利用二维同步荧光相关光谱研究温度对胶原溶液中分子聚集行为的影响

采用恒波长同步荧光法和二维相关分析技术研究了不同浓度Ⅰ型胶原溶液中胶原分子聚集行为随温度升高(10~70 ℃)的变化规律。选取0.2，0.4，1.6 mg·mL-1的胶原溶液，在初始温度下各浓度溶液中胶原分子分别处于单分子状态、较低程度和较高程度的聚集态。研究表明：波长差为9 nm的同步荧光光谱中，激发波长282和292 nm处荧光峰分别归属于未参与形成氢键的Tyr(酪氨酸)残基和参与形成氢键的Tyr残基。对升温过程同步荧光数据进行二维相关分析，得两荧光值对温度的响应顺序，进而推测得到：当温度低于30 ℃时，0.2 mg·mL-1溶液中出现了胶原分子间形成Tyr残基参与的氢键的趋势。0.4和1.6 mg·mL-1的溶液中原有聚集体可能发生进一步聚集，形成疏水微区。当逐步接近胶原变性温度(36~38 ℃)时，推测0.4和1.6 mg·mL-1胶原溶液中的疏水微区和聚集体有被破坏的趋势，而0.2 mg·mL-1胶原溶液保持分子间形成氢键的趋势。超过胶原变性温度时，各浓度溶液中胶原分子三股螺旋结构发生松散。当超过45 ℃时，胶原分子三股螺旋结构松散的趋势更为明显。

Effect of Temperature on the Aggregation Behavior of Collagen Solution by Two-Dimensional Synchronous Fluorescence Correlation Spectroscopy

The synchronous fluorescence spectroscopy and two dimensional correlation analysis method were applied to study the aggregation behavior of acid-soluble collagen solutions (0.2, 0.4 and 1.6 mg·mL-1) during the heating process of 10～70 ℃. It was found that the fluorescence excited at 292 and 282 nm (Δλ=9 nm) belongs to the tyrosine (Tyr) residues which participate in forming hydrogen bonds or not, respectively. The two dimensional correlation analysis with the temperature varying showed that with the temperature increased (10～30 ℃) hydrogen bonds among collagen molecular with Tyr residues formed in the 0.2 mg·mL-1 collagen solution, while the higher aggregations of collagen molecular and hydrophobic micro-domains appeared in the 0.4 and 1.6 mg·mL-1 collagen solutions. With approaching the denatured temperature of collagen (36～38 ℃), the hydrophobic micro-domain and aggregates seemed to be broken in the 0.4 and 1.6 mg·mL-1 collagen solutions, however the hydrogen bonds in the 0.2 mg·mL-1 were stable. Above the denaturation temperature of collagen, the triple-helix structure of collagen molecular in solution of each concentration tended to be loose. In the heating process of 45～70 ℃, this trend was more obvious.