Investigations On Structures And Properties Of Regenerated Bombyx Mori Silk Fibroin In Aqueous Solutions

A wonderful process of soluble silk fibroin transiting to solid silk catches great attentions for decades. Native silk, including spider silk and silkworm silk, as well as their proteins, hold great potential applications in the field of biomedicine, food and chemical industry due to their excellent properties. In this work, a variety of methods were used to study the structure and properties of Regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under diversely physical and chemical conditions. Thus, a further understanding of conformational transition for RSF in aqueous solution and adsorption for RSF at the interface was addressed. A preliminary exploration of self-assembly of RSF in solution was also fulfilled in this paper.Fluorescence and circular dichroism (CD) spectroscopy were used to monitor the conformational transition of RSF in aqueous solutions under different conditions. It is concluded: (1) According to the analysis of fluorescence spectra using Anilinonaphthalene-8-sulfonic acid Magnesium salt (ANS) as an exterior probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse might initiate the conformational transition from random coil to P-sheet for RSF; (2) The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism shows a reversible process upon heating and re-cooling, with the midpoint around 45℃, suggesting thatheat induced structural change of RSF is a partial unfolding process and a equilibrium exists between random coil state and β-sheet state; (3) Most of the Tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they would change their location in the highly ordered structure (e.g. becoming more homogeneous) with the conformational transition of silk fibroin; (4) Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin by facilitating the movement of molecular chain of RSF; (5) The secondary structure of RSF is very sensitive to acid. During acid conformational transition process, the protein aggregated near the isoelectric point of RSF (pH=4.22), whereas the conformation is relatively stable as pH value is about 6, which is accordance with the state and environment of fibroin in the gland of silkworm, showing that it is a essential way for silkworm controlling the state of fibroin by pH. On the other hand, the structure of RSF is rather stable during the whole alkaline conditions; (6) The existence of intermediate during the heat and pH induced unfolding process implied that the unfolding of RSF does not fit the simple two-state mechanism.The dynamic surface tensions of aqueous RSF solution were measured by using a pendant bubble experimental technique. The results show: (1) As a hetero-biomacromolecules, RSF has a significant surface activity with a critical micelle concentration of 2.2×10^-2 g/mL. The surface tension of RSF aqueous solution varies with bulk concentration and molecular weight of RSF; (2) The initial step of adsorption for RSF at the air-water interface is diffusion controlled. Beyond diffusion, the adsorption behavior deviates from diffusion mechanism to mixed diffusion-kinetic-controlled mechanism, indicating the existence of energy barriers in the adsorption process; (3) The analysis via a first-order rate equation suggest that the diffusion of RSF involves two distinct steps which could be characterized by two first-order rate constants (k₁ and k₂). Thereinto, the kinetic rate constant is concentration and molecular weight of RSF dependence. Moreover the first step of adsorption is quite faster than the second one; (4) Owing to the polydisperse RSFmolecular weight, the small RSF molecule adsorbs to the interface first and then is partial replaced by large RSF molecule; (5) The induction time observed in the adsorption of high molecular weight RSF indicates that large RSF molecule possesses a less flexibility and high stability compared to the small size RSF molecule, accordingly with high surface activity and low adsorption efficiency; (6) Due to alternating hydrophobic and hydrophilic side groups along RSF chain, the effect of heat on hydrophobic region and hydrophilic region is compensation, hereby there is almost no effect of heat on the surface activity of RSF. For the thermo-circular process, adsorption for RSF at the air-water interface is partial reversible owing to the heat structural change of RSF at interface; (7) As for ethanol-RSF mixing system, ethanol adsorbed to the interface first rising from its high adsorption efficiency, and then partial replaced by RSF molecule for the high surface activity RSF. The interaction between ethanol and RSF would decrease the surface pressure of RSF aqueous solution; (8) pH value has important effect on both surface activity and adsorption efficiency of RSF, which could be further increased in acidic environment. However, the low pH (e.g. pH lower than 3) would destroy molecular chain of RSF and consequently decrease the surface activity of RSF.The self-assembly of Bombyx mori RSF in aqueous solution was also involved in this work. We found: (1) The distinguishingly structure of RSF with alternating hydrophobic and hydrophilic side groups along chain as well as the motif of GAGAGS (crystal domain) and the other amino acids forming amorphous region facilitate the self-assembly of RSF into nanoparticle with diameter of 10～100nm; (2) Diameter size of nanoparticle would increase with time and finally RSF could aggregate into fibriform or membrane-like structure; (3) The structure of aggregates formed by self-assembly of RSF in aqueous solution is β-sheet.