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Separation of closely related peptide substrates of human proteinases by micellar electrokinetic chromatography with anionic and nonionic surfactants
Author(s) -
Lupi Anna,
Viglio Simona,
Luisetti Maurizio,
Zanaboni Giuseppe,
Cetta Giuseppe,
Iadarola Paolo
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(20000601)21:10<1985::aid-elps1985>3.0.co;2-z
Subject(s) - micellar electrokinetic chromatography , chemistry , micelle , chromatography , electrophoresis , peptide , critical micelle concentration , proteolytic enzymes , enzyme , biochemistry , organic chemistry , aqueous solution
In order to use micellar electrokinetic chromatography to determine the proteolytic activity of different proteinases simultaneously present in physiological fluids, the technique must be able to separate mixtures of substrates with closely related structures. In an attempt to determine the best electrophoretic conditions for resolving six p ‐nitroanilide peptides used as synthetic substrates of the elastolytic enzymes (human neutrophil elastase, cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in pulmonary diseases, we investigated the efficiency of ionic and nonionic surfactants in achieving the separation of this complex mixture. The results presented here show that, of all the electrophoretic systems tested, 30 m M sodium tetraborate, pH 9.3, containing 25 m M Brij 35 as micellar agent offered the best performance; the separation efficiency of peptides is greater than that obtained with other reagents and all peaks are baseline resolved and unambiguously identifiable. Analysis of the micelle‐solute interaction with the surfactants investigated allowed better definition of the mechanism involved in the distribution of these peptides to the micelles and identification of some structural features that determined the magnitude of the micelle peptide complex formation.