High-performance liquid chromatographic separation of modified and native melittin following transglutaminase-mediated derivatization with a dansyl fluorescent probe.
The 26-amino acid linear, amphiphilic peptide melittin was enzymatically modified with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase and a fluorescent derivative of stoichiometry 1:1 was obtained. Reversed-phase and size-exclusion high-performance liquid chromatographic modes were tested in order to resolve the labelled peptide and native species. The influence of several operational variables was analysed and the elution conditions were optimized so that a satisfactory resolution could be achieved in both instances in a rapid, easy manner. Both chromatographic modes offer the possibility of accurate monitoring of the time course of the enzyme-mediated conversion and more interestingly, can be applied to the semi-preparative purification of the labelled peptide.
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, Spain.
E
E
Perez-Paya
L
L
Braco
C
C
Abad
J
J
Dufourcq
D000595:Amino Acid Sequence; D000818:Animals; D002103:Cadaverine / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D006168:Guinea Pigs; D008555:Melitten / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D011503:Transglutaminases
D003619:Dansyl Compounds; D008555:Melitten; D011503:Transglutaminases; D002103:Cadaverine