Hattori S, Adachi E, Ebihara T, Shirai T, Someki I, Irie S
Nippi Research Institute of Biomatrix, Adachi-ku, Tokyo, 120-8601, Japan.
J Biochem. 1999 Apr;125(4):676-84. doi: 10.1093/oxfordjournals.jbchem.a022336.
Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35 degrees C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin alpha2beta1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4 h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30 degrees C.
碱处理是一种即使从老龄动物标本中也能高回收率提取胶原蛋白的好方法。然而，在碱性条件下处理的胶原蛋白的特性尚未得到很好的确立。用3%氢氧化钠和1.9%甲胺溶液处理后，由于天冬酰胺和谷氨酰胺转化为天冬氨酸和谷氨酸，I型胶原蛋白的等电点从9.3降至4.8。随着等电点的酸化，处理20天后胶原蛋白的变性温度从42℃降至35℃，但胶原蛋白特有的三螺旋构象得以维持。人角质形成细胞和成纤维细胞通过胶原蛋白受体整合素α2β1附着于碱处理的胶原蛋白。这表明碱处理的胶原蛋白保持了其作为生物粘附分子的特性。与酸溶性胶原蛋白不同，碱处理的胶原蛋白在生理条件下的中性pH值下失去了形成原纤维的能力。即使在碱性处理4小时后，当胶原蛋白的变性温度没有变化时，这种能力也丧失了。另一方面，碱处理的胶原蛋白在30℃酸性条件下形成了直径均匀为50-70nm的纤维状沉淀物。
