Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with immobiline membranes.
The purification of small glycopeptides (a Hepta-Tyr of the teicoplanin family, exhibiting broad activity against highly glycopeptide-resistant enterococci) by isoelectric focusing in multicompartment electrolyzers with buffering, isoelectric membranes, is described. The main obstacle to such a preparative technique, in common with all focusing methodologies, is the poor solubility of the analyte at the pI value with resultant precipitation and coprecipitation of all impurities with the main fraction. A good solubilizing power was obtained in hydro-organic solvents, particularly a mixture of 6 M urea and 20-25% trifluoroethanol. Best results, however, were obtained with mixtures of 8 M urea and zwitterionic detergents, notably the 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) family. A unique behavior of the peptide was found in concentration gradients of CHAPS: solubility increases up to 3.5% CHAPS, but the curve shows a maximum and then solubility decreases again at 5% CHAPS. In mixtures of 8 M urea and 3.5% CHAPS, sample loads of 500 up to 1000 mg Hepta-Tyr could be purified in a single run, with recoveries > 90% and purity in excess of 99%. The main glycopeptide fraction (pI 8.56) was collected into an isoelectric trap delimited by pI 8.46 and pI 8.65 membranes. Attempts at purifying the glycopeptide by most known reversed phase-high performance liquid chromatography (RP-HPLC) techniques failed completely.
Bossi
A
A
Department of Cell Biology, University of Calabria, Cosenza, Italy.
Righetti
P G
PG
Riva
E
E
Zerilli
L
L
D000900:Anti-Bacterial Agents / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002793:Cholic Acids; D003902:Detergents; D006020:Glycopeptides / Q000302:isolation & purification*; D007526:Isoelectric Point; D012995:Solubility; D017334:Teicoplanin; D014508:Urea
D000900:Anti-Bacterial Agents; D002793:Cholic Acids; D003902:Detergents; D006020:Glycopeptides; D017334:Teicoplanin; D014508:Urea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate