Abstract
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 β-alanyl-tyrosine (β-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 β-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 β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.
Highlights
Excessive use of antibiotics in human and veterinary medicine and agriculture farming in the last several decades almost all over the world caused the selection and spread of antibiotic-resistant microorganisms
capillary zone electrophoresis (CZE) Analysis of β-Ala-Tyr Isolated from the Hemolymph of N. bullata by reversed-phase high-performance liquid chromatography (RP-HPLC)
The dipeptide β-Ala-Tyr was isolated as a main component from one of the fractions obtained by the RP-HPLC separation of the solid phase extract of hemolymph of the larvae of Neobellieria bullata using the above procedure, see Section 2.2
Summary
Excessive use of antibiotics in human and veterinary medicine and agriculture farming in the last several decades almost all over the world caused the selection and spread of antibiotic-resistant microorganisms. The insect AMPs are especially strongly investigated as promising candidates for new antimicrobials because insects represent the largest and most biodiverse class of organisms in the animal kingdom [5,9] Their humoral immune response to microbial infection includes synthesis of AMPs and their release into the insect hemolymph. ZE for preparative separation of peptides and other (bio)Hmoowleecuvelers, iasplpimliciatetido.n PoafrtCiaZllEy, fdour eptroepmaroarteivceomsepplaicraatteiodnadofappteaptitoindeosf aanndalyottihcearl (CbZioE)mseotluepcsulteospisrelpimariatetidv.ePoanrteisaltlhya, nduine tthoemcaosree ocof mHpPlLicCa,tbedutamdaapintalytiobnecoafuasneaolfytthicealinChZeErseenttulypslotowpprreeppaarraatitviveeocnaepsatchiatyn oinf nthaerrcoawseboofrHe PcaLpCil,lbauritems waiinthlyinbencearudsieaomfettheer i≤nh1e0r0enμtmly The former problem (caused by dipping of both capillary ends into the solution in electrode vessels at high separation voltage during the whole CZE analysis) can be solved by short interrupting of electric field and using autosamplers as fraction collectors [19,20]. The background electrolyte (BGE) was prepared from the deionized Milli-Q water and was filtered through a 0.45 μm syringe filter (Millipore, Bedford, MA, USA) before use
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