DIRECT DETERMINATION OF SOLUTION BINDING CONSTANTS FOR NONCOVALENT COMPLEXES BETWEEN BACTERIAL-CELL WALL PEPTIDE ANALOGS AND VANCOMYCIN GROUP ANTIBIOTICS BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

We report a method based on electrospray ionization (ESI) mass spectro metry to determine solution association constants (KA) for complexes b etween glycopeptide antibiotics (vancomycin and ristocetin) and severa l peptide ligands. The measured KA values are in good agreement with p reviously reported values obtained by standard spectroscopic titration techniques. The pH stability of the ristocetin-diacetyl-L-lysyl-D-ala nyl-D-alanine complex was investigated by ESI mass spectrometry and ci rcular dichroism (CD) spectroscopy. The two methods produce very simil ar results, demonstrating that the ESI mass spectra reflect the pH sta bility of the complex. In solution, the antibiotics bind stereospecifi cally to peptides containing a C-terminal D-Ala-D-Ala sequence, wherea s no complex formation is observed with peptides containing the L-Ala- L-Ala stereoisomer. To investigate whether electrospray ionization is able to reflect the structurally specific interaction between antibiot ics and D-Ala-D-Ala peptide ligands, an equimolar mixture of vancomyci n, diacetyl-L-lysl-D-alanyl-D-alanine, and the deuterium-labeled stere oisomer diacetyl-L-lysyl-L-alanyl-L-alanine-d(6) was analyzed by ESI m ass spectrometry. In agreement with solution-phase behavior, only ions corresponding to a complex between diacetyl-L-lysyl-D-alanyl-D-alanin e and vancomycin are observed.