Mono-dimensional, 1H-NMR for the authentication of food allergens

INTRODUCTION: Food allergens are major tools as reagents for allergy diagnosis, consequently, their quality and authenticity are essential. A library of 31 well characterized food allergenic proteins has been established within the Europrevall project and one-dimension 1H-NMR analysis applied to contribute to their authentication. This method is proposed as a high throughput low cost method to asses the conformation of allergenic proteins. METHODS: Purification methods were developed to obtain highly pure and biologically active allergens. Due to the variable concentrations of allergen, broad molecular weight range (from 9 to 78kDa), natural abundance of 15N and 13C, and variable buffer conditions, the NMR protocol was modified on a case by case basis. Two High-Resolution, 1D-1H-NMR experiments were carried out, using a 700MHz field (presaturation to minimize the water signal and gradient-based water suppression by excitation sculpting); 256 scans at 298K were usually programmed. The spectra were evaluated considering the dispersion of the NMR signals in the regions of the methyl protons (-.5-1.5 ppm), alpha-protons (3.5–6.0 ppm), and amide protons (6.0–10 ppm). The consistency of the allergen’s spectra with structural information already available was routinely checked, otherwise the calculation of a modeled structure was attempted. If no reliable model was possible the sequence of the allergen was used to predict the degree of folding of the tertiary structure of the protein. RESULTS AND DISCUSSION: The analysis of the 1D-1H-NMR spectra allowed the classification of the allergens under study into three classes. 1) Molecules whose spectra showed the unquestionable feature of a rigid and extended tertiary structure (Api g 1, Api g 4, Ara h 2, Ara h 6, Bos d 4 , Bos d 5 , Cor a 1, Cor a 2, Cor a 8, Cor a 11, Cyp c 1, Gad m 1, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5, Mal d 1, Mal d 2, Mal d 3, Mal d 4, Pru p 1, Pru p 3). 2) Molecules whose spectra did not show rigid tertiary structure (Bos d 8, Whole Caprin Casein, Pen a 1). 3) Allergens whose spectra showed both a rigid tertiary structure and flexible and/or mobile regions (Cor a 9, Ara h 1). The allergens Ara h 3/4, Api g 5 were mixtures of very high molecular weight proteins. All the spectra were consistent with the experimental or modeled structures already known. CONCLUSIONS: 1D-1H-NMR is a highly useful tool for the structural authentication of allergens even when there is a limited quantity of protein available and the experimental conditions are not optimized for NMR analysis.