Abstract
Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.
Keywords: Chlorophyll biosynthesis, Tetrapyrroles, Flourescence detector, High Performance Liquid Chromatography, Synechocystis 6803
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. Representative chromatograms of separated precursors extracted from the cyanobacterium Synechocystis PCC 6803. A. Chromatogram recorded by the first fluorescent detector (FLD). B. Chromatogram recorded simultaneously by the second FLD. Settings of excitation and emission wavelengths for FLD #1 and #2 are shown above the each chromatogram. The number in round brackets indicates the FLD sensitivity (gain; the maximum = 16) set for the given wavelengths (see Note 3). Copro III = Coproporphyrin IX; DV-PChlide = divinyl-protochlorophyllide; MV-PChlide = monovinyl-protochlorophyllide; DV-Chlide = divinyl-chlorophyllide; MV-Chlide = monovinyl-chlorophyllide; Proto IX - Protoporphyrin IX; Mg-Proto = Mg-protoporphyrin IX; Mg-Proto ME = Mg-protoporphyrin IX monomethylester.
Notes
Recipes
Acknowledgments
This research project was supported by projects Algatech (CZ.1.05/2.1.00/03.0110) and Algain (EE2.3.30.0059) of the Czech Ministry of Education, Youth and Sports.
References
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