Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization
Graphical abstract
Introduction
In the past decades, a kind of novel glycosaminoglycan isolated from the body wall of sea cucumber has appeared as a potentially useful therapeutic for antithrombotic applications.1 It has a chondroitin sulfate-like structure containing large numbers of sulfated α-l-fucopyranose branched to position 3 of the β-d-glucuronic acid residues.1, 2, 3 The complex biosynthesis of glycosaminoglycan from the body wall of sea cucumber leads to a much different chemical composition of sequences containing different ratios of N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), fucose (Fuc), and ester sulfate.2, 3 Recently, we isolated a new glycosaminoglycan with a molecular weight of around 70 kDa from the body wall of sea cucumber Thelenata ananas (holothurian glycosaminoglycan from T. ananas, THG), which consists of GalNAc, GlcUA, fucose, and ester sulfate in a ratio of 1:1:1:3.7, respectively.4 Its chemical composition and molecular weight are different from those of glycosaminoglycan isolated from Ludwigothurea grisea2 and from Stichopus japonicas.3
In the antithrombotic applications, due to an undesirable effect of platelet aggregation,5 the depolymerization of glycosaminoglycan from sea cucumber is essential.3, 4, 5, 6, 7 In a preliminary study, a low molecular weight glycosaminoglycan fraction was obtained after depolymerization from a sea cucumber S. japonicas.6 However, there were no data concerning the depolymerization of glycosaminoglycan from T. ananas, but the physico-chemical characteristics of its different molecular weight fractions are currently available.
For the degradation of polysaccharides, free radical depolymerization is an interesting route because it enables, with reproducibility and constant composition, the controlling of the extent of depolymerization.8 Moreover, compared with acid hydrolysis, there is no preferential cleavage of side-chains9 and the primary structure is retained after chemical depolymerization.10 The reaction also has potential to achieve some selectivity of cleavage by the appropriate choice of metal ion catalyst.11 The conditions of the reaction are mild and it can be quenched at any point by removal of the metal ions by a chelating agent. The reagents are inexpensive and suitable for use on a large scale.12 Thus, free radical depolymerization has considerable promise for the industrial production of low molecular weight THG.
However, in practice, it is often difficult to make large quantities of molecular weight fractions of biopolymers with narrow molecular weight distribution (MWD).13 This adds to difficulties in characterizing the polymer and studying the molecular weight (Mw) dependence of its properties.
This work first dealt with free radical depolymerization using cupric ion catalyst to obtain a series of new low molecular weight THGs. The main parameters were investigated by high-performance gel permeation chromatography and low-angle laser light scattering (HPGPC-LALLS). Partially depolymerized THG (DTHG) samples were analyzed using HPGPC and dilute solution viscosity, the chain conformation was evaluated according to known theories for unperturbed wormlike cylinders, and molecular morphologies of THG were observed by atomic force microscopy.
Section snippets
Materials
The sea cucumber T. ananas was collected in Sanya of Hainan province of China. Diastase vera (EC 3.3.21.4) was obtained from Aolipharm, Inc. (Chongqing, China). Hydrogen peroxide (30 wt % solution in water), copper(II) acetate monohydrate, sodium acetate, and disodium ethylenediamine tetra-acetate dihydrate (EDTA) were purchased from DamaoChem., Ltd (Tianjin, China). All other chemicals were of reagent grade and were obtained commercially.
Analysis of molecular weight
The molecular mass, weight average molecular mass (Mw),
Analysis of free radical depolymerization
First, the effect of pH on the depolymerization process was observed. During the addition of metallic cation to THG solutions, a sudden decrease in pH immediately occurred due to the chelation process.8 The pH of the solution then remained stable with values ranging from 6.3 to 6.8 at 35 °C depending on the initial concentration of the added cation. This was also observed by Petit et al.8 in the depolymerization of an exopolysaccharide produced by a bacterium isolated from a deep-sea
Conclusions
We have isolated a new glycosaminoglycan from the body wall of the sea cucumber T. ananas, and prepared with high reproducibility, low molecular weight, and size-defined DTHG derivatives in the molecular weight range from 6500 to 45,000 Da, depending on the experimental conditions by the free radical depolymerization with hydrogen peroxide. To control the free radical reaction, and then, the efficiency and reproducibility of the process, most parameters were studied. Under the conditions of a
Acknowledgments
We are grateful to Mrs. W. Zeng and Miss H. Liang for their skilful technical assistance. Dr.Ing M. Huang is acknowledged for performing the AFM experiments.
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