Five μL of purified mutacins, diluted in acidified (10 mM HCl) di

Five μL of purified mutacins, diluted in acidified (10 mM HCl) distilled water to promote solubility of the peptides, were deposited on the lawn and allowed to Quisinostat order dry before appropriate incubation. Mutacin activity was expressed in AU/mL and corresponds to the reciprocal of the highest dilution showing a noticeable inhibition zone on the lawn [14]. Amino acid sequencing procedure Alkaline ethanethiol GS-1101 datasheet derivatisation as described by Meyer et al. [26] was performed prior to sequencing of mutacins. Briefly, the purified sample was vacuum dried and

was dissolved in 30 μL of a derivatisation mixture composed of 1.4 M ethanethiol and 0.5 M NaOH in 46% aqueous ethanol. The sample was then incubated for 60 min at 50°C in limited oxygen atmosphere. The reaction was stopped by the addition of 2 μL of glacial acetic acid (Sigma-Aldrich, St Louis, MO, USA) just before sequencing. Pure

mutacin B-Ny266 was used as control for the Edman degradation with the alkaline ethanethiol derivatisation procedure [39]. Automated Edman degradation was performed on a protein sequencer (ABI Procise cLC, Applied Biosystems, Foster City, CA, USA) at the Biotechnology Research Institute (Montréal, QC, Canada). Amino acids were identified by capillary HPLC on a C18 0.8 × 150 mm column. Characterisation of mutacins by bioinformatic analyses Homology searches were carried out with the National Center of Biotechnology Information (NCBI) using the basic local alignment search tool for protein (BLAST-P) Megestrol Acetate with default parameters [41]. The constraint-based Roscovitine research buy multiple alignment tool

(COBALT) from NCBI was used with the default parameters to perform alignment. The primary and secondary structures of the mutacin F-59.1 were analyzed by the ExPASy Proteomics Server http://​ca.​expasy.​org/​tools/​#proteome[42] and the SCRATCH protein predictor http://​scratch.​proteomics.​ics.​uci.​edu/​[43]. Molecular mass analysis The molecular masses of mutacins (D-123.1 and F-59.1) were determined from pure HPLC fractions by MALDI-TOF MS analyses at the Mass Spectrometry Laboratory of Molecular Medicine Research Centre (University of Toronto, Toronto, ON, Canada). A saturated β-cyano-4-hydroxycinnamic acid in 70% acetonitrile and 0.1% TFA was used as the matrix solution. One μL of peptide sample was spotted on the sample target, and then 1 μL of saturated matrix solution was added on the top. After the crystal was formed, the sample target was inserted into the mass spectrometer. MALDI MS was acquired in linear mode at positive on Applied Biosystems Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a 337 nm laser. Acceleration voltage was set at 20 kV, grid voltage at 94%, guide wire at 0.05%, and delay time at 175 nsec. The mass spectra were externally calibrated by the molecular weights of a mixture of standard peptides. The mass accuracy is typically 0.05%.

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