Introduction: Tolyporphins are unusual tetrapyrrole macrocycles produced by a non-axenic filamentous cyanobacterium (HT-58-2). Tolyporphins A-J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a. Identification and accurate quantitation of the various tolyporphins in these chlorophyll-rich samples presents challenges.
Objective: To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a.
Methodology: Chromatographic fractionation of large-scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small-scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a, and by inference the amounts of tolyporphins A-M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a. The reported absorption spectral data for the various tolyporphins required re-evaluation for quantitative purposes.
Results and discussion: The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a. Under soluble-nitrogen deprivation after 35-50 days, tolyporphin A represents 1/3-1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8-fold that of chlorophyll a.
Conclusions: The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd.
Keywords: HPLC; absorption spectroscopy with multicomponent analysis; growth curve; mass spectrometry; nitrogen deprivation.
Copyright © 2017 John Wiley & Sons, Ltd.