Chlorophyll Biosynthesis: Spectrofluorometric Estimation of Protoporphyrin IX, Mg-Protoporphyrin and Protochlorophyllide

Abstract

The chlorophyll (chl) molecules are synthesized from 5 aminolevulinic acid (ALA) via various intermediates. It is already demonstrated that chl biosynthesis takes place via monovinyl and divinyl monocarboxylic routes (2,4–6). To elucidate the chl biosynthetic pathways and to monitor the amount of over-accumulated porphyrins to induce photodynamic damage to the plants (1,3), the various pools of intermediates of chl biosynthetic pathway like protoporphyrin IX (Proto IX), mg-protoporphyrin monoester (MPE) or protochlorophyllide (Pchlide) etc. need to be quantified (4). In small amount of tissues and especially in isolated etioplasts or chloroplasts the pools of above intermediates are too small to be estimated spectrophotometrically. As Proto IX, MPE and Pchlide fluoresce, and spectrofluorometry is a very sensitive tool, the fluorescence of these compounds can be measured for their quantification. However, as these components especially Proto IX, Pchlide and Chlide are having overlapping fluorescence spectra, it would be incorrect to quantify the above components from their mixtures by measuring fluorescence amplitudes at their respective peaks. Therefore it is essential to correct the fluorescence amplitude of the compound to be quantified (say Proto IX) for the contribution due to other fluorescing compounds (say Pchlide and Chlide) at the measured wavelength.