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Monitoring the heat-induced structural changes of alkaline phosphatase by molecular modeling, fluorescence spectroscopy and inactivation kinetics investigations

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Abstract

The heat induced conformational changes of calf alkaline phosphatase (ALP) were analyzed using different methods, based on fluorescence spectroscopy, molecular modeling and inactivation studies. Experimental studies were conducted in buffer solution in the temperature range between 25 and 70 °C. Molecular dynamic (MD) simulation provided details on thermally induced changes in ALP structure, highlighting that heating favored the hydrophobic exposure and important alteration of the catalytic site above 60 °C. Additional information to MD data were obtained by using different fluorescence spectroscopy methods, which revealed a complex mechanism of thermal denaturation. Therefore, the emissive properties indicated an unfolding of ALP at temperatures below 60 °C, whereas at higher temperatures, the polypeptides chains fold leading to a higher exposure of Trp residues. In order to establish a structure-function relationship, the results were correlated with inactivation studies of ALP in buffer at pH 9.0. The inactivation data were fitted using a first-order kinetic model, resulting in an activation energy value of 207.26 ± 21.68 kJ · mol−1.

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Acknowledgments

The work has been funded by the Sectoral Operational Programme Human Resources Development 2007–2013 of the Ministry of European Funds through the Financial Agreement POSDRU/159/1.5/S/132397.

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Authors and Affiliations

  1. Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, Domneasca Street 111, Building E, Room 304, 800201, Galati, Romania

    Loredana Dumitrașcu, Nicoleta Stănciuc, Iuliana Aprodu, Ana-Maria Ciuciu, Petru Alexe & Gabriela Elena Bahrim

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  1. Loredana Dumitrașcu
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  2. Nicoleta Stănciuc
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  3. Iuliana Aprodu
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  4. Ana-Maria Ciuciu
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  5. Petru Alexe
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  6. Gabriela Elena Bahrim
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Correspondence to Nicoleta Stănciuc.

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Dumitrașcu, L., Stănciuc, N., Aprodu, I. et al. Monitoring the heat-induced structural changes of alkaline phosphatase by molecular modeling, fluorescence spectroscopy and inactivation kinetics investigations. J Food Sci Technol 52, 6290–6300 (2015). https://doi.org/10.1007/s13197-015-1719-1

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  • DOI: https://doi.org/10.1007/s13197-015-1719-1

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