Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

Nguyen, Kathy‐Uyen; Zhang, Ran; Taniguchi, Masahiko; Lindsey, Jonathan S.

doi:10.1111/php.13474

Title: Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

Journal Article · 15 July 2021 · Photochemistry and Photobiology

DOI: https://doi.org/10.1111/php.13474 · OSTI ID:1836396

Nguyen, Kathy‐Uyen ^[1]; Zhang, Ran ^[1]; Taniguchi, Masahiko ^[1];

^[1]

Department of Chemistry North Carolina State University Raleigh NC

Abstract 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 (CH ₂ Cl ₂ /2‐propanol, 1:1) of HT‐58‐2 cultures with NaBH ₄ in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17‐dioxobacteriochlorins) exhibit a bathochromic shift ( λ _abs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 13 ¹ ‐oxochlorin) exhibits a hypsochromic shift ( λ _abs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λ _em 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 μ m ), 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.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: FG02-05ER15661

OSTI ID:: 1836396

Journal Information:: Photochemistry and Photobiology, Journal Name: Photochemistry and Photobiology Journal Issue: 6 Vol. 97; ISSN 0031-8655

Publisher:: Wiley-BlackwellCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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