Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide beta-alanyl-tyrosine (beta-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of beta-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of beta-Ala-Tyr and its admixtures have been converted into preparative purification of beta-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the beta-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Automated summary

The growing resistance of bacteria to conventional antibiotics has led to the search for new antimicrobial agents, with insect antimicrobial peptides (AMPs) showing promise as potential therapeutics. A dipeptide beta-alanyl-tyrosine (beta-Ala-Tyr) in insect toxins has exhibited antibacterial activity, but requires purification before testing for other antimicrobial activities. A method using capillary zone electrophoresis (CZE) was developed for analysis, and a preparative purification of beta-Ala-Tyr was achieved using free-flow zone electrophoresis (FFZE).