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Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles
Institution:1. Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands;2. Department of Molecular Physics, Agricultural University, 6703 HA Wageningen, The Netherlands;3. Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, UK;1. Beijing Key Laboratory of Multiphase Flow and Heat Transfer for Low Grade Energy Utilization, North China Electric Power University, Beijing, 102206, PR China;2. Key Laboratory of Power Station Energy Transfer Conversion and System (North China Electric Power University), Ministry of Education, Beijing, 102206, PR China;1. Food Research and Development Unit, National Technology of Mexico/Technological Institute of Veracruz, Ver., Mexico;2. Physiology Department, Faculty of Medicine, Autonomous University of Madrid, Madrid, Spain;3. Faculty of Bioanalysis, Veracruz University, Veracruz, Ver., Mexico
Abstract:The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV vircular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.
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