Department of Chemistry, North Carolina State University, Raleigh, NC, 27695-8204, USA.
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695-8204, USA.
Phytochem Anal. 2018 Mar;29(2):205-216. doi: 10.1002/pca.2735. Epub 2017 Nov 6.
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.
To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a.
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.
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.
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.
托利卟啉是一种不依赖于共生体的丝状蓝藻（HT-58-2）产生的不寻常的四吡咯大环化合物。托利卟啉 A-J、L 和 M 具有共同的二氧杂苯并卟啉核心，在周围取代基上有所不同，并表现出与优势蓝藻色素叶绿素 a 重叠的吸收光谱。在这些富含叶绿素的样品中，鉴定和准确定量各种托利卟啉具有一定挑战性。
开发用于定量测定在各种生长条件下产生的托利卟啉相对于叶绿素 a 的方法。
大规模（440 L）培养物的色谱分离提供了分离的单个托利卟啉。小规模（25 mL）培养物的亲脂性提取、带内部标准的 HPLC 分离和吸收检测使托利卟啉 A 和叶绿素 a 的定量成为可能，并推断出托利卟啉 A-M 的量。对 2 毫升培养物的亲脂性提取物进行多组分吸收光谱分析，可获得所有托利卟啉与叶绿素 a 的比值。为了定量目的，需要重新评估各种托利卟啉的报告吸收光谱数据。
光照 50 天后，硝酸盐存在时托利卟啉 A 的量范围为 0.13 nmol/mg 干细胞（含硝酸盐）至 1.12 nmol/mg（不含硝酸盐），最大量为叶绿素 a 的 0.23 倍。在 35-50 天后可溶性氮缺乏的情况下，托利卟啉 A 占总托利卟啉的 1/3-1/2，总托利卟啉的量高达叶绿素 a 的 1.8 倍。
本文开发的定量方法应有助于研究托利卟啉（和其他四吡咯）的生物合成以及其他菌株生产托利卟啉的情况。版权所有© 2017 年 John Wiley & Sons, Ltd.
