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. 2021 Feb 2;11:586705. doi: 10.3389/fphar.2020.586705

FIGURE 1.

FIGURE 1

Purification steps and molecular mass characterization. (A) Chromatographic profile of Duvernoy's gland secretion. Sample was subjected to a Jupiter® C18 semipreparative column (10 μm, 300 Å, 10 × 250 mm, Phenomenex™), with a 0–80% linear gradient of ACN in acidified water for 80 min at 2 ml/min flow rate. Absorbance was monitored at 280 and 225 nm. The fractions indicated by the numbers (2, 31 and 33) had antimicrobial activity. (B) Second step of purification by RP-HPLC. The chosen fraction (33) was subjected to an analytical column Jupiter® C18 (10 μm, 300 Å, 4.60 mm × 250, Phenomenex™) to check the homogeneity. The prominent peak was hand collected and submitted to a test of the inhibition of microbial growth in a liquid medium and mass analyzing. The SDS-PAGE analysis under reducing conditions shows a single band, with a mass of 25 kDa approximately, and confirms the fraction homogeneity. (C) Mass spectrometry analyses. The deconvolution of the ions revealed an m/z of 24,848.8 Da. The spectrum was submitted to the MagTran® software.