Hukmani P, Tripathy B C
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Anal Biochem. 1992 Oct;206(1):125-30. doi: 10.1016/s0003-2697(05)80021-1.
A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fluorescence emission spectra is developed. At room temperature (293 degrees K) protoporphyrin IX is monitored from its emission maximum, 633 nm, when excited at 400 nm (E400/F633). Protochlorophyllide is estimated at 638 nm, while being excited at 440 nm (E440/F638). Mg-protoporphyrin+Mg-protoporphyrin monoester pool has emission around 589-592 nm. Therefore the integration value of the emission band that extends from 580 to 610 nm is taken to calibrate its concentration. This spectrofluorometric method designed for the determination of protoporphyrin IX, esterified and nonesterified Mg-protoporphyrin pool, and protochlorophyllide is far superior to available spectrophotometric methods and estimates as low as 1 nM concentration of plant pigments. As minute quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates analytical uncertainties due to recovery losses caused by chromatography. However, only dilute samples can be estimated by this spectrofluorometric method as the quantitative relation between fluorescence and concentration deviates from linearity at high, i.e., above 150 nM, concentrations of pigment to be quantified.
开发了一种高灵敏度的荧光分光光度法，用于从具有重叠荧光发射光谱的植物四吡咯混合物中定量估计叶绿素生物合成的某些前体。在室温（293开尔文）下，当在400纳米处激发时（E400/F633），从其633纳米的发射最大值监测原卟啉IX。原叶绿素酸酯在440纳米激发时，在638纳米处进行估计（E440/F638）。镁原卟啉+镁原卟啉单酯池在589 - 592纳米左右有发射。因此，取580至610纳米范围内发射带的积分值来校准其浓度。这种设计用于测定原卟啉IX、酯化和未酯化的镁原卟啉池以及原叶绿素酸酯的荧光分光光度法，远远优于现有的分光光度法，能够估计低至1纳摩尔浓度的植物色素。由于可以从混合物中对微量的单个色素进行定量分析，该方法消除了因色谱法导致的回收损失所引起的分析不确定性。然而，这种荧光分光光度法只能估计稀释样品，因为在高浓度（即高于150纳摩尔）待定量色素的情况下，荧光与浓度之间的定量关系会偏离线性。
