Functional material features of Bombyx mori silk light versus heavy chain proteins

Abstract

Bombyx mori (BM) silk fibroin is composed of two different subunits: heavy chain and light chain fibroin linked by a covalent disulfide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fiber mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibers formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fiber formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications, and potential applications of these materials in biomedical and industrial applications.

Figure 1

Condition of silk a). After degumming b). Formic acid insoluble c). Formic acid soluble.

Figure 2

Percent soluble and insoluble silk fractions over a range of 0.01 – 8% total silk in formic acid.

Figure 3

SDS-PAGE analysis of regenerated fibroin and fractions

Figure 4

Photon correlation spectroscopy number size distribtion analysis of the isolated fractions and combined fibroin at stable (pH 9) and insolubles aggregating pH (pH 7).

Figure 5

Photon correlation spectroscopy (left) and corresponding zeta potential data (right).

Figure 6

Calculated pKa’s (upper black numbers) and charge states at pH 7 (lower red numbers) for heavy chain and light chain components of silk fibroin as determined by software made available by the Scripps Research Institute, blue zones denote basic properties (+ve at neutral pH) and red zones acidic properties (−ve at neutral pH). Amino acid residue numbers in green.

Figure 7

Flow chart of fractions generated based on SDS-PAGE, zeta potential, mass balance and calculations using peptide calculator and the known amino acid sequences. Red boxes refer to formic acid solubles isolated, blue boxes insoluble isolates, green boxes fragments lost through dialysis. Blue values are absolute % of original mass and based on weight measurements, black values refer to content calculated from known amino acid sequences and the grey value represents an estimate based on the difference between mass found and known light chain content. The key refers back to Figure 6.

Figure 8

Shear rheology of silk fibroin fractions and mixtures. a) Total silk fibroin regenerated, b) soluble fraction regenerated (and formic acid blank), c) Insoluble fraction regenerated, d) Soluble/insoluble blends (total silk concentration 20%). The legends in a)–c) show silk content and d) percentage contribution of the insoluble fraction.

Figure 9

Mats produced by electrospinning of solutions of the regenerated fibroin isolates, total fibroin and blends. Individual isolates and total fibroin solutions were 5 – 20% formic acid solutions. Blends were 20% by weight silk protein comprising 0 – 100% insoluble fraction by composition All scale bars represent 5μm

Figure 10

Correlation of fraction and concentration with viscosity (red line), mean fibre diameter and electrospin character.

Figure 11

Comparison of insoluble and soluble blends a) viscosity at shear stress above 1 Pa and b) fibre diameter and uniformity with that of total silk fibroin.