Phytochemistry 57 (2001) 633–642 www.elsevier.com/locate/phytochem Vanadium haloperoxidases from brown algae of the Laminariaceae family M. Almeidaa, S. Filipea, M. Humanesa, M.F. Maiaa, R. Melob, N. Severinoa, J.A.L. da Silvac, J.J.R. Frau´ sto da Silvac,*, R. Weverd aCentro de Electroquı´mica e Cine´tica, Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias da Universidade de Lisboa, Edifı´cio C1-5 piso, Campo Grande, 1749-016 Lisbon, Portugal bInstituto de Oceanografia, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal cCentro de Quı´mica Estrutural, Complexo 1, Instituto Superior Te´cnico, Av. Rovisco Pais,1, 1049-001 Lisbon, Portugal dInstitute for Molecular Chemistry, University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS, Amsterdam, The Netherlands Received 25 July 2000; received in revised form 21 February 2001 Abstract Vanadium haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae — Laminaria saccharina (Linne´ ) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis–Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Laminaria saccharina; Laminaria hyperborea; Laminaria ochroleuca; Laminariaceae; Vanadium in biology; Vanadium-dependent haloperoxidases 1. Introduction lyse halogenation reactions of several substrates. These enzymes are found in many brown, in some red and in The bioinorganic chemistry of vanadium has aroused one green marine alga (Butler and Walker, 1993) and in much interest in recent years due to the curious roles the lichen Xantoria parietina (Plat et al., 1987). and states of this metal in various lower organisms (see Recently, vanadium dependent haloperoxidases were reviews by Chasteen, 1990; Wever and Kustin, 1990; also found in some fungi (van Schijndel et al., 1993; Butler and Carrano, 1991; Redher, 1991; Sigel and Sigel, Barnett et al., 1998). 1995; Tracey and Crans, 1998). One case which received All the vanadium haloperoxidases isolated to date more attention in the last few years is that of the vanadium share some common features: they are composed of one haloperoxidases, a new class of enzymes that contain or more subunits of relative molecular mass around 67 vanadium(V), as vanadate, in the active site and cata- kDa; they can be inactivated by dialysis against EDTA at low pH; their activity is only restored by addition of vanadium (as vanadate) and they seem to have similar * Corresponding author. Tel.: +351-1841-9000; fax: +351-2136- coordination of vanadium in the active site (Messersch- 12510. midt and Wever, 1996; Messerschmidt et al., 1997; E-mail address: [email protected] (J.J.R. Frau´ sto da Silva). Weyand et al., 1999). 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00094-2 634 M. Almeida et al. / Phytochemistry 57 (2001) 633–642 Vanadium haloperoxidases have been traditionally 2. Results and discussion classified as chloro, bromo and iodoperoxidases. Vana- dium chloroperoxidases have only been detected in ter- 2.1. Extraction of the enzymes restrial organisms, whereas bromo and iodoperoxidases are dominant in the marine environment. The algae from the Laminariaceae family are extre- Marine organisms, particularly seaweeds, produce mely rich in alginates and polyphenolic compounds that large quantities of halometabolites, which are held complicate the extraction and purification of these pro- within algal membrane bound vesicles and are thought teins. An aqueous salt/polymer two-phase system was to result from the catalytic activity of haloperoxidases. employed for the extraction. We have used poly- These halometabolites probably act as hormones or as ethyleneglycol (PEG) and potassium carbonate in this repellents in biological defence mechanisms (Jordan and system, since previous experience has shown that this Vilter, 1991). Interestingly in this respect, thirty years salt is the most adequate for the extraction of this par- ago, Siegel and Siegel (1970) proposed that an anom- ticular type of seaweed (Jordan and Vilter, 1991). From alous substrate specificity of haloperoxidases might be several compositions tested, the medium with 22.5% (w/ the reason why seaweeds are not lignificated. More v) K2CO3 and 15% (w/v) PEG 1500 in water was found recently, Ku¨ pper et al. (1998) reported that haloperox- to be the most efficient for the extraction of the halo- idases might be involved in the uptake of iodide from peroxidases from these algae. The vanadium haloperox- seawater, a process that, according to these authors, may idases (V-HPOs) were mainly found in the top-phase, be related to conditions of oxidative stress. In the same bounded to the polymer (PEG); the bottom aqueous year, ten Brink et al. (1998) reported that marine vana- phase, rich in salts, accumulated most of the alginates and dium haloperoxidases were able to catalyse enantio- hydrophilic compounds. The separation of the enzyme selective sulfoxidation reactions. Despite these many from the polymer was easily achieved by the addition of studies, the reason for the selection of vanadium for these acetone. After centrifugation the pellet thus obtained enzymes remains elusive since other more common bio- was dissolved in the minimum amount of 50 mM Tris– logical metals, e.g. iron (heme) or manganese could also HCl (pH 9.0). Since the pH is quite high, a partial loss have been used (Frau´ sto da Silva and Williams, 1991). of the prosthetic group occurred; activity was fully The isolation of more than one form of vanadium restored by addition of vanadate (20 mM). haloperoxidase has been reported for Ascophyllum nodosum (Krenn et al., 1989). Recently, two papers from 2.2. Purification of the enzyme — isoforms our laboratory were published showing that it is also possible to purify more than one form of the enzyme The extracts from the three seaweeds were subjected (Almeida et al., 1998; Almeida et al., 2000) from the to several chromatographic steps to purify the enzymes. brown seaweeds Sacchoriza polyschides and Pelvetia The first stage was a hydrophobic interaction step with canaliculata. Histochemical tests are a very important phenyl-sepharose. For L. saccharina and L. ochroleuca control tool in this kind of studies, since contamination two sets of fractions with activity (named Ls1 and Ls2; of the collected seaweeds with symbiont organisms Lo1 and Lo2, respectively) were obtained, whereas for living in and on the seaweeds is very common (Pede´ rsen the L. hyperborea extract only one set of active fractions and Fries, 1977). These controls were performed on A. was obtained. When subjected to electrophoresis under nodosum and indicated that the enzyme activity was non-denaturing conditions all these fractions revealed located on the surface of the cell wall of the thallus and several bands (results not shown); hence, a second chro- inside the alga between the cortex and the medulla and matographic step was necessary. especially around the conceptacles (Krenn et al., 1989). Two different approaches were used for the second Similar experiments in P. canaliculata revealed that purification step; a further hydrophobic interaction enzyme activity was located, on the surface cuticle of the chromatography with butyl-sepharose, for L. saccharina thallus (Almeida et al., 2000). and L. hyperborea, and an ion exchange chromato- Strangely, the seaweeds collected along the Portu- graphy with DEAE-Sephacel for L. ochroleuca. guese coast, which were previously studied, exhibited The active fraction from L. hyperborea yielded, after lower haloperoxidase activities than most of the algae this second chromatographic step two active bands from northern regions of the Atlantic Ocean (Almeida (named Lh1 and Lh2), while for L. saccharina this sec- et al., 1998; Almeida et al., 2000). To confirm if this is a ond step provided separation from most of the con- general trend, which would suggest an environment taminants. A chromofocusing chromatography was used dependence of the activity, we decided to carry out a as the final purification step and yielded the two forms of comparative study of the properties of the haloper- the vanadium-haloperoxidases (V-HPO) from L. sac- oxidases extracted from L. saccharina, L. hyperborea charina and L. hyperborea species. For L. ochroleuca the and L. ochroleuca since the Portuguese coast is the ion exchange chromatographic step with DEAE-Sepha- southern European habitat limit for these species. cel resolved the Lo2 fraction into a major fraction M. Almeida et al. / Phytochemistry 57 (2001) 633–642 635 (named a) and a minor fraction (named b). The Lo1 extraction procedure used by de Boer et al. (1986) is not fraction lost its activity very rapidly and could not be so selective; thus the fact that we have used the two- reactivated. phase aqueous system may explain why we found two Table 1 shows the specific activities of the purified forms isoforms in L. saccharina. The reason for the existence of from the three Laminariaceae studied.