仿刺参胶原蛋白的提取及理化性质

Extraction and the physical and chemical characterization of collagen in Stichopus japonicus

对仿刺参(Stichopus japonicus)胶原蛋白的提取及理化性质进行了研究。结果表明,低温提取可得到胃蛋白酶促溶且去除端肽(telopeptide)的酸溶性胶原蛋白(PSC)。紫外-可见扫描测得PSC的最大吸收峰位于220nm;氨基酸分析发现,在1 000个总氨基酸残基中,甘氨酸为329个,羟脯氨酸为66个,羟脯氨酸与脯氨酸之比为0.69,酪氨酸、苯丙氨酸及组氨酸的含量较低,可能不含胱氨酸,这些特性均符合Ⅰ型胶原的特征。差示量热扫描法(DSC)测定仿刺参PSC的热稳定性温度(Ts)为57℃,低于牛皮Ⅰ型胶原5℃;苯酚-硫酸及次甲基蓝法测得PSC的总糖及粘多糖含量分别为0.61%及0.48%;经DEAE-52纯化后,可得到除去粘多糖的蛋白纯品。SDS-PAGE显示,本研究所提取的仿刺参PSC不含杂蛋白,主要成分为胶原β及α链,含少量γ链;其中α链类似于脊椎动物Ⅰ型胶原α1链,PSC分子组成为(α1)3。

Sea cucumbers （ Stichopus japonicus） belonging to the phylum Echinodermata are traditional tonic consumed by Chinese and Japanese. The major edible parts of sea cucumber are the body wall mainly consisting of collagen and mucopolysaccharides. More than 21 types of collagen have so far been identified in various tissues and their roles have been investigated. In invertebrate, at least 2 types of collagen, type Ⅰ and type Ⅴ are found to exist. There is little information about the collagen of sea cucumber except for few reports on S. japonicus and Cucumaria frondosa, respectively. However, results of the former findings were not consistent with that of the latter about the molecular composition of the sea cucumber collagen. In this paper, the intact collagens were isolated from the body wall of S. japonicus by a new method and their physical and chemical characterization were studied. The results showed that pepsin-solubilized collagens（PSC） without telopeptide could be prepared at low temperature. Through the ultraviolet scanning spectra from 190 to 400 nm, the PSC presented maximum absorption at 220 nm which was consistent with that of type Ⅰ collagen. The compositional features of the amino acid were with the high contents of glycine（329 residues/1000） and hydroxyproline（66 residues/ 1000）, but with low contents of tyrosine, phenylalanine and histidine. No cysteine was detected. The ratio of hydroxyproline to proline was 0.69. It was concluded that PSC was typical type Ⅰ collagen. This was the first report about the thermal stability temperature （Ts） of sea cucumber collagen which was 57 ℃ as measured by DSC, about 5 ℃ lower than that of typeⅠ collagen of The content of total polysaccharide determined with phenol-sulfuric acid method was 0.61% and the glycosaminoglycan measured with cationic dye of methylene blue was 0.48%. The collagen could be separated from glycosaminoglycan by DEAE-52.Based on SDS polyacrylamide gel electrophoresis（SDS-PAGE）, the purities of PSC were higher. Judging from their electrophoretic patterns, PSC contained β, α and a few γ chains, while α chain resembled α1 chain of type- Ⅰ collagen of vertebrate, however, the presence of （α1）3 trinters was evident.