Multi-PEGylation of Melittin – Structural Characterization and Hemostatic Effects

Multipégylation de la mélittine – caractérisation structurale et effets hémostatiques

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Authors
  1. Peng, H.T.
  2. Huang, H.
  3. Shek, P.N.
  4. Charbonneau, S.
  5. Blostein, M.D.
Corporate Authors
Defence R&D Canada - Toronto, Toronto ONT (CAN)
Abstract
Melittin is a peptide that can be applied for modeling protein-membrane interactions and biomedical applications. To explore its possible use to promote hemostasis and to understand its structure-property relationship, we conducted a series of studies to modify melittin, using 4-arm poly(ethylene glycol) (PEG) with N-hydroxysuccinimide ester end group under different conditions. The PEGylated melittin was characterized by FTIR, MALDI-MS, NMR, a bicinchoninic acid (BCA) assay, circular dichroism (CD), hemolysis assay and thromboelastography (TEG). FTIR and MS confirmed the successful PEGylation and removal of free melittin. The modification produced a mixture of products with different yields as quantified by MS, NMR and BCA, and is influenced by the concentration of reactants, reaction solvents, pH, and melittin: PEG ratio. These conditions affected the extent of the modification and the numbers of melittin conjugated to PEG arms. As indicated by MS, the reaction in pH 9.2 phosphate buffer at a high melittin to PEG ratio, resulted in the highest modification. Reactions in DMSO resulted in more multi-arm coupled melittin (i.e., loading of melittin per PEG), reaching a maximum of 4 melittin per PEG. In addition, the solvent and buffer pH also influenced the secondary structure of PEGylated melittin as indicated by CD measurements; the helicity of the modified peptide, relative to the native peptide, was essentially maintained in DMSO, but substantially lost in pH 9.2. Lastly, h

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Keywords
Melittin;Hemostasis;PEGylation
Report Number
DRDC-TORONTO-SL-2008-157 — Scientific Literature
Date of publication
01 Jan 2010
Number of Pages
23
DSTKIM No
CA034603
CANDIS No
534132
Format(s):
Electronic Document(PDF)

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