Comparative Biochemistry and Physiology Part C 131 (2002) 439–446 1532-0456/02/$ - see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S1532-0456 Ž 02 . 00033-9 Tetracycline–Cu(II) photo-induced fragmentation of serum albumin Mateen A. Khan , Javed Musarrat * a b, Interdisciplinary Biotechnology Unit, AMU, Aligarh, 202 002 UP, India a Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh, 202 002 UP, India b Received 26 January 2001; received in revised form 1 July 2001; accepted 3 February 2002 Abstract The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 mM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0–1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals ( OH) in tetracycline– • Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.  2002 Elsevier Science Inc. All rights reserved. Keywords: Tetracycline; Bovine serum albumin; Superoxide anions; Hydroxyl radicals; SDS-PAGE; Protein fragmentation; Reactive oxygen species (ROS); Phototoxicity 1. Introduction Tetracycline (TC) undergoes oxidative degra- dation upon photoexcitation and yields metastable quinone derivatives (Davies et al., 1979; Moore et al., 1983). These photo-induced products evoke biological responses manifested as a tingling, burn- ing sensation (Frank et al., 1971), oncholysis (Orentreich et al., 1961; Frank et al., 1971) and papular eruptions (Frost et al., 1971). Upon pho- tosensitization, tetracycline loses a dimethyl-amino group (Moore et al., 1983) and eventually triggers the production of free radicals (Davies et al., 1979; *Corresponding author: Tel: q91-571-502283; fax: q91- 571-701081. E-mail address: musarratj1@yahoo.com (J. Musarrat). Green and Hill, 1984). Tetracycline-induced reac- tive species have been reported to inhibit fibroblast growth (Bjellerup et al., 1985), damage the mon- ocytes (Hassan et al., 1984) and inactivate plant and animal viruses (Murphy, 1975; Novo and Esparza, 1979). This antibiotic is known to affect DNA synthesis in prokaryotic cells upon interac- tion with the cell membrane (Pato, 1977). It also forms photo-adducts with ribosomal proteins (Goldman et al., 1983) and induces single-strand breaks in bacteriophage w X 174 DNA (Piette et al., 1984). Other intracellular targets susceptible to tetracycline-induced damage in higher organisms are erythrocytes (Nilsson et al., 1975), ribosomes (Rebout et al., 1982; Goldman et al., 1983) and macromolecules in cartilage (Monboisse et al.,