Effect of detergents, trypsin and unsaturated fatty acids on latent loquat fruit polyphenol oxidase: Basis for the enzyme’s activity regulation Susana Selle ´s-Marchart, Juan Casado-Vela 1 , Roque Bru-Martı ´nez * Grupo de Proteo ´ mica y Geno ´ mica Funcional de Plantas, Departamento de Agroquı ´mica y Bioquı ´mica, Facultad de Ciencias, Universidad de Alicante, Campus de San Vicente del Raspeig, Apdo, 99, E-03080 Alicante, Spain Received 9 February 2007, and in revised form 25 April 2007 Available online 15 May 2007 Abstract The effects of detergents, trypsin and fatty acids on structural and functional properties of a pure loquat fruit latent polyphenol oxi- dase have been studied in relation to its regulation. Anionic detergents activated PPO at pH 6.0 below critical micelle concentration (cmc), but inhibited at pH 4.5 well above cmc. This behavior is due to a detergent-induced pH profile alkaline shift, accompanied by changes of intrinsic fluorescence of the protein. Gel filtration experiments demonstrate the formation of PPO–SDS mixed micelles. Par- tial PPO proteolysis suggest that latent PPO losses an SDS micelle-interacting region but conserves an SDS monomer-interacting site. Unsaturated fatty acids inhibit PPO at pH 4.5, the strongest being linolenic acid while the weakest was c-linolenic acid for both, the native and the trypsin-treated PPO. Down-regulation of PPO activity by anionic amphiphiles is discussed based on both, the pH profile shift induced upon anionic amphiphile binding and the PPO interaction with negatively charged membranes. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Polyphenol oxidase; Alkyl sulfates; Alkyl sulfonates; Chlorogenic acid; Fluorescence; Latency; Linolenic acid; Loquat fruit; Regulation; Trypsin Polyphenol oxidases (PPOs) 2 (EC 1.14.18.1 or EC 1.10.3.2) are ubiquitous plant enzymes that catalyze the OB 2B -dependent oxidation of mono- and di-phenols to o-quinones. The oxidation of phenolic substrates by PPO is thought to be the major cause of the brown discoloration of many fruits and vegetables during ripening as well as handling, storage and processing [1–4]. This problem, known as enzymatic browning, is of considerable impor- tance to the food industry as it affects nutritional quality and appearance, reduces consumer acceptability and there- fore causes significant economic impact, both to primary food producers and to the food processing industry [1,2]. Thus the detailed knowledge of the regulation of this ubiq- uitous enzyme has practical implications in the food and agriculture industry. Although sub-cellular compartimen- tation is a generally accepted mechanism of enzymatic browning control [3], substantial experimental evidence point to additional mechanisms of PPO activity regulation. Many plant PPO preparations from different tissues including leaf [5,6], fruit [7–9] and root [10,11] are said to be latent since an increase in enzyme activity at pH 6.0 or higher occurs when treated with activating agents. Acti- vation of PPO can be achieved either by long exposure to acidic pH [12,13], to sub-cmc concentrations of SDS 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.04.023 * Corresponding author. Fax: +34 965903880. E-mail address: Roque.Bru@ua.es (R. Bru-Martı ´nez). 1 Present address: Protein Technology Unit, Biotechnology Programme, Spanish National Cancer Centre (CNIO), Madrid, Spain. 2 Abbreviations used: CA, chlorogenic acid; cmc, critical micellar concentration; CTAB, cetyltrimethylammonium bromide; DPHT, 1,6- diphenyl-1,3,5-hexatriene; Li, linoleic acid; a-Ln, a-linolenic acid; c-Ln, c-linolenic acid; Ol, oleic acid; PPO, polyphenol oxidase; SDS, sodium dodecyl sulfate; SDSA, sodium 1-dodecanesulfonic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SOS, sodium octyl sulfate; SOSA, sodium 1-octanesulfonic acid; TBC, tert-butylcate- chol; THF, tetrahydrofurane; TX-100, Triton X-100. www.elsevier.com/locate/yabbi ABB Archives of Biochemistry and Biophysics 464 (2007) 295–305