Calorimetric and thermodynamic analysis of cowpea β-vignin: unveiling effects of seed germination on the conformation of a storage protein

Storage proteins have been generally considered as a mere nitrogen reserve that supports seedling growth during germination [1]. Nevertheless, new findings indicate that several biological activities become manifest because of the action of intermediate polypeptide fragments that are formed upon a selective breakdown of specific peptide bonds occurring through a regulated mechanism [2]. β-vignin is the most represented storage protein in cowpea seeds (Vigna unguiculata, L. Walp) and belongs to the vicilin-like family [3]. It is synthesized during seed development and consists of two main differently glycosylated isoforms. Moreover, it exists in a monomer-to-trimer equilibrium that depends on the environment pH: the higher the pH, the greater the trimer-to-monomer ratio [4]. Up to now, cowpea β-vignin molecular properties have been object of few studies, and many aspects have not been addressed yet. The full understanding of cowpea β-vignin structural and physicochemical properties is crucial to disclose possible biological roles and to approach applicative uses in various fields, including eco-friendly plant defense, nutrition and nutraceutics. In this frame, the present work was aimed at gaining insights on the thermodynamic stability of both the cowpea native β-vignin and the structures produced during seed germination. Therefore, purified β-vignin was subjected to a limited proteolysis by using cowpea proteases extracted from germinating seeds to simulate the early germination process, and a calorimetric investigation was performed through high-sensitivity DSC on both the undigested and digested β-vignin in different buffered environments (pH 9.0, 8.5 and 6.5). A thermodynamic analysis was also accomplished on the β-vignin undigested form to assess the protein stability and thermal denaturation mechanism. The results revealed that the storage protein behaviour is rather peculiar if compared to proteins with other biological functions.