EXTRACTION OF FIBROIN AND STUDY OF ITS MOLECULAR SIZES

АННОТАЦИЯ

В данной исследовательской работе представлена информация о структуре, медицинском применении, биологической активности и методах производства белка фиброина. С использованием современных методов исследования изучены термостабильность, морфологическая структура, качественный и количественный аминокислотный состав фиброина, выделенного из натурального шелкового волокна. Экспериментальные результаты изучения молекулярных размеров фиброина, выделенного из шелкового волокна, доказали, что он полностью очищен от посторонних веществ и пригоден для медицинских целей.

ABSTRACT

In this research paper, the information on the structure, medical use, biological activity and obtaining methods of fibroin protein has been presented. The thermal stability, morphological structure and qualitative and quantitative composition of amino acids of the fibroin protein extracted from natural silk fiber have been studied using modern research methods. The experimental results of studying the molecular sizes of fibroin extracted from silk fiber proved that it has been completely purified from foreign substances and suitable for medical purposes.

Ключевые слова: фиброин, белок, шелковое волокно, аминокислота, размер молекул.

Keywords: fibroin, protein, silk fiber, amino acid, molecular size.

Introduction. Fibroin belongs to the class of fibrillar proteins and it is characterized by multiple repetitions of certain amino acids in its primary structure. The main recurrent amino acids consist of the sequence glycine-serine-glycine-alanine-glycine-alanine (Gly-Ser-Gly-Ala-Gly-Ala) (Figure 1). The rest of fibroin has an amorphous structure, consisting mainly of hydrophilic amino acid residues. This molecular structure leads to the homogeneity of the secondary structure of fibroin. Amorphous areas of the protein form α-helces, and their increase in number depends on protein hydration. In the tertiary structure of fibroin, 2 chains - molecular weights of 390 kDa and 26 kDa are formed by connecting each other with disulfide bonds in a 1:1 ratio [1].

Figure 1. Primary amino acid sequence in fibroin

The structure and physico-chemical properties of silk fibroin make it possible to use it to obtain medical biomaterials. For example, fibroin undergoes rapid phase changes under the influence of alcohols and other factors, quickly passing from aqueous solutions to an insoluble state. This feature is very important in obtaining tools that are robust and flexible at the same time. In addition, silk fibroin is heat resistant, and products made from it can be sterilized by processing at temperatures up to 150°C [2].

A medically and biologically important property of fibroin is its biological compatibility with the body. Absence of immune response to biomaterials based on silk fibroin has been found in invivo and invitro models. However, in some literature, the issue of the biocompatibility of fibroin remains open. In particular, there are conclusions that the amount of fibroin in the medical device can cause an immune reaction depending on the location, size and morphology of the implant, depending on its long-term interaction with tissues, and these conclusions show that the level of immunity against the biomaterial depends on the conformation of silk fibroin [3].The level of immune response to biomaterial based on silk fibroin in α-helical conformation is lower than that of fibroin in β-helical conformation. However, the response of the immune system to biomaterials based on fibroin containing sericin is high. Therefore, it can be concluded that the immune response to fibroin-containing agents is often associated with incomplete purification of fibroin from sericin and other impurities [4].

Biodegradability of fibroin has several advantages over other synthetic materials. For instance, as a result of the decomposition of some synthetic materials, foreign products are formed. In this respect, fibroin is considered safe, as a result of its biological decomposition, amino acids necessary for the recovery and development of tissues and cells of the body are formed. Furthermore, some medical materials cannot maintain their mechanical properties for a certain period of time, on the contrary, the relatively slow biodegradation of fibroin in the implanted area has the possibility of maintaining its properties for a certain period of time [5]. In addition, the presence of a large number of free chemical groups in the chemical structure of the fibroin macromolecule makes it possible to change some of its physicochemical properties by easy chemical modification [6].

In our research, we have set the task of extracting fibroin completely purified from foreign substances from local natural silk fibers and studying its molecular sizes and the qualitative and quantitative composition of amino acids.

Object and methods of research . The following reagents were used in the research: natural silk fibers (Bombyx mori), sodium carbonate (SS 83-79), lithium bromide (CAS №: 7550-35-8).

For fibroin extraction, ground natural silk fibers were treated in 0.02 M sodium carbonate solution for 1 hour to remove impurities, sericin and residual oils. Then, the raw silk was washed in water for 30 minutes, then kept in distilled water at a temperature of 85-90°C for 2 hours, and then dried at room temperature. In the next step, it was stored in 9 M lithium bromide solution at 70°C for 1 hour until complete dissolution. The resulting solution was first centrifuged, then filtered and dialyzed for 72 hours to remove mineral salts (with dialysis water replacement every 8 hours). The dialyzed product was dried in a lyophilizer.

Thermo-analytical studies of fibroin protein was carried out using a STA-409 PG TG-DSK-analyzer device manufactured by NETZSCH, using a K (Low RG Silver) type thermocouple and aluminum crucibles. 5-6 mg of samples were taken for experiments. All measurements  were carried out in an inert atmosphere of nitrogen which is driving speed is 50 ml/min. The heating rate in measurements was 10 K/min. In the conditions, the temperature range was +20...+600°C. The measuring system was calibrated using standard substances - indium, bismuth, tin, zinc and cesium chloride.

Molecular properties of fibroin samples were investigated by scanning electron microscopy (SEM). To do this, the sample was covered with carbon in a Q 150 RES (QUORUM. USA) device under vacuum at a voltage of 15 kV, and its morphological structures were studied in EVOMA 10 (Zeis, Germany).

The qualitative and quantitative amount of amino acids in fibroin was checked by high-performance liquid chromatography (HPLC). HPLC conditions for qualitative and quantitative analysis of amino acids: chromatography equipped with a 1200 series DAD detector from Agilent Technologies; column 75x4.6 mm; Discovery HS C18; mobile phase A; 0.14M CN3SOONa + 0.05% TEA rN 6.4, V:CH3CN; flow rate - 1.2 ml/minute; wavelength 269 nm. Gradient % B/minute: 1-6%/0-2.5 minutes; 6-30%/2.51-40 minutes; 30-60%/40.1-45 minutes; 60-60%/45.1-50 minutes; 60-0%/50.1-55 minutes. The set of amino acids from “SERVA” company was used as a standard. To do this, a mixture of standard amino acids with a concentration of 0.01 mg/ml was prepared. PTC derivatives of these amino acids were also synthesized by the method of Steven A., Cohen Daviel [7].In the identification of amino acids in the samples, the elution times of the amino acids in the standard sample were used, and for the quantitative analysis, the area of the peaks of each amino acid in the chromatogram was used.

Results and their discussion.Obtaining soluble fibroin protein is very difficult. Dissolving silk fibroin in a mixture of CaCl₂-C₂H₅OH-H₂O in the ratio 1:2:8 is the most simple method. In this method, transparent solutions of light yellow color with almost no precipitate are obtained. The main drawback of this method is the presence of ethanol in the system and the constant change in the concentration of the solution due to high temperature processing [8]. It is possible to obtain fibroin for a short time by processing silk fibers under the influence of alkali, however, it is known from the literature that proteins are unstable to the influence of alkali solutions, and even low-concentration alkali can lead to complete hydrolysis of the protein under the influence of time and temperature. Therefore, if the temperature is not strictly controlled when dissolving fibroin in alkali, the yield of fibroin extraction may decrease dramatically [9].

Fibroin was extracted dry from natural silk fibers by our proposed method. It is possible to prepare solutions with specific concentration on the basis of fibroin obtained by this method. The results of the experiment to determine the thermal stability of the fibroin protein extracted in dry form are presented in Fig. 2.

Figure 2. Thermal analysis of fibroin protein

The results of thermal analysis of fibroin protein showed that, unlike other fibrillar proteins, the denaturation temperature of fibroin is relatively high. In the process of heating fibroin, it was found that an endothermic effect was observed between 160-300°C and the mass decreased by 25%. It was also observed that with increasing temperature, the fibroin protein disintegrates without liquefaction and turns into an amorphous state.

The micro and macro structure of fibroin protein was studied by SEM in comparison with natural silk fiber (Fig. 3).

Figure 3. SEM images: 1) - natural fibres, 2) - fibroin

From the results presented in Figure 3, it can be seen that in the SEM images of natural silk fibers (1), there are large superimposed fibers. Also, the size of the fibers is different. In the SEM images of the fibroin sample (2), it was observed that the fibers are relatively small and uniform in thickness. So, the fibroin protein isolated by us from silk fibers has preserved its fibrous structure and has the same molecular size.

Also, the qualitative and quantitative composition of amino acids in fibroin protein was studied with HPLC.

Table 1.

Qualitative and quantitative analysis of amino acids in fibroin protein

The results of qualitative and quantitative analysis of amino acids in fibroin protein showed that glycine (42.86%), alanine (31.42%) and serine (12.14%) were the main amino acids in fibroin extracted from silk fiber. Also, these results are not significantly different from fibroin obtained for medical purposes [10], indicating that the isolated fibroin has a high degree of purity.

Conclusion.Thus, as a result of our research, fibroin protein was isolated from natural silk fiber (Bombyx mori) and its molecular parameters were determined. In particular, it was determined that the denaturation temperature is higher than 100°C, the fiber size is uniform, and glycine-alanine-serine is 86.42% of the total amino acids amount. At present, researches on obtaining a composition based on fibroin and inulin derivatives and studying its physico-chemical properties are ongoing.

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