Peridinin as the Major Biological Carotenoid Quencher of Singlet Oxygen in Marine Algae Gonyaulax polyedra Ernani Pinto,* Luiz Henrique Catalani,† Norberto Peporine Lopes,‡ Paolo Di Mascio,* and Pio Colepicolo* *Departamento de Bioquı ´mica and †Departamento de Quı ´mica Fundamental, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, C.P. 26077, CEP 05599-970, Sa ˜ o Paulo, Brazil; and ‡Departamento de Quı ´mica e Fı´sica, Faculdade de Cie ˆncias Farmace ˆuticas, Universidade de Sa ˜ o Paulo, Av. Do Cafe ´, s/n, CEP 14040-903, Ribeira ˜ o Preto, Brazil Received December 14, 1999 Carotenoids in light-harvesting proteins and reac- tion centers increase the overall efficiency of photo- synthesis by transferring absorbed light energy to chlorophylls. Peridinin and -carotene were isolated from Gonyaulax polyedra in a one-step purification protocol using the preparative circular chromatogra- phy (Chromatotron), performed on silica gel under N 2 atmosphere and n-hexane/acetone 8:2 as mobile phase and characterized by extensive 1 H NMR, infrared, and electrospray ionization mass spectrometry analyses. The quenching of singlet molecular oxygen [O 2 ( 1  g )] was evaluated by NIR-emission assays using singlet oxygen generated by sensitization of either perinaph- thenone or methylene blue. The NIR-emission assay showed that peridinin quench as singlet oxygen (k q  9.5  10 8 M 1 s 1 ) 5-fold less efficiently than -carotene (52  10 8 M 1 s 1 ). A method, based on the use of high- performance liquid chromatography with UV–VIS de- tection, was then developed for the sensitive quantifi- cation of peridinin (55% of total carotenoids) and -carotene (4.1% of total carotenoids). Thus, since peri- dinin is 10-fold more abundant than -carotene, it is expected to be the major protector against the delete- rious effects of O 2 ( 1  g ) in Gonyaulax polyedra. © 2000 Academic Press Key Words: Gonyaulax polyedra; peridinin; carote- noids; time-resolved NIR emission; singlet oxygen. Carotenoids are pigments widely distributed in na- ture, from bacteria, yeast and algae to plants, animals, and humans (1). They play an important role in pro- tecting cells and organisms. In photosynthetic organ- isms, the carotenoids protect the light-harvesting pig- ments (LHC) in the antenna complexes against photochemical damage caused by excited triplet states (2). Antioxidant activity is another role played by caro- tenoids (3). The photoprotection role of -carotene as singlet molecular oxygen [O 2 ( 1  g )] quencher was es- tablished in the photosystem II reaction center isolated from Pisum sativum (4). The peridinin (Fig. 1), an allenic oxi-carotenoid uniquely present in dinoflagel- lates, extends the range of absorption for light harvest- ing. It is associated with a protein and chlorophyll a, this protein complex is called peridinin– chlorophyll– protein (PCP) (5). Peridinin can quench O 2 ( 1  g ) and there are no reports on the rate constant of physical quenching of this carotenoid. In the dinoflagellate Am- phidinium carterae, each polypeptide of the PCP trim- mer contains 4 peridinin molecules per chlorophyll. This structure provides an efficient excitonic energy transfer from peridinin to chlorophyll (6). Previous work with the marine dinoflagellate Gonyaulax poly- edra indicated the presence of the carotenoids: peri- dinin, diadinoxanthin, dinoxanthin, diatoxanthin, and -carotene (7, 8). Furthermore, peridinin is an impor- tant biological compound that can suppress electroni- cally excited molecules. Since O 2 ( 1  g ) has been shown to be capable of inducing DNA damage and to be mu- tagenic (9), it is of great importance to determine the quenching ability of biological compounds as peridinin to establish what role they may play in protection against the photosensitization induced by their own chlorophyll. In this work, we isolated the most abun- dant carotenoids from G. polyedra and structurally characterized peridinin. In addition, we evaluated the physical quenching constant of this purified peridinin against generated O 2 ( 1  g ) and compared it with -carotene and lycopene using a time-resolved NIR- sensitized emission technique. Perinaphthenone or methylene blue were used as sensitizers (Sens) to pro- duce O 2 ( 1  g ) by transfer (Scheme 1, Eqs. [a] and [b]). Peridinin was isolated using circular chromatogra- phy, and characterized using high-performance liquid chromatography, 1 H NMR, infrared, and electrospray ionization mass spectrometry analyses. Concentration of peridinin in G. polyedra is very high compared to Biochemical and Biophysical Research Communications 268, 496 –500 (2000) doi:10.1006/bbrc.2000.2142, available online at http://www.idealibrary.com on 496 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.