Doutoramento em Bioquimica
Labelling of Proteinaceous Binders in Art
Ooi Su Yin

Orientação:  Ana Teresa Caldeira & João Paulo Prates Ramalho & António Manuel Deométrio Rodrigues Lourenço Pereira

Easel paintings are important Cultural Heritage assets with significant historic and cultural value. They usually possess a multi-tiered structure, composed of different layers, some of which may present protein binders. Proteins have been commonly used as paintings medium, adhesives and coating layers in easel paintings. Hence, their recognition is a crucial step for easel painting’s conservation and restoration processes. The present work presents a novel fluorescent labelling methodology, using a coumarin derivative chromophore, C392STP (sodium (E/Z)-4-(4-(2-(6,7-dimethoxycoumarin-3-yl)vinyl)benzoyl)-2,3,5,6-tetrafluorobenzenesulfonate) as a fluorophore probe to bond proteinaceous binders used in paintings. The method was developed and optimized using commercial proteins and proteins extracted from hen’s egg yolk and white, bovine milk, and rabbit skin. In order to mimic the real conditions, paint models of easel paintings have been prepared by mixing proteins such as ovalbumin, casein and rabbit glue with different pigments (lead white, chrome yellow and black bone) and the fluorescent labelling method was miniaturized and tested. The results revealed that proteins in concentration as low as 6.0 μg/ml could be detected.Finally, for validation methodology, real micro samples of easel paintings were analyzed. The extracted proteins were submitted to the fluorescent labelling method developed and clearly identified in electrophoretic profiles. The results evidence the applicability of this methodology as an effective and useful analytical tool for the identification of protein binders obtained from easel paintings and, possibly in other art work.Additionally, theoretical quantum chemical calculations based on the Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TD-DFT) have been performed in the C392STP coumarin and in a related coumarin derivative ((E/Z)-4-(2-(6,7- dimethoxycoumarin-3-yl)vinyl)-N-propylbenzamide), that mimics the coumarin bonded to lysine. The calculations confirm the experimental trends in absorption wavelengths and are in good agreement with the experimental absorption spectra, providing a comprehensive characterization of the main spectral features of the studied compounds.

Easel paintings are important Cultural Heritage assets with significant historic and cultural value. They usually possess a multi-tiered structure, composed of different layers, some of which may present protein binders. Proteins have been commonly used as paintings medium, adhesives and coating layers in easel paintings. Hence, their recognition is a crucial step for easel painting’s conservation and restoration processes. The present work presents a novel fluorescent labelling methodology, using a coumarin derivative chromophore, C392STP (sodium (E/Z)-4-(4-(2-(6,7-dimethoxycoumarin-3-yl)vinyl)benzoyl)-2,3,5,6-tetrafluorobenzenesulfonate) as a fluorophore probe to bond proteinaceous binders used in paintings. The method was developed and optimized using commercial proteins and proteins extracted from hen’s egg yolk and white, bovine milk, and rabbit skin. In order to mimic the real conditions, paint models of easel paintings have been prepared by mixing proteins such as ovalbumin, casein and rabbit glue with different pigments (lead white, chrome yellow and black bone) and the fluorescent labelling method was miniaturized and tested. The results revealed that proteins in concentration as low as 6.0 μg/ml could be detected.Finally, for validation methodology, real micro samples of easel paintings were analyzed. The extracted proteins were submitted to the fluorescent labelling method developed and clearly identified in electrophoretic profiles. The results evidence the applicability of this methodology as an effective and useful analytical tool for the identification of protein binders obtained from easel paintings and, possibly in other art work.Additionally, theoretical quantum chemical calculations based on the Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TD-DFT) have been performed in the C392STP coumarin and in a related coumarin derivative ((E/Z)-4-(2-(6,7- dimethoxycoumarin-3-yl)vinyl)-N-propylbenzamide), that mimics the coumarin bonded to lysine. The calculations confirm the experimental trends in absorption wavelengths and are in good agreement with the experimental absorption spectra, providing a comprehensive characterization of the main spectral features of the studied compounds.

KEYWORDS:  fluorescent labelling method, coumarins, protein binders, easel paintings.