Site-specific deprotonation/sodiation of gramicidin S studied by electrospray ionization/mass spectrometry

The functioning of many enzymes is metal ion dependent. In this work, deprotonation/sodiation of gramicidin S (GS) was studied by electrospray ionization mass spectrometry. Electrospray mass spectra for 10 (-5) M of GS in water/methanol with and without the addition of 1 mM NaOH, AcONa, NaCl, NH4Cl, AcONH4 and 1% AcOH were measured. It was found that one and two amide N-H bonds for [GS + H](+) and [GS + 2H](2+), respectively, were preferably deprotonated to form [GS + H - H + Na](+) and [GS + 2H - 2H + 2Na](2+). The extent of sodiation was found to be in the order of NaOH > AcONa > NaCl. With the addition of 1 mM NaOH, [GS + 2H - nH + nNa](2+) with n up to 8 were observed indicating that all of the eight amide N-H bonds in GS were deprotonated. Sodiation sites are likely to be the negatively charged amide enolate C-O- to form salt-like C-O- center dot center dot center dot center dot Na+ bonds. In the negative mode, one and two amide N-H bonds are preferably deprotonated to form [GS- H], [GS - 2H](2-) and [GS - 2H + Na](-) for the solution with the addition of 1 mM NaOH.