Heme quantification was conducted as described previously (65 (link), 107 (link), 108 (link)). Briefly, frozen mycelia were ground into fine powder with liquid nitrogen. Samples were homogenized in phosphate-buffered saline, and protein contents were determined by the Bradford method (109 (link)). A portion of extract was mixed with an equal volume of acetone and concentrated HCl (97.5/2.5; vol/vol). After centrifuging, the supernatant was analyzed by reversed-phase HPLC with an ultraviolet detector.
The analysis was carried out using a Shimadzu UFLC HPLC system (Shimadzu, Japan). To prepare solvent A, 0.1 M ammonium phosphate solution (pH adjusted to 3.5 with phosphoric acid) was filtered with a 0.45-µm membrane filter (Millipore, USA). The filtered solution was mixed with methanol (56:44; vol/vol), and the pH of the solution was adjusted to 3.4 with phosphoric acid. Pure methanol was used for solvent B. Samples were eluted on a C18 HPLC column (Shimadzu, Japan) at 35°C at a flow rate of 1.5 mL/min. In gradient elution, the composition of solvent B was changed from 30% to 100% for 15 min. The spectra of the samples were monitored at 405 nm.
The analysis was carried out using a Shimadzu UFLC HPLC system (Shimadzu, Japan). To prepare solvent A, 0.1 M ammonium phosphate solution (pH adjusted to 3.5 with phosphoric acid) was filtered with a 0.45-µm membrane filter (Millipore, USA). The filtered solution was mixed with methanol (56:44; vol/vol), and the pH of the solution was adjusted to 3.4 with phosphoric acid. Pure methanol was used for solvent B. Samples were eluted on a C18 HPLC column (Shimadzu, Japan) at 35°C at a flow rate of 1.5 mL/min. In gradient elution, the composition of solvent B was changed from 30% to 100% for 15 min. The spectra of the samples were monitored at 405 nm.
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