Inoue Naoki, Bessho Masahiko, Furuta Masakazu, Kojima Takao, Okuda Shuichi, Hara Masayuki
Department of Applied Biochemistry, Graduate School of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka, Japan.
J Biomater Sci Polym Ed. 2006;17(8):837-58. doi: 10.1163/156856206777996835.
We made a new type of collagen gel by gamma-ray irradiation of an acidic solution of type-I collagen, and performed comparative studies on a conventional gel and the new type of gel. The neutral gel, a conventional 0.3% (w/v) collagen gel, was formed at neutral pH and then irradiated by gamma-rays. The acidic gel, a 0.3% (w/v) collagen gel, was formed directly from the acidic solution of collagen by y-ray irradiation. Both types of gel were prepared, swollen in water and then dried for the measurement of specific water content. The neutral gel showed a relatively high specific water content and shrunk moderately, depending on the dose, while the acidic gel showed lower specific water content and shrunk clearly by y-ray irradiation. A three-dimensional tangled network of microfibrils was clearly observed in the neutral gels by scanning electron microscopy, but not in the acidic gels. From these results, we concluded that the acidic gel was quite different from a conventional collagen gel. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that the alpha1 subunit and alpha2 subunit of the collagen molecule were cross-linked. The triple-helical structure of collagen was only partially perturbed, but not denatured completely, because the circular dichroism spectrum of the collagen solution irradiated at 1.3 kGy was similar to that of native collagen solution. Amino-acid analysis revealed that tyrosine, phenylalanine and histidine decreased by irradiation in the neutral gel. In the case of the acidic gel, these three amino acids and methionine decreased. We considered that these amino acids were cross-linking points between the collagen subunits during the gamma-ray irradiation.
我们通过对 I 型胶原蛋白的酸性溶液进行γ射线辐照制备了一种新型胶原蛋白凝胶，并对传统凝胶和新型凝胶进行了比较研究。中性凝胶是一种传统的 0.3%（w/v）胶原蛋白凝胶，在中性 pH 条件下形成，然后进行γ射线辐照。酸性凝胶是一种 0.3%（w/v）胶原蛋白凝胶，通过γ射线辐照直接从胶原蛋白的酸性溶液中形成。两种类型的凝胶均制备好后，在水中溶胀，然后干燥以测量比含水量。中性凝胶显示出相对较高的比含水量，并根据剂量适度收缩，而酸性凝胶显示出较低的比含水量，并且通过γ射线辐照明显收缩。通过扫描电子显微镜在中性凝胶中清晰地观察到微纤维的三维缠结网络，但在酸性凝胶中未观察到。从这些结果中，我们得出结论，酸性凝胶与传统胶原蛋白凝胶有很大不同。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明胶原蛋白分子的α1 亚基和α2 亚基发生了交联。胶原蛋白的三螺旋结构仅受到部分扰动，但并未完全变性，因为在 1.3 kGy 辐照下的胶原蛋白溶液的圆二色光谱与天然胶原蛋白溶液的相似。氨基酸分析表明，在中性凝胶中，酪氨酸、苯丙氨酸和组氨酸因辐照而减少。在酸性凝胶的情况下，这三种氨基酸以及蛋氨酸减少。我们认为这些氨基酸是γ射线辐照期间胶原蛋白亚基之间的交联点。
