Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples.
Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples.
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DOI：
10.1186/1746-4811-9-19
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发表时间：
2013-06-19
期刊：
影响因子：
5.1
通讯作者：
Tanaka R
中科院分区：
文献类型：
--
作者：
Hu X;Tanaka A;Tanaka R
在进行植物研究时，光合色素的测量可以提供有关植物生理状态的基本信息。高压液相色谱法（HPLC）正被广泛用于这一目的，因为它提供了一个准确的测定多种光合色素的同时。然而，与传统的光谱技术相比，该技术具有缺点，因为其在提取期间更倾向于颜料的结构改变，从而潜在地产生错误的结果。在用丙酮或乙醇提取色素的过程中，叶绿素的叶绿醇侧链有时会被除去，形成叶绿酸，这会影响使用HPLC的叶绿素测量。我们通过比较不同的提取方法与野生型和突变体拟南芥叶片，缺乏叶绿素酶的主要亚型，叶绿素提取过程中的人工叶绿素生产进行了评估。几种提取方法进行了比较，以提供替代方案的研究人员谁利用高效液相色谱法分析叶绿素水平。因此，推荐以下三种方法。在第一种方法中，叶子在提取之前被简单地煮沸。在第二种方法中，在零度以下的温度下进行叶片的研磨和均质化。在第三种方法中，N，N '-二甲基甲酰胺（DMF）用于提取色素。比较前两种方法，几乎消除了拟南芥、皂角、豌豆中所有的叶绿素形成活性。和紫叶稠李Prunus sargentii sp.然而，DMF有效地抑制叶绿素酶的活性，只有在拟南芥叶片。叶提取物中叶绿素的产生主要是人为因素。本研究中评价的所有三种方法都减少了叶绿素的人为产生，因此适用于HPLC分析的色素提取。当叶子不太厚时，煮沸方法将是一个实用的选择。然而，它可以将一小部分叶绿素a转化为脱镁叶绿素a。虽然在零度以下的温度下提取适合于本研究中检查的所有植物物种，但这种方法对于大量样品可能很复杂，并且需要液氮和叶片研磨设备。用DMF作为提取剂，方法简单，适用于拟南芥样品的提取。然而，这种溶剂不能完全阻止较厚叶片中叶绿素的形成。
When conducting plant research, the measurement of photosynthetic pigments can provide basic information on the physiological status of a plant. High-pressure liquid chromatography (HPLC) is becoming widely used for this purpose because it provides an accurate determination of a variety of photosynthetic pigments simultaneously. This technique has a drawback compared with conventional spectroscopic techniques, however, in that it is more prone to structural modification of pigments during extraction, thus potentially generating erroneous results. During pigment extraction procedures with acetone or alcohol, the phytol side chain of chlorophyll is sometimes removed, forming chlorophyllide, which affects chlorophyll measurement using HPLC. We evaluated the artifactual chlorophyllide production during chlorophyll extraction by comparing different extraction methods with wild-type and mutant Arabidopsis leaves that lack the major isoform of chlorophyllase. Several extraction methods were compared to provide alternatives to researchers who utilize HPLC for the analysis of chlorophyll levels. As a result, the following three methods are recommended. In the first method, leaves are briefly boiled prior to extraction. In the second method, grinding and homogenization of leaves are performed at sub-zero temperatures. In the third method, N, N’-dimethylformamide (DMF) is used for the extraction of pigments. When compared, the first two methods eliminated almost all chlorophyllide-forming activity in Arabidopsis thaliana, Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd. However, DMF effectively suppressed the activity of chlorophyllase only in Arabidopsis leaves. Chlorophyllide production in leaf extracts is predominantly an artifact. All three methods evaluated in this study reduce the artifactual production of chlorophyllide and are thus suitable for pigment extraction for HPLC analysis. The boiling method would be a practical choice when leaves are not too thick. However, it may convert a small fraction of chlorophyll a into pheophytin a. Although extraction at sub-zero temperatures is suitable for all plant species examined in this study, this method might be complicated for a large number of samples and it requires liquid nitrogen and equipment for leaf grinding. Using DMF as an extractant is simple and suitable with Arabidopsis samples. However, this solvent cannot completely block the formation of chlorophyllide in thicker leaves.
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