Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

• Published in: Photochemistry and photobiology Vol. 97; no. 6; pp. 1507 - 1515
• Main Authors: Nguyen, Kathy‐Uyen, Zhang, Ran, Taniguchi, Masahiko, Lindsey, Jonathan S.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.11.2021; Wiley-Blackwell
• Subjects: Chlorophyll; Chlorophyll - chemistry; Chlorophyll A; Cyanobacteria - chemistry; Dichloromethane; Environmental stress; Excitation spectra; Fluorescence; Fractionation; Germfree; Microorganisms; Natural products; Propanol; Reduction; Sample preparation; Spectrometry, Fluorescence; Spectroscopy
• ISSN: 0031-8655; 1751-1097
• DOI: 10.1111/php.13474

Tolyporphins are distinctive tetrapyrrole natural products found singularly in a filamentous cyanobacterial‐microbial holobiont (termed HT‐58‐2) from Micronesia. The absorption and fluorescence features of tolyporphins resemble those of chlorophyll a, complicating direct analysis of culture samples. Treatment of the crude (unfractionated) organic extract (CH2Cl2/2‐propanol, 1:1) of HT‐58‐2 cultures with NaBH4 in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17‐dioxobacteriochlorins) exhibit a bathochromic shift (λabs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 131‐oxochlorin) exhibits a hypsochromic shift (λabs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λem 710 nm) enabled detection of reduced tolyporphins amidst abundant reduced chlorophyll a (1:19 ratio), a detection sensitivity >5 times that without reduction. The resulting assay combines simple sample preparation from non‐axenic cultures at microscale quantities (2 mL, 2 μm), absence of any fractionation procedures, and fluorescence detection. Tolyporphins were readily detected in cultures of HT‐58‐2 at reasonable growth periods in the absence of environmental stressors, which was not possible previously. Tolyporphins—a mixture of 18 unusual tetrapyrrole macrocycles comprised chiefly of dioxobacteriochlorins—were easily detected in the presence of chlorophyll sans fractionation of lipophilic extracts of a non‐axenic cyanobacterial culture. The assay relies upon reduction of the respective ketones with NaBH4 followed by fluorescence excitation spectroscopy, enabling facile spectroscopic detection of these unusual and incongruent macrocycles.