对胶原分子聚集行为的研究, 不仅能改善其理化特性, 同时也为其在食品、 组织工程和生物医药等领域的应用提供理论指导。 基于胶原分子中苯丙氨酸(Phe)和酪氨酸(Tyr)的内源荧光特性, 采用常规波长、 同步荧光和二维(2D)荧光光谱技术研究了不同浓度和温度下胶原分子的聚集行为。 研究结果表明: (1)在激发波长275 nm条件下, 胶原分子仅在发射波长303 nm处出现了归属于Tyr的特征峰; 选取波长差(Δ λ)为15 nm的同步荧光扫描胶原分子, 发现其在261和282 nm处出现了分别归属于Phe和Tyr的特征峰。 (2)特征峰的荧光强度与胶原浓度呈现良好的线性关系, 表明了基于常规波长和同步荧光光谱技术对胶原定量分析的可行性。 (3)随着胶原浓度的增加, Tyr和Phe的含量逐渐增大, 且胶原分子间距逐渐降低并聚集成纤维束, 使得Tyr和Phe相互靠近并参与形成大量的氢键, 从而导致荧光强度不断增大。 然而随着温度的升高, 荧光基团与溶剂碰撞的猝灭机会增大, 且胶原分子中Tyr和Phe的荧光量子产率逐渐降低, 同时胶原分子动能增大, 其聚集体逐渐松散, 其三股螺旋结构逐渐坍塌, Tyr和Phe参与形成的氢键被破坏, 从而导致荧光强度随温度的升高不断降低。 (4)275 nm常规波长的2D荧光光谱分析表明, 胶原分子在297, 303和310 nm处出现了相关峰, 其中303 nm归属于Tyr, 297 nm归属于胶原分子聚集过程中参与氢键形成的Tyr; 310 nm可能归属于Tyr的激发态, 其不断的蓝移形成稳定的基团, 以便参与氢键的形成, 从而促进了胶原分子的聚集。 以浓度为外扰的基团响应顺序为303 nm>297 nm>310 nm; 以温度为外扰的基团响应顺序为297 nm>310 nm>303 nm。 (5)2D同步荧光光谱分析表明, 随着胶原浓度和温度的升高, Phe均比Tyr优先响应。 综上, 采用常规波长、 同步荧光光谱技术均能较好的研究胶原分子在不同浓度和温度下的聚集行为, 且为胶原的定量分析提供了一种新的方法, 但同步荧光光谱技术可将量子产率较低的Phe显现出来, 体现了其具有窄化谱带和提升分辨率的优点。 此外, 结合2D荧光分析技术, 可进一步研究胶原分子基团的响应顺序。

The aggregation behavior of collagen molecules not only improves its physicochemical characteristics, but also provides theoretical guidance for its application in the fields of food, tissue engineering and biomedicine. In this paper, the aggregation behavior of collagen molecules at various concentrations and temperatures was analyzed by using the conventional wavelength, the synchronization fluorescence and the two-dimensional (2D) fluorescence spectroscopy technology based on the intrinsic fluorescence characteristic of the tyrosine (Tyr) and the phenylalanine (Phe). The results showed that: (1) At the excitation wavelength of 275 nm, the characteristic peak at 303 nm could be found, which was attributed to Tyr. Under the synchronous fluorescence scanning (Δ λ=15 nm), two auto-peaks at 261 and 282 nm could be found, which were mainly assigned to Phe and Tyr, respectively. (2) It could find a good linear relationship between the fluorescence intensity and collagen concentration, indicating the feasibility of the quantitative analysis of collagen based on the conventional wavelength and the synchronous fluorescence spectroscopy. (3) With the increase of collagen concentrations, the amount of Tyr and Phe increased gradually, collagen molecular distance reduced and collagen molecules aggregated into fibrils, then Tyr and Phe were close to each other and participated in forming many hydrogen bonds, which led to the increase of the fluorescence intensity. When the temperature was increased from 10 to 70 ℃, the quenching opportunity between the fluorescence group and solvent increased and the fluorescence quantum yield of Tyr and Phe in collagen molecules decreased. Meanwhile, collagen molecular kinetic energy increased, then the collagen aggregate became loose and the trip-helix structure of collagen was destroyed gradually. Finally, the hydrogen bonds involved by Tyr and Phe were destroyed. Therefore, the fluorescence intensity of collagen decreased with the increase of temperatures. (4) The results of 2D conventional wavelength (275nm) fluorescence spectrum demonstrated that there were three relation peaks, which were located at 297, 303 and 310 nm. The peak at 303 nm was attributed to Tyr; the peak at 297 nm was recognized by Tyr, which participated in the formation of hydrogen bonds. Additionally, the peak at 310 nm might be assigned to an excimer-like species, which exhibited a blue shift to form stable Tyr with the aim of forming hydrogen bonds and then promoted the aggregation of collagen molecules. Finally, concentration-dependent and temperature-dependent 2D conventional wavelength correlation spectroscopy showed that the response order was 303 nm>297 nm>310 nm and 297 nm>310 nm>303 nm, respectively. (5) 2D synchronization fluorescence correlation spectroscopy demonstrated that Phe changed before Tyr. In a word, both the conventional wavelength and the synchronization fluorescence spectroscopy technology can investigate the aggregation behavior of collagen excellently at various concentrations and temperatures, and provide a new method for the quantitative analysis of collagen. Although the quantum yields of Phe is much lower than that of Tyr, the characteristic peak could be found by the synchronization fluorescence spectroscopy technology, demonstrating that the synchronization fluorescence spectroscopy technology has the advantages of narrowing the band and improving resolution. Combined with 2D fluorescence spectroscopy technology, the respond order of groups of collagen molecules can be illustrated further.