Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts.

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 (CH2 Cl2 /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.