Aggregation Behavior of Collagen Molecules in Aqueous Solution Based on Fluorescence Spectroscopy Technology
LI Cong-hu1,2,3, WU Yan1,2,3, MA Xing-hong1, FANG Yi-fan1, ZHANG Ying1, LI Wen-juan1,2, TIAN Hui-lin4
1. College of Life Science, Anqing Normal University, Anqing 246133, China
2. The Province Key Laboratory of the Biodiversity Study and Ecology Conservation in Southwest Anhui, Anqing 246133, China
3. Research Center of Aquatic Organism Conservation and Water Ecosystem Restoration, Anqing 246133, China
4. School of Leather Chemistry and Engineering, Qilu University of Technology (Shandong Academy of Sciences)，Ji’nan 250353, China
Abstract：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 (Δλ=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.
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