Interaction of Chlorin p6 with bovine serum albumin and photodynamic oxidation of protein Biplab Bose * , Alok Dube Biomedical application section, Block-D, Center for advanced Technology, Indore 452013, India Received 29 December 2005; received in revised form 25 March 2006; accepted 4 April 2006 Available online 9 June 2006 Abstract The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern–Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62 ± 0.27 · 10 5 M 1 and 1.086 ± .019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein car- bonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS–polyacrylamide gel electropho- resis (SDS–PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroper- oxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H 2 O was replaced by D 2 O. The results show that chlorin p6 induced photodynamic oxi- dation of BSA was mainly mediated by singlet oxygen. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Serum albumin; Chlorin; Protein oxidation; Binding; Carbonyl compounds 1. Introduction Photodynamic therapy (PDT) is an emerging modality for treatment of cancer and premalignant conditions. PDT is based on selective accumulation of photosensi- tizer in transformed tissue and irradiation with light of appropriate wavelength that produces cytotoxic reactive oxygen species (ROS) [1]. PDT may damage proteins, lipids, nucleic acids and a variety of small molecules in the cell. Proteins are major targets for photodynamic damage due to their relative high abundance in cell. Reactive oxygen species may lead to biophysical, bio- chemical and functional changes in the proteins [2]. Pro- tein oxidation may generate new reactive species (e.g., protein-bound reactive moieties and hydroperoxides) that can give rise to additional radicals under physiological condition [3,4]. Chlorins are molecules having a number of favorable characteristics to be a PDT drug. These molecules have strong absorption in the therapeutic window, high singlet oxygen quantum yield, low dark toxicity and high tumor selectivity [5,6]. Chlorin p6 is an amphiphilic molecule, having basic tetrapyrrole structure that may get distrib- uted in membrane and cytoplasm. In both regions func- tionally active proteins are present that might bind to chlorin p6 and alter its functional activity. Under photo- dynamic treatment condition this may lead to damage to the protein that may contribute to efficacy of PDT. Photodynamic activity depends on the photosensitizer 1011-1344/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.04.005 * Corresponding author. Tel.: +91 731 2488487; fax: +91 731 2488430. E-mail address: bbose@cat.ernet.in (B. Bose). www.elsevier.com/locate/jphotobiol Journal of Photochemistry and Photobiology B: Biology 86 (2006) 49–55