Küpper Hendrik, Seibert Sven, Parameswaran Aravind
Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany.
Anal Chem. 2007 Oct 15;79(20):7611-27. doi: 10.1021/ac070236m. Epub 2007 Sep 14.
Quantification of pigments in complex mixtures is an important task in the physiology of photosynthetic organisms, because pigment composition differs depending on the species, tissue, and physiological state. Currently available methods, however, are either limited to very few pigments (classical UV/vis spectroscopic methods), or they are time-consuming, labor intensive, or costly (e.g., HPLC). Here we describe a UV/vis spectrophotometric method that is capable of a rapid (approximately 1 min/sample) and inexpensive (<1 euro/sample) quantification of more than a dozen pigments in a crude extract, which means it is suitable for high-throughput screening applications. A detection limit of <1 pmol for each pigment allows for determining the pigment composition in only 0.5 microg of lyophilized leaves or algae. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss peak spectra" including an automatic correction of wavelength inaccuracy, baseline instability, sample turbidity, and effects of temperature/water content. Here we present the Gauss peak spectra from 350 to 750 nm for acetone solutions of all chlorophyll and carotenoid derivatives that are abundant (including conditions of Cd, Cu, or Zn stress) in leaves of higher plants, Euglena, brown algae, and various cyanobacteria like Anabaena and Trichodesmium: [Mg]-Chl a, b, c1, c2; pheophytin a, b; [Cd]-Chl a, b; [Cu]-Chl a, b; [Zn]-Chl a, b; antheraxanthin, aurochrome, beta-carotene, beta-cryptoxanthin, cis- and trans-canthaxanthin, diadinochrome (=diadinoxanthin 5,6-epoxide), cis- and trans-diadinoxanthin, diatoxanthin, cis- and trans-echinenone, fucoxanthin, lutein, myxoxanthophyll, neoxanthin, violaxanthin, and all three stereoisomers of zeaxanthin in acetone. We present extensive tests of our new quantification method for determining optimal and limiting conditions of its performance and for comparison with previous methods. Finally, we show application examples for Thlaspi fendleri (Chlorophyta), Euglena gracilisc (Euglenophyta), Ectocarpus siliculosus (Phaeophyta), and Trichodesmium erythraeum IMS101 (cyanobacteria).
对光合生物生理学而言，复杂混合物中色素的定量分析是一项重要任务，因为色素组成因物种、组织和生理状态而异。然而，目前可用的方法要么仅限于极少数色素（经典紫外/可见光谱法），要么耗时、费力或成本高昂（例如高效液相色谱法）。在此，我们描述了一种紫外/可见分光光度法，该方法能够快速（约1分钟/样品）且廉价（<1欧元/样品）地对粗提物中的十几种色素进行定量分析，这意味着它适用于高通量筛选应用。每种色素<1皮摩尔的检测限使得仅用0.5微克冻干叶片或藻类就能确定色素组成。该方法基于用一系列高斯峰描述每种色素光谱。然后通过这些“高斯峰光谱”的线性组合拟合样品光谱，包括自动校正波长不准确、基线不稳定、样品浊度以及温度/含水量的影响。在此，我们给出了高等植物、裸藻、褐藻以及各种蓝细菌（如鱼腥藻和束毛藻）叶片中所有丰富的叶绿素和类胡萝卜素衍生物（包括镉、铜或锌胁迫条件下）在丙酮溶液中350至750纳米的高斯峰光谱：[Mg]-叶绿素a、b、c1、c2；脱镁叶绿素a、b；[Cd]-叶绿素a、b；[Cu]-叶绿素a、b；[Zn]-叶绿素a、b；花药黄质、金藻色素、β-胡萝卜素、β-隐黄质、顺式和反式角黄素、二乙二烯色素（=二乙二烯色素5,6-环氧化物）、顺式和反式二乙二烯色素、硅藻黄质、顺式和反式海胆酮、岩藻黄质、叶黄素、粘藻叶黄素、新黄质、紫黄质以及玉米黄质的所有三种立体异构体在丙酮中的光谱。我们对新的定量方法进行了广泛测试，以确定其性能的最佳和极限条件，并与以前的方法进行比较。最后，我们展示了对费氏遏蓝菜（绿藻门）、纤细裸藻（裸藻门）、丝状褐藻（褐藻门）和红海束毛藻IMS101（蓝细菌）的应用实例。
