Synthesis of tubular nanostructures from wheat bran albumins during proteolysis with V8 protease in the presence of calcium ions
Introduction
Albumins are the preferred proteins for the fabrication of nanoparticles with intended use in the delivery of drugs and bioactive compounds. This is because they are non-toxic, biodegradable, water soluble, and contain relatively large amounts of the charged amino acids Asp and Glu, along with a variety of side groups that can be functionalized with other molecules (Elzoghby, Samy, & Elgindy, 2012). The common strategies used for protein nanoparticle fabrication are emulsification, desolvation and self-assembly. The latter strategy is based on the spontaneous and reversible association of molecules leading to the formation of supramolecular entities via a bottom-up approach. During this process, the self-assembly of building blocks leads to the formation of nanoparticles, such as nanofibers and nanotubes, of uniform size and shape (Gazit, 2007).
Earlier studies on the mechanisms of protein self-assembly have primarily used synthetic peptides as building blocks (Zhang, 2002). A limited number of reports have described peptide building blocks released by controlled proteolysis, particularly in the presence of calcium ions, of albumins isolated from agroindustrial by-products (Balandrán-Quintana et al., 2013, Graveland-Bikker and de Kruif, 2006). It has been reported that protein concentration is a determining factor in the nucleation process that initiates the growth of peptide nanostructures during proteolysis. For example, an earlier study reported that a concentration of 30 g/L of α-lactalbumin was required for the formation of nanotubes (Graveland-Bikker, Ipsen, Otte, & de Kruif, 2004). Thus, purity and solubility of proteins are key criteria to be considered in the self-assembly of peptides. However, as in directed self-assembly, other compounds, when present in the reaction mixture, could act as templates during the nucleation and growth processes (Brown et al., 2002, Thakur et al., 2013). Such an approach would lower the critical concentration of proteins and permit the use of those proteins that can be isolated from agroindustrial by-products by aqueous extraction.
This is the case with wheat bran (WB), a by-product of the milling industry, which consists of a set of histological tissues surrounding the wheat grain. WB has a protein content of 13–19% w/w (USDA., 2014). These proteins, which perform a variety of functions, have been identified from the more than 600 spots that they produce in 2D electrophoresis (Jerkovic et al. (2010)). The presence of these proteins implies that there is large structural diversity and hence a high probability of non-covalent intermolecular interactions in WB protein extract, making the WB proteins interesting in a nanotechnological context, particularly as sources of building blocks for self-assembly processes (Balandrán-Quintana, Mercado-Ruiz, & Mendoza-Wilson, 2015). The proteins present in the outermost layers of WB, which share the features of albumins in general, can be easily extracted with water as the albumin fraction and shows good functional properties, such as emulsification, foaming, and gelation (Idris, Babiker, & El Tinay, 2003). These properties enable the use of WB proteins as candidates for the fabrication of nanocarriers. However, during the isolation of WB proteins, other components, mainly water-soluble polysaccharides, are co-extracted. These contaminants are difficult to separate unless expensive procedures are employed.
We hypothesized that after partial hydrolysis of the extracts, the non-protein constituents of the aqueous crude extracts of WB could drive the self-assembly of albumins contained in the extracts. The objective of the present work was to study the capacity of WB albumins to form nanostructures after hydrolysis with V8 protease in the presence of calcium ions. The V8 protease was selected because of antecedents of nanoparticle production upon its use (Balandrán-Quintana et al., 2013) and because of its specificity for Glu and Asp residues (Houmard & Drapeau, 1972) that are present in significant amounts in WB proteins.
Section snippets
Raw material and chemicals
The WB was obtained from a commercial mill located in Hermosillo Sonora, Mexico and was immediately stored at 5 °C in polyethylene bags until use. All chemicals used, unless specified, were purchased from Sigma–Aldrich (St. Louis, MO).
Extraction of the albumin fraction of wheat bran (AFWB)
The WB was sieved through a 40-mesh sieve to remove fine particles and some of the adhering endosperm. The fraction retained in the mesh was washed quickly with HPLC grade water to remove most of the residual endosperm according to the procedure described by
Proximate analysis, elemental composition and amino acid profile of AFWB
Table 2 shows the results of the proximate analysis, the determination of elemental composition and the amino acid content of AFWB. An earlier report is not available for comparison. The similarity of the values of total carbohydrate and total fiber indicates that most of these are water-soluble arabinoxylans. The presence of ash content, even after dialysis, suggests that some of the minerals are not dialyzable.
Elemental composition shows the presence of divalent cations, monovalent potassium,
Conclusions
A lyophilized form of AFWB, with 45% protein (w/w), whose molecular masses are in the range 14 to 65 kDa, was prepared. Proteolysis of this AFWB with V8 protease in the presence of calcium at specific molar ratios led to the formation of three-way tubular nanostructures with inner and outer diameters of approximately 100 and 200 nm, respectively. These nanostructures were more than 35 μm in length. Ultrasound released the nanotubes from a matrix made primarily of arabinoxylans. The methods
Acknowledgments
We acknowledge Consejo Nacional de Ciencia y Tecnología (CONACYT), México, for financing the project CB2011-169839 and the scholarship for the doctoral studies of Chaquilla-Quilca. We also acknowledge the participation of the Laboratorio de Química de Materiales CINVESTAV-IPN Unidad Mérida Yucatán, as well as the laboratories LANNBIO, projects FOMIX-YUCATAN 2008-108160 and CONACYT LAB-2009-01 N° 123913. Special thanks to the staff of the different departments of CIAD as well as to Dra. Patricia
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