紫细菌光合色素指纹图谱的建立与色素分析

【目的】探索快速高效的色素样品制备方法;为建立紫细菌全色素TLC和HPLC标准指纹图谱和数据库提供研究方法和思路;为色素代谢与调控等研究提供快速的色素分析方法。【方法】选择沼泽红假单胞菌(Rhodopesudomonas palustris CQV97)为材料,采用改良丙酮甲醇提取法、TLC重复展层法、图像灰度分析法、吸收光谱法、HPLC和MS法进行色素分析。【结果】甲醇或丙酮可选择性地提取样品的细菌叶绿素和类胡萝卜素,通过对丙酮甲醇法的改良,使色素提取总量提高了13.5%。建立了CQV97菌株全色素的TLC和HPLC指纹图谱,二者均含有11种色素组分。图像灰度分析法估算了TLC指纹图谱各色素组分的表观相对含量。以TLC图谱的各色素组分为外标物,明确了HPLC图谱中各色素组分的保留时间(Rt)与TLC图谱中各色素组分的迁移率(Rf)之间的对应关系。结合色素代谢途径、光谱分析和MS,定性分析了指纹图谱中11种色素组分。TLC或HPLC分析结果表明,理论样品与对照样品色素组分和含量一致,而实际样品与对照样品色素组分一致,但含量不同,重复测定3次,样品中色素相对含量的RSD均小于5%。【结论】改良的丙酮甲醇法可以快速高效地提取光合细菌的色素。采用TLC重复展层法和HPLC法建立的全色素指纹图谱色素组成一致,重复稳定性好,各具特色。TLC和HPLC两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫细菌色素合成途径中主要积累色素的组分和含量变化规律研究。

Fingerprinting analysis of photopigments in purple bacteria

Objective]Photopigments,including carotenoid and bacteriochlorophyll a,are the most important functional units of photosynthesis in purple bacteria.We developed rapid qualitative and quantitative methods to determine photopigments.Methods] Using Rhodopesudomonas palustris CQV97 as a reference,we used image gray intensity analysis,absorption spectrophotometry,thin layer chromatography(TLC),HPLC and mass spectrometry(MS) for photopigment analysis.Results] The total amount of photopigments increased by 13.5% by using modified acetone-methanol extraction.We developed two types of photopigment fingerprintings by TLC and HPLC,estimated the apparent relative content of each photopigment of fingerprintings,and determined the corresponding relationships between Rf value of each photopigment on TLC fingerprinting and retention time of each photopigment elution in HPLC fingerprinting.Based on the data from the absorption spectra,MS and related photopigment biosynthetic pathway analysis,we identified 11 photopigments in CQV97 strain.Using this strain as a standard,we analyzed photopigments of the tested samples by TLC or HPLC.It was shown that(1) the relative standard deviation(RSD) of the two methods was less than 5%;(2) the compositions and contents of the theory sample were consistent with that of the standard sample;(3) the photopigment compositions of the real sample was the same as the standard sample,but the photopigment content was different.Conclusion] Both of TLC and HPLC analyses for photopigment determination have good stability and repeatability.The fingerprintings analyses are suitable for rapid determination of photopigments of purple bacteria and have important application in control of regulation mechanism for photopigment synthesis.