, 1.9 ± 0.1 and 4.1 ± 0.1 mm were observed by conductivity measurements for phosphate buffers with concentrations of 0.1, 0.05 and Fig. 1. Activation kinetics of hemocyanin by SDS. Eurypelma hemocyanin (0.7 mgÆmL )1 ) was activated with SDS in 0.1 M phos- phate buffer (pH 7.0) at 30 °C by incubating hemocyanin with 5 m M SDS for periods between 15 s and 8 min. The reaction was started by the addition of 2 m M dopamine, and the activity was determined by measuring the absorption increase at 475 nm for 10 s. E. Jaenicke and H. Decker Catecholoxidase activity of tarantula hemocyanin FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS 1519 0.01 m, respectively (Fig. 3). For the activation of hemocyanin, a sigmoidal concentration dependence was observed, and the concentration of half-maximum activation always coincided with the critical micellar concentration of the respective buffer concentration (Fig. 2). This observation proves that hemocyanin is activated by SDS micelles, as suggested recently [20]. The lowest SDS concentration at which full activation is reached under all conditions is 5 mm. Consequently, this concentration was used for further experiments. Under these conditions, hemocyanin is activated by SDS, which induces a conformational switch in cheli- cerate hemocyanins, without any destructive effect on protein structure [20]. Stability of the activated state Many hemocyanins, especially those of crustaceans, exhibit only very weak or transient activity when acti- vated by SDS. This can be attributed to the fact that, unlike chelicerate hemocyanins, the conformational change evoked by activation by SDS harms crustacean hemocyanin and leads to a loss of activity within a few minutes [35]. To ensure that the catecholoxidase activ- ity of activated tarantula hemocyanin remains con- stant, the activity of hemocyanin was measured after exposure to 5 mm SDS for several hours (Fig. 4). The enzymatic activity remained almost unchanged within a 12 h period in the presence of SDS, indicating a stable activated state. 100 Activity (%) 80 60 012345 SDS concentration (m M) 67 40 20 0 Fig. 2. Dependence of activation on the SDS concentration. The dependence of activation on the SDS concentration and the critical micellar concentration of SDS was assayed in phosphate buffer (pH 7.0) at 20 °C. The hemocyanin concentration was 0.25 mgÆmL )1 and 1 mM dopamine was used as substrate. First, hemocyanin was preincubated with SDS for 5 min, and then the reaction was started by the addition of substrate. The reaction was followed for 5 min by measuring the absorption at 475 nm. The critical micellar concentration of SDS was varied using sodium phosphate buffer at three concentrations: , 0.01 M; , 0.05 M; d, 0.1 M. The activity is given as the percentage of the maximum activity measured at any SDS concentration in the experiment. Fig. 3. Determination of the critical micellar concentration of SDS. The critical micellar concentration was determined by conductivity measurements in phosphate buffer (pH 7.0). Critical micellar concentrations of 1.3 ± 0.1, 1.9 ± 0.1 and 4.1 ± 0.1 m M were observed for phosphate buffers with concentrations of 0.1 M (A), 0.05 M (B) and 0.01 M (C), respectively. The critical micellar concen- tration (broken line) is defined as the intersection between the extrapolated conductivity slope of monomers and micelles (full lines), which differ in their electrophoretic mobility because of their different size. Catecholoxidase activity of tarantula hemocyanin E. Jaenicke and H. Decker 1520 FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS Substrate turnover and substrate specificity of activated hemocyanin After establishing the activation conditions, the cat- echoloxidase activity of activated Eurypelma hemocya- nin was characterized more closely with respect to substrate turnover and substrate specificity. The experi- ments concerning substrate specificity used only diphenolic substrates, as monophenolase activity was never observed under the experimental conditions used (results not shown). The concentration dependence of substrate turn- over was characterized using dopamine as substrate and 5 mm SDS for activation (Fig. 5). At high dopa- mine concentration, a decrease in activity was observed, indicating substrate inhibition. The highest activity was observed at a dopamine concentration of 6 mm. The Michaelis–Menten constant (K M ) was determined by nonlinear fitting using normal Micha- elis–Menten kinetics in the concentration range below 4mm where no substrate inhibition was observed (Fig. 5). In this way, the K M value was determined to be 1.45 ± 0.16 mm, the maximum velocity (V max ) 5.5 ± 0.4 lmÆs )1 and the resulting turnover number (k cat ) 6.0 ± 0.4 s )1 . When the data were fitted to the model for substrate inhibition (Eqn 2), similar values were obtained, i.e. K M = 1.22 ± 0.20 mm, V max = 5.25 ± 0.34 lmÆs )1 and k cat = 5.7 ± 0.3 s )1 (Fig. 5). The inhibition constant ( K I ) was deter- mined to be 29.64 ± 4.98 mm, thus revealing that the substrate molecules at the active site bind with a 25-fold higher affinity than inhibitory substrate molecules. Substrate specificity To investigate the substrate specificity of activated hemocyanin, the turnover of several diphenols was compared using an oxygen electrode (Fig. 6). For this comparison, a set of physiologically important diphe- nols, such as dopamine, l-dihydroxyphenylalanine (l-DOPA), N-acetyldopamine (NADA), epinephrine and norepinephrine, was used. Catechol, although normally not of physiological significance, was also included, because it is the smallest diphenol. The high- est substrate concentration tested was limited by the solubility of the respective substrate. Thus, because of their low solubility, l-DOPA, NADA, epinephrine and norepinephrine could not be measured at concentra- tions at which the maximum velocity is expected; con- sequently, some of the curves did not approach maximum velocity closely enough to yield a reliable nonlinear fit of K M and V max . Thus, it was only possi- ble to determine the catalytic efficiency (k cat ⁄ K M ) (Eqn 1). The best substrate was dopamine, with a catalytic efficiency at least five-fold higher than that of the other substrates (Table 1). Dopamine is an important metabolite for sclerotization and melanization of the 1.0 0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 50 60 Incubation time (min) v 0 (M·min –1 ) × 10 –4 200 400 600 Fig. 4. Stability of the activated state of hemocyanin. Eurypelma hemocyanin (0.19 mgÆmL )1 ) was preincubated with 5 mM SDS for periods between 5 min and 12 h in 0.1 M phosphate buffer (pH 7.0) at 25 °C. The reaction was started by the addition of 1 m M dopa- mine as substrate, and the activity was determined by measuring the absorbance at 475 nm. 300 250 200 150 100 50 0 0 10 20 Dopamine concentration (m M) 30 40 50 v 0 (µM·min –1 ) Fig. 5. Substrate inhibition of activated Eurypelma hemocyanin. The enzymatic activity of Eurypelma hemocyanin (0.4 mgÆmL )1 ) was assayed at an SDS concentration of 5 m M in 0.1 M phosphate buffer (pH 7.0). To measure at a constant rate, Eurypelma hemo- cyanin was incubated with SDS for 5 min before addition of the substrate (Fig. 1). Data analysis in the substrate range 0.3–4 m M (broken line) revealed a K M value of 1.45 ± 0.16 mM and a k cat value of 6.0 ± 0.4 s )1 . At substrate concentrations above 4 mM, substrate inhibition was observed and the data were fitted to a sim- ple model for substrate inhibition (Eqn 2). K M = 1.22 ± 0.20 mM, k cat = 5.7 ± 0.3 s )1 and K I = 29.64 ± 4.98 mM were obtained for the inhibitory binding site (full line). E. Jaenicke and H. Decker Catecholoxidase activity of tarantula hemocyanin FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS 1521 cuticle, giving physiological importance to this obser- vation [10,36]. NADA, epinephrine, norepinephrine and l-DOPA had quite similar but lower catalytic effi- ciencies. The lowest catalytic efficiency was observed for catechol. This is probably a result of the fact that catechol is the smallest diphenol and is therefore diffi- cult to maintain in a stable position at the active site needed for oxidation. Inhibition of activated hemocyanin The effect of typical inhibitors of phenoloxidase on activated tarantula hemocyanin was assayed (Fig. 7). For this experiment, dopamine was used as substrate and l-mimosine, kojic acid, tyramine, phenythiourea and azide were used as inhibitors. All five inhibitors are known to inhibit phenoloxidases, which bind to the active site or in the substrate binding pocket. As a result of their structural similarity to the substrates, this is easily rationalized for l-mimosine, kojic acid and tyramine, which inhibit by competitive inhibition [37]. It should be noted that tyramine is also a sub- strate for tyrosinase. However, as the tyrosinase activ- ity of activated hemocyanin was not observed under our assay conditions, we were able to use tyramine as a substrate analog (i.e. competitive inhibitor). Phenyl- thiourea is a strong inhibitor, binding with its sulfur atom between the copper atoms of the active site, as observed in the crystal structure of sweet potato cat- echoloxidase [8]. Azide interacts with type 3 copper centers and inhibits tyrosinase, although the mode of inhibition has not yet been clearly established [38,39]. All five inhibitors inhibited the phenoloxidase activity of activated Eurypelma hemocyanin (Fig. 7). Fitting the enzymatic data using a competitive mecha- nism (Eqn 3) yielded K I values of 11.9 ± 1.3 and 47.7 ± 5.2 lm for kojic acid and l-mimosine, respec- tively. Inhibition by tyramine was one order of magni- tude weaker, with a K I value of 278.2 ± 30.6 lm. Fitting attempts revealed that inhibition by phenylthio- urea and azide was not in accordance with a competi- tive binding model. Thus, the binding affinity of the inhibitors could not be determined by fitting the data. 300 1000 800 600 400 200 0 250 200 150 100 50 0 0123 Substrate concentration (m M) Dopamine L-dopa NADA Epinephrine Norepinephrine Catechol (m M) Catechol 4 5 6 7 8 9 0 20406080100 v 0 (µM·min –1 ) v 0 (µM·min –1 ) Fig. 6. Substrate specificity of activated hemocyanin. The turnover of several different diphenols by activated Eurypelma hemocyanin (0.42 mgÆmL )1 ) was measured with a Clark-type oxygen electrode in 0.1 M phosphate buffer (pH 7.0). Hemocyanin was preincubated with 5m M SDS for 5 min before addition of the substrate (Fig. 1) to ensure measurement at a constant rate. The highest substrate concentration used was limited by the solubility of the respective substrate. Catechol, which is much more soluble than the other diphenols, was mea- sured to concentrations up to 100 m M (inset). For each substrate concentration, two independent measurements were made and the mean is given in the graph. Data were analyzed by fitting the catalytic efficiency (k cat ⁄ K M ) in the linear part of the curve (Table 1). The lines con- necting the data are shown to facilitate the identification of the different substrate curves and do not represent a fit. Table 1. Catalytic efficiency of activated hemocyanin for different substrates. Data from the comparison of different substrates (Fig. 6) were analyzed using linear fitting, as described in the text, to obtain the catalytic efficiency (k cat ⁄ K M ). Substrate k cat ⁄ K M (mM )1 Æs )1 ) Dopamine 3.91 ± 0.55 NADA 0.68 ± 0.08 Norepinephrine 0.66 ± 0.10 Epinephrine 0.60 ± 0.09 L-DOPA 0.59 ± 0.08 Catechol 0.20 ± 0.03 Catecholoxidase activity of tarantula hemocyanin E. Jaenicke and H. Decker 1522 FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS Nevertheless, phenylthiourea is obviously the strongest inhibitor by far as it inhibits strongly at the micro- molar concentrations used. The binding affinities of l-mimosine and kojic acid to the active site were quite similar, but they inhibited the phenoloxidase activity of hemocyanin to a much lesser degree than did phen- ylthiourea. The binding affinity of the monophenol tyramine as an inhibitor was much weaker than that of l-mimosine and kojic acid, and azide was the weak- est inhibitor of all five inhibitors tested. Therefore, activated Eurypelma hemocyanin is inhibited by the same inhibitors and in the same way as other phenol- oxidases. Discussion Phenoloxidase, especially catecholoxidase, activity has been observed in many arthropod hemocyanins. Sev- eral chelicerate species, such as tarantulas, horseshoe crabs and scorpions, seem to lack a phenoloxidase, although considerable effort has been made to identify one. Given the fact that these animals need a phenol- 3.0 L-Mimosine A D E B C Phenylthiourea Azide Kojic acid Tyramine 2.5 2.0 1.5 1.0 0.5 0.0 0 1 2 Dopamine concentration (m M) Dopamine concentration (mM) Do p amine concentration ( mM ) Do p amine concentration ( mM ) Dopamine concentration (m M) 3 4 5 6 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 0 1 2 3 4 5 6 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 v 0 (M·min –1 ) × 10 –4 v 0 (M·min –1 ) × 10 –4 Fig. 7. Inhibition of activated hemocyanin. The inhibition of SDS-activated hemocyanin (0.42 mgÆmL )1 ) by five inhibitors of catecholoxidase was assayed in 0.1 M phosphate buffer (pH 7.0). Before initiation of the reaction by the addition of the substrate dopamine, hemocyanin was preincubated for 5 min with 5 m M SDS and the indicated concentration of inhibitor. For each inhibitor ⁄ substrate concentration, one experiment was performed. The data for the three competitive inhibitors L-mimosine, kojic acid and tyramine were fitted in parallel according to Eqn (3). Kojic acid and L-mimosine inhibited enzymatic activity with K I values of 11.9 ± 1.3 and 47.7 ± 5.2 lM respectively. Inhibition by tyramine was one order of magnitude weaker with K I = 278.2 ± 30.6 lM. Note that tyramine is not a substrate of activated hemocyanin under the conditions used. Data for the inhibitors phenylthiourea and azide were not analyzed further because of the lack of a suitable inhibi- tion model. (A) L-Mimosine: d,0mM; , 100 lM; , 250 lM; r, 500 lM; (B) kojic acid: d,0mM; ,20lM; ,50lM; r, 150 lM; (C) tyra- mine: d,0m M; , 0.2 mM; , 0.6 mM; r, 1.6 mM; (D) phenylthiourea: d,0mM; ,3lM; ,6lM; r,9lM; (E) azide: d,0mM; , 3.0 mM; , 4.5 mM; r, 6.0 mM. E. Jaenicke and H. Decker Catecholoxidase activity of tarantula hemocyanin FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS 1523 oxidase, as all known arthropods do, it seems that their hemocyanin replaces the phenoloxidase in their hemolymph, as shown for the horseshoe crab Tachyp- leus [18,19]. Arthropod phenoloxidases generally are produced as inactive prophenoloxidases, which can be activated by limited proteolysis [11,40,41]. Interaction with fatty acids and phospholipids can also activate prophenoloxidase, although the physiological signifi- cance of this method of activation has not been estab- lished in arthropods [32,42]. Hemocyanin can also be considered as a propheno- loxidase, as it is activated by the same mechanisms as those which activate phenoloxidase, and can be inhib- ited by the same inhibitors [25,35]. An additional method of activation was found for the hemocyanin of the horseshoe crab Tachypleus by binding of anti- microbial peptides and proteins of the hemolymph coagulation cascade to hemocyanin [18,19]. In the laboratory, activation by SDS mimics the activation by fatty acids and phospholipids, and is accepted as the standard method for prophenoloxidase and hemocyanin activation [21–31]. At first sight, SDS, as an unnatural synthetic agent, may not seem to be the compound of choice to study the physiological function of a protein. Nevertheless, for practical rea- sons (i.e. solubility, availability), it has been used to activate proenzymes, including even the proteasome [26,27]. In addition, the real physiological activators are often unknown, and thus the use of an activator such as SDS is warranted to learn more about the functional properties of an enzyme. Generally, binding of SDS during activation causes a conformational change, as shown for the proteasome, phenoloxidases and, recently, for hemocyanin [20,26,27,31,43]. In our experiments, the activation curves for hemo- cyanin by SDS showed a sigmoidal behavior, with a midpoint which always coincided with the critical micellar concentration of SDS. Recently, the hypothe- sis that SDS micelles are needed to induce a conforma- tional change, which activates hemocyanin, was suggested [20]. Our observation that the critical micel- lar concentration and SDS midpoint concentrations of activation strictly coincide confirms this hypothesis, and indicates that free SDS monomers are not suffi- cient for activation under our experimental conditions (Figs 2 and 3). The nature of the interaction between the hemocyanin multimer and SDS micelles is not known, and it is unclear why the conformational change induced by this interaction does not occur instantly but requires several minutes for completion (Fig. 1). However, the activated state is stable, as only small variations in activity were observed over the time course of several hours (Fig. 4). Although tyrosinase and catecholoxidase from plants and fungi have been characterized in great detail, only very few enzymatic data are available on phenoloxidases from arthropods, where most studies have focused only on establishing the enzymatic activ- ity. Even less is known about the enzymatic parame- ters of activated hemocyanin. Most studies on the phenoloxidase activity of hemocyanin in the past have focused on the activity in the species investigated and ⁄ or the way in which hemocyanin is activated. Only for a few crustacean hemocyanins have the enzy- matic properties been reported to a small extent [17,44,45]. In all of these cases, the activity was very weak and the activated state was not stable, therefore limiting the enzymatic analysis. Therefore, the enzy- matic properties of the phenoloxidase activity of a chelicerate hemocyanin have not been investigated thoroughly previously. We have attempted to close this gap with this study. The enzymatic parameters of acti- vated hemocyanin and selected parameters reported in the literature are compared in Table 2. Activated hemocyanin is able to turn over a wide range of diphenolic substrates. Activated hemocyanin only accepts o-diphenols as substrates (i.e. catecholase activity) under our experimental conditions, and lacks the ability to hydroxylate monophenols in the ortho- position (i.e. monophenolase activity). The reason why Table 2. Enzymatic parameters of activated hemocyanin in com- parison with other phenoloxidases. Activated hemocyanin Other phenoloxidases K M 1.45 ± 0.16 mM (dopamine) 0.72 ± 0.08 m M (dopamine, Agaricus [53]) 0.28 ± 0.01 m M (L-DOPA, Agaricus [53]) 1.04 m M (L-DOPA, Neurospora [54]) 8.9 m M (L-DOPA, Streptomyces [37]) 0.54 ± 0.05 m M (tyramine, Agaricus [53]) k cat 6.0 ± 0.4 s )1 (dopamine) 475 ± 17 s )1 (dopamine, Agaricus [53]) 107 ± 2 s )1 (L-DOPA, Agaricus [53]) 1070 s )1 (L-DOPA, Neurospora [54]) 25 ± 1 s )1 (tyramine, Agaricus [53]) K I (L-mimosine) 47.7 ± 5.2 lM 30 ± 3 lM (Streptomyces [37]) K I (kojic acid) 11.9 ± 1.3 lM 3.4 ± 0.3 lM (Streptomyces [37]) Catecholoxidase activity of tarantula hemocyanin E. Jaenicke and H. Decker 1524 FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS activated hemocyanin is unable to catalyze the mono- hydroxylation reaction is not known. With regard to diphenolic substrates, the best substrate by far is dopa- mine, whereas other diphenols are converted at a much lower catalytic efficiency, with almost identical values for all diphenols except catechol (Table 1). Dopamine is an important substrate in arthropods for cuticle sclerotization after molting [46,47]. This observation is in good agreement with the fact that, in the tarantula Eurypelma, hemocyanin has been identified as part of the protein components of the cuticle, and similar observations have been made in crustaceans [44,48,49]. The substrate affinity for dopamine is within the nor- mal range observed for other phenoloxidases, which varies over almost one order of magnitude depending on the substrate (Table 2). Unfortunately, no data are available on physiological diphenol levels in chelicer- ates, and thus a comparison of how substrate affinity and physiological substrate levels relate to one another is not possible. The turnover rate for dopamine is almost two orders of magnitude lower than the dopa- mine turnover in other phenoloxidases. Nevertheless, turnover rates as low as 25 s )1 have been reported for the monophenol tyramine, and this turnover rate is only four-fold higher than the dopamine turnover by activated hemocyanin. At first sight, the low turnover rate observed for activated hemocyanin seems to make a physiological role for this molecule less likely. How- ever, it must be considered that hemocyanin, unlike other phenoloxidases, is present at concentrations of at least 40 mgÆmL )1 in the hemolymph. These high con- centrations, which are orders of magnitude higher than the concentrations found for other phenoloxidases, could make up for the lower turnover rates. Unexpectedly, strong substrate inhibition was observed for the substrate dopamine, which has not been reported previously for other phenoloxidases. Recently, the first study on the detailed enzymatic parameters of a hexameric arthropod phenoloxidase has also reported substrate inhibition [50]. Thus, it is possible that substrate inhibition is common amongst arthropod phenoloxidases, but has not been reported previously simply because of a lack of studies. The inhibition of activated hemocyanin by typical inhibi- tors of phenoloxidase was similar to the inhibition observed in phenoloxidase (Table 2). In conclusion, it has been shown that tarantula hemocyanin exhibits the properties of a common phe- noloxidase, and consequently may function as such when activated appropriately in the animal. In the future, further efforts will be made to determine the physiological activators and regulatory mechanisms in vivo. Experimental procedures Reagents All reagents used were of the highest purity and were pur- chased from Sigma (Steinheim, Germany). Ultrapure water (Milli-Q-Plus-PF; Millipore, Eschborn, Germany) was used for all solutions. Purification of hemocyanin Tarantulas (Eurypelma californicum) were obtained from the North Carolina Biological Supply (Charlotte, NC, USA). Hemolymph was collected by dorsal puncturing of the pericard and immediately diluted 1 : 1 with stabilization buffer (0.1 m Tris ⁄ HCl, 5 mm MgCl 2 ,5mm CaCl 2 , pH 7.8). On average, 100 lL of hemolymph was collected from one spider. Therefore, it was necessary to pool the hemolymph of five or six spiders to obtain a sufficient hemolymph volume for the purification procedure. The hemolymph was centrifuged at 15 000 g for 10 min at 4 °C to remove cellular debris. The supernatant containing hemocyanin was applied to a Sephacryl S-300 16 ⁄ 60 HR size exclusion column (GE Healthcare Biosciences, Uppsala, Sweden). The column was eluted with stabilization buffer at a flow rate of 0.6 mLÆmin )1 at room temperature. Hemocyanin-containing fractions were identified by their absorbance at 340 nm and stored at 4 °C. Only fractions containing 24-meric hemocyanin were used for the experiments. The protein concentration of hemocyanin samples was determined by measuring the absorbance at 278 nm using the molar extinction coefficient [e 278 (nm) = 1.1 mLÆmg )1 Æcm )1 ] for chelicerate hemocyanin. Hemocyanin samples were concentrated in Biomax30K centrifugal filters (Millipore), when necessary. Determination of the critical micellar concentration The critical micellar concentration of SDS was determined by conductivity measurements using a Model 712 Conduc- tometer (Metrohm, Herisau, Switzerland) [34]. During the measurement, the temperature of the sample was kept con- stant at 20 °C in a water bath. Enzymatic assays The phenoloxidase activity of hemocyanin was followed spectroscopically or with a Clark-type oxygen electrode. For the spectroscopic assay, the formation of dopa- chrome was measured at 475 nm with a Hitachi U-3000 photometer (Hitachi, Tokyo, Japan) [51]. Quartz cuvettes (Hellma, Mu ¨ llheim, Germany) with an optical path length of 1 cm were used. Dopachrome is unstable and reacts to E. Jaenicke and H. Decker Catecholoxidase activity of tarantula hemocyanin FEBS Journal 275 (2008) 1518–1528 ª 2008 The Authors Journal compilation ª 2008 FEBS 1525 derivatives after a few minutes; thus, only the first 5 min after initiation of the reaction were measured. All measure- ments were performed at 25 °C in 0.1 m phosphate buffer (pH 7.0) unless noted otherwise. Three different protocols were used. For the first protocol, hemocyanin and dopamine were mixed and the reaction was initiated by the addition of activator (SDS). For the second protocol, hemocyanin was preincubated with activator (SDS) for 5 min and the reaction was then initiated by the addition of dopamine. The third protocol was used for inhibition experiments. The inhibitor was preincubated with hemo- cyanin and activator (SDS) for 5 min and the reaction was initiated by the addition of dopamine. The dead time between initiation of the reaction and the measurement was less than 15 s in all protocols. The unstable dopamine solutions were prepared fresh daily and kept on ice in the dark to minimize the auto-oxidation of dopamine. A Clark-type oxygen electrode (Hansatech Oxygen-Elec- trode DW1, Saur Laborbedarf, Reutlingen, Germany) was used to compare the turnover of different substrates by fol- lowing the oxygen consumption in the reaction mixture. The same conditions and protocols as described for the spectroscopic measurements were used. In all experiments, dopamine was used as substrate unless noted otherwise, as it was the best substrate when the sub- strate specificity was tested; furthermore, its good solubility allowed experiments at high substrate concentrations. At the beginning of the enzymatic measurements, the experimental error of the enzymatic assays was determined for both the spectroscopic and oxygen electrode assays by measuring the turnover ten times under identical condi- tions. The experimental error amounted to ± 5%. Data analysis Enzyme kinetic data were analyzed using the program Sigma-Plot 2000 (Systat Software, Erkrath, Germany). Data for uninhibited kinetics were fitted by nonlinear regression to uninhibited Michaelis–Menten kinetics. Some of the substrates showed poor solubility, and there- fore the kinetic data only covered the part of the curve at low concentration relative to the respective K M value, where saturation had not yet been reached. In these cases, K M and k cat could not be determined with confidence. However, at substrate concentrations lower than K M , the catalytic efficiency (k cat ⁄ K M ) could be determined from the initial slope of the curve: v 0 ¼ k cat K M ½E T ½Sð1Þ where v 0 is the initial enzymatic rate, [S] is the substrate concentration, k cat is the turnover number and [E T ] is the enzyme concentration. Substrate inhibition was fitted according to the simplest model, assuming that a second substrate molecule can bind to the enzyme–substrate complex and render it inactive [52]. Data were fitted according to: v 0 ¼ v max ½S ½SþK M þ ½S 2 K I ð2Þ where K I is the binding constant for the inhibitory substrate molecule. The data for the three competitive inhibitors kojic acid, l-mimosine and tyramine were fitted in parallel for all three inhibitors according to: v 0 ¼ V max ½S ½SþK M 1 þ ½I 1  K IM þ ½I 2  K IK þ ½I 3  K IT  ð3Þ where K IM is the inhibition constant for l-mimosine, K IK is the inhibition constant for kojic acid and K IT is the inhibi- tion constant for tyramine. Although no combinations of inhibitors were tested, all the enzymatic data for the three competitive inhibitors were fitted in parallel to ensure that only one K M value and one V max value were obtained for the uninhibited reaction. Fitting the data for the three inhibitors individually would have resulted in three K M and three V max values. For the calculation of the enzyme concentration, it was assumed that only subunit types b and c, i.e. four of the 24 subunits which make up the native hemocyanin molecule, possess enzymatic activity [16,25]. 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To ensure that the catecholoxidase activ- ity of activated tarantula hemocyanin remains con- stant, the activity of hemocyanin was measured after exposure