Purification and Identification of Selenium-containing C-phycocyanin from Spirulina: Implications for Bioaccumulation and Ecotoxicity A Thesis Submitted to the Committee on Graduate Studies in Partial Fulfillment of the Requirements of the Degree of Master of Science in the Faculty of Arts and Science TRENT UNIVERSITY Peterborough, Ontario, Canada (c) Copyright by Jana Farell 2014 Environmental and Life Sciences M.Sc. Graduate Program January 2015 Abstract Purification and Identification of Selenium-containing C-phycocyanin from Spirulina: Implications for Bioaccumulation and Ecotoxicity Jana Farell Selenium is an essential trace nutrient to many organisms, yet in high concentrations it is toxic. Organic selenium is more bioavailable to aquatic biota than inorganic selenium, but is usually found in much lower concentrations. Algae are known to biotransform inorganic selenium into several organo-selenium compounds, but it is unknown whether any of these bioaccumulate in the food chain. In this study, selenium was incorporated into the methionine residues of an algal photosynthetic protein, c- phycocyanin from Spirulina spp. The extent of selenium incorporation was quantified by inductively coupled plasma-mass spectrometry (ICP-MS), and the protein was identified using electrospray mass spectrometry (ES-MS). C-phycocyanin was isolated and purified from Spirulina with a final recovery of 20-30 % of the total c-phycocyanin present. Selenomethionine replaced 92.8% ± 1.22 of the methionine residues in c-phycocyanin when grown in 2.5 ppm sodium selenite. ES- MS was used to obtain protein spectra, and pure c-phycocyanin was identified. Data of full scans provided estimated masses of both protein subunits—α-chain measured at 18,036 Da; β-chain measured at 19,250 Da—close to the theoretical masses. Protein fragmentation by collision-induced dissociation and electron capture dissociation provided approximately 52 % amino acid sequence match with c-phycocyanin from Spirulina platensis. This study demonstrates the incorporation of selenium into an algal protein, and the identification of c-phycocyanin using electrospray ionization-mass spectrometry. ii Keywords: organo-selenium, selenomethionine, c-phycocyanin, electrospray mass spectrometry iii Acknowledgements I would like to thank everyone who has contributed to my thesis. For their guidance and support, I would first like to thank my supervisor, Dirk Wallschläger, and my supervisory committee, Steven Rafferty and Neil Emery. A big thank you to Jacqueline London for help in the lab, particularly with temperamental nebulizers. Thanks to Naomi Stock for instrument training, troubleshooting, and protein mass spectrometry suggestions. A thank you to Bastian Georg and Michael Doran for instrument training and assistance. I would like to thank Katherine Kellersberger, without whose help obtaining protein mass spectra at Bruker Daltonic, this thesis would be woefully short. A special thanks to Sarah D’Amario for having patience with the trials of method development, and for having enthusiasm in the face of bleak results. Thanks to Kelly LeBlanc for algal culturing help, and other tips throughout my thesis. And thanks to Stephanie Jackman for CE tutorials, even though I never got around to using the instrument. iv Table of Contents Page number Abstract ii Acknowledgements iv List of Figures and Tables vii I. Introduction 1 1.1 Selenium as a trace element in natural waters 1.2 Selenium ecotoxicity and biogeochemistry 1.3 Selenium incorporation into proteins 1.4 Protein biodegradation 1.5 C-phycocyanin in Spirulina 1.6 Recombinant proteins 1.7 Protein mass spectrometry 1.8 Experimental objectives II. Methods 15 2.1 Recombinant c-phycocyanin expression 2.2 Algal growth conditions 2.3 C-phycocyanin purification from Spirulina 2.4 Inductively coupled plasma-mass spectrometry 2.5 Electrospray mass spectrometry III. Results and Discussion 22 3.1 Recombinant c-phycocyanin 3.2 C-phycocyanin yield and recovery from Spirulina 3.3 Selenium incorporation into c-phycocyanin 3.4 Evidence of c-phycocyanin 3.4.1 SDS-PAGE 3.4.2 Electrospray mass spectrometry 3.4.3 Selenium-containing c-phycocyanin v VI. Conclusions and outlook 47 References 49 vi List of Figures Page number Figure 1: Selenium biotransformation 3 Figure 2: C-phycocyanin crystal structure 9 Figure 3: Phycocyanobilin structure 10 Figure 4: Spirulina growth curve 17 Figure 5: SDS-PAGE of recombinant protein expression 23 Figure 6: SDS-PAGE of purified algal c-phycocyanin 32 Figure 7: ES-MS full scan of denatured c-phycocyanin 36 Figure 8: ES-MS fine structure 37 Figure 9: ES-MS full scan of non-denatured c-phycocyanin 40 Figure 10: ECD of non-denatured c-phycocyanin 41 Figure 11: CID of both side chains showing phycocyanobilin 42 Figure 12: ES-MS of a selenium-containing protein 45 Figure 13: ES-MS full scan of non-denatured Se-CPC and CPC 46 List of Tables Table 1: Algal growth and protein concentration 25 Table 2: Protein mass balance 28 Table 3: Selenium incorporation into c-phycocyanin 29 Table 4: Approximate c-phycocyanin mass 33 Table 5: Fragmentation sequencing 39 vii 1 I. Introduction 1.1 Selenium as a trace element in natural waters Over the past several decades, selenium has garnered increasing attention from the environmental community due to toxic effects to aquatic biota even at low concentrations. Selenium in most ecosystems is found in low concentrations. It is naturally occurring in the environment in shale deposits and other sedimentary rock formations (Presser et al., 1994), as well as in some sulfidic ores. Natural leaching by weathering is usually a slow process and so not a large factor in selenium toxicity. Conversely, anthropogenic activity can cause large selenium influxes to the environment. Some sources of anthropogenic selenium include irrigation, agricultural, and industrial wastewaters (Martin et al., 2011; Petrov et al., 2011). Additionally, commercial and pharmaceutical plants can be a source of selenium-contaminated wastewaters (Young et al., 2010). Dissolved selenium is primarily found in natural waters as selenate (SeVI) or selenite (SeIV), and to a lesser extent, selenide (Se-II) (Maher, et al., 2010). Selenium speciation in aqueous environments is predominantly determined by the reduction potential in the water (Cutter, 1982; Dungan and Frankenberger, 1999). Aside from reduction potential, chemical speciation is generally determined by microbially mediated transformations of mostly inorganic selenium into both inorganic and organic selenium species. This influences trophic transfer and bioaccumulation (Maher, et al., 2010). Since selenium ecotoxicity is determined by a number of factors, such as the chemical and biological species present, selenium contamination is assessed on a site- specific basis (Maher et al., 2010). Some selenium-contaminated sites have been reported 2 to have significantly higher than background concentrations; background concentrations for most sites are generally defined as < 1 µg/L. For example, in the Elk River Valley, total selenium concentrations in surface waters were reported to be over 20 µg/L (Martin et al., 2011). Such sites have been the focus of bioremediation efforts (Jones et al., 2009). 1.2 Selenium ecotoxicity and biogeochemistry Selenium shares many chemical properties with sulfur, which, due to sulfur’s biological prevalence, makes selenium an ecotoxicological concern. Selenium’s similar chemistry with sulfur means that it can compete with sulfur in biological uptake. Selenium is an essential trace element for many living organisms, yet it has a narrow nutritional margin. Indeed, its biological essentiality is in particular proteins, specifically as selenocysteine, the selenium analogue of sulfur containing cysteine. In most aquatic environments, selenium concentrations are low enough that they do not pose a threat to aquatic biota (Young et al., 2010). Aquatic organisms are known to take up selenium analogues of sulfur molecules; some aquatic plants can convert selenate and selenite into organic selenium compounds. In most aquatic environments, concentrations of these inorganic selenium compounds are too low to pose a toxicity threat to higher organisms. However, since organic selenium is much more bioavailable, it likely poses a larger bioaccumulation risk, even though it is found in much lower concentrations than its inorganic counterparts (Besser et al., 1993). Selenium bioconcentration occurs most dramatically in the first trophic transfer: from water to algae, selenium concentrations can increase by up to six orders of magnitude (Young et al., 2010). Algal selenium uptake is an active process, and, due to selenium’s chemical similarities to sulfur, selenium can be transported intracellularly in 3 place of sulfur through sulfate channels. Some algae are able to resist selenium toxicity even at high concentrations by producing volatile selenium compounds or by reduction to the relatively less toxic elemental selenium (Janz et al., 2010). Conversion to selenides happens rapidly after uptake of inorganic selenium, which is then converted to selenocysteine or selenomethionine (see Figure 1). Figure 1. Biotransformation of inorganic selenium into selenocysteine and selenomethionine. Algae in particular have been shown to significantly influence the chemical speciation. Organisms lower on the food chain, such as algae and macroinvertebrates, have high selenium tolerances compared to vertebrates. Aquatic organisms, such as fish, and their predators, such as birds,