International Journal of Biological Macromolecules 45 (2009) 181–187 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Inﬂuence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin Krassimira Idakieva a,∗ , Nurul Islam Siddiqui b , Filip Meersman c , Marc De Maeyer b , Irena Chakarska a , Constant Gielens b,∗∗ a Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str. Bl. 9, Soﬁa 1113, Bulgaria b Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200 G, 3001 Leuven-Heverlee, Belgium c Division of Molecular and Nanomaterials, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200 F, 3001 Leuven-Heverlee, Belgium article info Article history: Received 13 March 2009 Received in revised form 28 April 2009 Accepted 29 April 2009 Available online 6 May 2009 Keywords: Hemocyanin Rapana thomasiana Phenoloxidase activity abstract The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by lim- ited treatment with subtilisin and by incubation of RtH with 2.9 mM sodium dodecyl sulphate (SDS), suggesting an enhanced substrate access to the active sites. The highest artiﬁcial induction of o-diPO activity in RtH, however, was obtained by lyophilization of the protein. This is ascribed to conformational changes during the lyophilization process of the didecameric RtH molecules, affecting the accessibility of the active sites. These conformational changes must be very small, since Fourier-transform infrared and circular dichroism spectroscopies did not reveal any changes in secondary structure of lyophilized RtH. The difference in accessibility of the copper containing active site for substrates between catechol oxidase and functional unit RtH2-e was demonstrated by molecular modeling and surface area accessi- bility calculations. The low level of intrinsic PO activity in the investigated hemocyanin is related to the inaccessibility of the binuclear copper active sites to the substrates. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Hemocyanin (Hc) is a member of the type-3 copper protein family, which further includes tyrosinases (Tys) (EC 1.14.18.1) and catechol oxidases (COs) (EC 1.10.3.1). Type-3 copper proteins con- tain a dinuclear copper active site that is comprised of two closely spaced copper atoms each coordinated by 3 histidine nitrogen atoms of the polypeptide chain. Oxygen is reversibly bound to the active site as - 2 : 2 peroxide [1,2]. Although the dinuclear site is highly conserved as witnessed by its characteristic spectroscopic properties, sequence homology and the available crystal structures of Hcs [3–6], CO [7], and Ty [8], the functionality of these proteins is different. Hcs function as oxygen carriers and oxygen storage pro- teins in several species of arthropods and molluscs, Tys catalyze both the o-hydroxylation of monophenols to o-diphenols (tyrosi- nase or monophenoloxidase activity) and subsequent oxidation of o-diphenols to o-quinones (catecholase or diphenoloxidase activ- ity) and COs catalyze only the second reaction. ∗ Corresponding author. Tel.: +359 2 9606190; fax: +359 2 8700225. ∗∗ Corresponding author. Tel : +32 16 327319; fax: +32 16 327978. E-mail addresses: idakieva@orgchm.bas.bg (K. Idakieva), constant.gielens@chem.kuleuven.be (C. Gielens). It is generally accepted that one of the main reasons for the absence or low level of phenoloxidase activity in Hcs is related to the inaccessibility of the type-3 center to potential substrates [9]. It has been shown that the oxygen-binding function of Hc can be converted to phenoloxidase (PO) activity and furthermore that PO activity can be induced in Hcs by in vivo and in vitro activa- tion [10]. Compared to the numerous studies of arthropodan Hcs, the enzymatic activity of molluscan Hcs has been less investigated. Hcs isolated from the cephalopods Octopus vulgaris and Sepia ofﬁc- inalis as well as from the gastropods Helix pomatia and Rapana venosa (synonym of Rapana thomasiana) have been demonstrated to exhibit o-diphenoloxidase activity (o-diPO) [11–13]. The functional units (FUs) mainly responsible for both the intrinsic PO activity and the enhanced PO activity after induction by limited proteolysis have been recently identiﬁed for the ﬁrst time for S. ofﬁcinalis and H. pomatia Hc [12]. In this study we further investigate the intrinsic and inducible PO activity of R. thomasiana hemocyanin (RtH) and its substructures to gain further insight into the enzymatic properties of molluscan Hcs. The inﬂuence of limited proteolysis and of sodium dodecyl sul- phate (SDS), known as an artiﬁcial activator of phenoloxidases and of phenoloxidase activity in arthropodan Hcs [14], is studied. Also the effect of lyophilization of the protein on the enzyme activity is investigated. 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.04.022