温度对鱼鳞胶原蛋白二级结构的影响

Effect of Temperature on the Secondary Structure of Fish Scale Collagen

从草鱼鱼鳞中提取酶溶性胶原蛋白(PSC),通过SDS-PAGE电泳分析为典型Ⅰ型胶原蛋白且达到电泳纯。在此基础上利用傅里叶变换红外光谱(FTIR)、拉曼光谱(Raman)和圆二色谱(CD)研究了温度对鱼鳞胶原蛋白二级结构的影响。FTIR分析表明:鱼鳞胶原蛋白具有典型的胶原蛋白特征吸收带,酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1658,1552和1238cm-1处。随温度升高,酰胺A和酰胺B峰位向低波数移动,1658cm-1处吸收峰裂解成多个吸收峰;1552cm-1处的吸收峰在35℃微略红移,随后发生明显蓝移;1238cm-1处吸收峰随温度升高向低波数移动。在拉曼光谱中,胶原蛋白的酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1669,1557和1245cm-1处,都较红外光谱的波数高;此外,921和855cm-1处脯氨酸的特征谱峰在拉曼光谱中体现出来。圆二色谱分析表明,胶原蛋白溶液在221.6和204.4nm分别有一正、负峰,具有典型胶原蛋白三螺旋结构的特征圆二色谱峰型。胶原蛋白冻干品的FTIR光谱和Raman谱线大都在35～60℃时发生波数和强度改变,而胶原蛋白乙酸溶液的CD谱线在20～35℃之间发生剧烈改变。由此可以判断胶原蛋白在固态和溶液状态下,变性温度存在一定差异,胶原蛋白冻干品比其乙酸溶液更稳定。

Pepsin-soluble collagen （PSC） was extracted from fish scale of grass carp and was analyzed by SDS-PAGE, which confirmed that PSC are typical type I collagen and reach electrophoretic purity. Effect of temperature on the secondary structure of collagen was studied by FTIR, Raman and CD. FTIR indicated that the fish scale PSC had typically characteristic absorptions of collagen, and 1 659, 1 552 and 1 238 cm^-1 were assigned to be amide Ⅰ ,Ⅱ and Ⅲ respectively. When the temperature increased, amide A and amide B shifted to low frequency, the absorption of i 658 cm^-1 split into several absorption peaks, the absorption at 1 552 cm^-1 had a slight red-shift followed by a distinct blue-shift, and the frequency of 1 238 cm^-1 declined. Raman spectra showed that the absorptions of amide Ⅰ , amide Ⅱ and amide Ⅲ appeared at 1 669, 1 557 and 1 245 cm^-1 respectively, which were higher than those in FTIR spectra. Furthermore, the characteristic absorptions of proline at 921 and 855 cm^-1 only appeared in Raman spectra. CD spectra demonstrated a rotatory maximum at 221.6 nm and a negative peak at 204. 4 nm of PSC solution, which were typical spectral characteristics of the collagen triple helix structure. The structure changes of the lyophilized PSC appeared mainly between 35 and 60℃ in FTIR and Raman spectra, yet CD spectra demonstrated that the configurational changes of PSC in acidic solution appeared in the range of 20 to 35℃, indicating that the lyophilized PSC was more stable than the acidic solution of PSC.