DETERMINATION OF ACCURATE EXTINCTION COEFFICIENTS AND SIMULTANEOUS-EQUATIONS FOR ASSAYING CHLOROPHYLL-A AND CHLOROPHYLL-B EXTRACTED WITH 4 DIFFERENT SOLVENTS - VERIFICATION OF THE CONCENTRATION OF CHLOROPHYLL STANDARDS BY ATOMIC-ABSORPTION SPECTROSCOPY
DETERMINATION OF ACCURATE EXTINCTION COEFFICIENTS AND SIMULTANEOUS-EQUATIONS FOR ASSAYING CHLOROPHYLL-A AND CHLOROPHYLL-B EXTRACTED WITH 4 DIFFERENT SOLVENTS - VERIFICATION OF THE CONCENTRATION OF CHLOROPHYLL STANDARDS BY ATOMIC-ABSORPTION SPECTROSCOPY
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DOI：
10.1016/s0005-2728(89)80347-0
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发表时间：
1989-08-03
期刊：
影响因子：
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通讯作者：
KRIEDEMANN, PE
中科院分区：
文献类型：
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作者：
PORRA, RJ;THOMPSON, WA;KRIEDEMANN, PE
叶绿素a和B在乙醚中的消光系数（Smith，J. H.C.和Benitez，A.（1955）在现代植物分析方法（Paech，K.和特蕾西，MV编辑），Vol. 4，pp. 143-196，Springer-Verlag，柏林），在本文中用作一级标准品，通过使用原子吸收分光光度法测定镁，验证其误差小于1%。我们还报道了叶绿素a和B在N，N“-二甲基甲酰胺、甲醇或缓冲的80%丙酮水溶液中的精确消光系数的测定。高度纯化的叶绿素和方法，不仅尽量减少由于挥发性溶剂的蒸发，在他们的估计，但也消除了可变的微污染叶绿素降解产物，这三种溶剂中获得的消光系数之间的不一致的潜在来源。使用这些新的系数，表示为毫摩尔系数和比系数，我们导出了新的联立方程，以获得叶绿素浓度分别为nmol/ml和μ g/ml。将这些方程应用于用三种指定溶剂提取的菠菜和Flindersia brayyyana的叶圆片获得的数据，并应用于加入到三种溶剂系统中的叶绿素a+B混合物的浓缩溶液（在N，N“-二甲基甲酰胺中）。叶绿素测定值和叶绿素a/B比值的一致性证明了这些方程的有效性。新的联立方程，兼容的方程推导出的三种溶剂，叶绿素a和B转化为环状羟基内酯衍生物的测定与碱性吡啶试剂（2.1 M吡啶在0.35 M NaOH）萃取。高等植物中的大多数叶绿素分析，包括植物类囊体和叶绿素-蛋白质复合物的叶绿素含量和叶绿素a/B比，已经在80%丙酮水溶液中用Arnon（Arnon，D.I.（1949）Plant Physiol.24，1-15）。为此，我们包括转换因子，纠正这些早期的数据，使其与本文提出的联立方程计算的数据兼容。这些校正的重要性，制定有意义的模型的光合机构的证明。我们的研究结果还表明，用N，N“-二甲基甲酰胺研磨叶盘是一种比用这种溶剂振荡24小时提取所有叶绿素更可靠的方法。
The extinction coefficients for chlorophylls a and b in diethylether (Smith, J. H.C. and Benitez, A. (1955) in Modern Methods of Plant Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143-196, Springer-Verlag, Berlin), used in this paper as primary standards, were verified, to within an error of less than 1%, by magnesium determination using atomic absorbance spectrophotometry. We also report the determination of accurate extinction coefficients for chlorophyll a and b in N,N''-dimethylformamide, methanol or buffered 80% aqueous acetone. Highly purified chlorophylls were used and methods were employed which not only minimize errors due to evaporation of the volatile solvents employed in their estimation but also eliminate variable micro-contamination by chlorophyll degradation products, a potential source of inconsistency between the extinction coefficients obtained in each of these three solvents. Using these new coefficients, expressed as both millimolar and specific coefficients, we have derived new simultaneous equations to obtain chlorophyll concentrations as nmol/ml and .mu.g/ml, respectively. These equations were applied to data obtained with leaf discs from spinach and Flindersia brayleyana extracted with the three specified solvents and to a concentrated solution (in N,N''-dimethylformamide) of a chlorophyll a+b mixture added to the three solvent systems. The validity of these equations is proven by the consistency of the chlorophyll determinations and of the chlorophyll a/b ratios. New simultaneous equations, compatible with the equations derived for the three solvents, are presented for the assay of chlorophylls a and b converted to their cyclic hydroxylactone derivatives by extraction with alkaline pyridine reagent (2.1 M pyridine in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including the chlorophyll content and chlorophyll a/b ratios of plant thylakoids and chlorophyll-protein complexes, have been obtained in 80% aqueous acetone with the much used simultaneous equations of Arnon (Arnon, D.I. (1949) Plant Physiol. 24, 1-15). For this reason we include conversion factors which correct these earlier data and make it compatible with data calculated with the simultaneous equations presented in this paper. The importance of these corrections to the formulation of meaningful models of the photosynthetic apparatus is demonstrated. Our results also indicate that grinding leaf discs with N,N''-dimethylformamide is a more reliable method for extracting all chlorophylls than shaking with this solvent for 24 h.