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Separation and Analysis of Honeybee Venom Components

Separation and Analysis of Honeybee Venom Components. Levi Blazer Liz Denning Laura Rhodes Juniata College Research Advisors: Dr. Lorraine Mulfinger and Dr. Michael Boyle. Honeybee Venom Components. Melittin: Our Primary Interest. Comprises 50% of raw honeybee venom

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Separation and Analysis of Honeybee Venom Components

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  1. Separation and Analysis of Honeybee Venom Components Levi Blazer Liz Denning Laura Rhodes Juniata College Research Advisors: Dr. Lorraine Mulfinger and Dr. Michael Boyle

  2. Honeybee Venom Components

  3. Melittin: Our Primary Interest • Comprises 50% of raw honeybee venom • Has antibacterial and lytic properties • Melittin tetramer (4 protein chains)

  4. Gel Filtration Chromatography SEPHADEX® G-50 (MW 30,000 – 1,500) • Stationary phase consists of porous beads • Beads composed of cross linked dextran (Sephadex) • Degree of crosslinking determines the size of the pores of the beads • Our column optimized for melittin

  5. Separation According to Size • Small molecules enter the pores of the beads and flow through the column more slowly • Large molecules will not be able to enter the beads and will flow through more quickly

  6. Sephedex Gel Chromatography FRACTIONS UV MONITOR COLUMN

  7. Honeybee Venom Sample: 20mg/mL Melittin Phospholipase ??????? Hyaluronidase

  8. Lyophilization-Freeze Drying Process • Purpose:ability to reconstitute peptide into varied solvents as necessary for certain experimentation

  9. Lyophilization Process • Lowering the temperature and pressure draws out solvent vapor leaving behind frozen faction sample • Solvent removed via sublimation • Solid phase Gas phase

  10. Analysis of Column Fractions • Gel Electrophoresis • SELDI-TOF Mass Spectrometry

  11. Purpose of Gel Electrophoresis • Determine purity of column separation • Compare with whole bee venom • Identify protein components of whole venom Figure 1: Shows electrophoresis components

  12. Polyacrylamide Gel Electrophoresis • Samples placed in 20% sucrose solution • Bands separated by charge • Stained in Rapid Reagent Figure: Shows PAGE gel results. (In lane 6: Whole Bee Venom, in lane 5: Melittin Standard, and in lane 1-4: Melittin Fractions)

  13. Polyacrylamide Gel Results 1 2 3 4 5 6 • Top Gel: • Lane 1: Whole Bee Venom • Lane 2: Melittin Standard • Lane 3-6: Melittin Fractions • Bottom Gel: • Lane 1: Whole Bee Venom • Lane 2: Melittin Standard • Lane 3-6: Phospholipase A Fractions Melittin Fractions 1 2 3 4 5 6 Phospholipase A Fractions

  14. Dr. Lorraine MulfingerAssistant professor, Juniata College Dept. of Chemistry Dr. Marielena McGuire Field Scientist, Mid-Atlantic Region, Ciphergen Biosystems, Inc. Dr. Michael Boyle Von Lebig chair in Biomedical Sciences, Juniata College Dept. of Biology Dr. Tom Lyons Fisher Professor of Chemistry Juniata College Dept. of Chemistry Acknowledgements

  15. References Altmann F, Kubelka V, Staudacher E, Uhl K, Marz L. 1991. Characterization of the isoforms of Phospholipase A2 from honeybee venom. Insect Biochem 21(5) 467-72. Kemeny DM, Dalton N, Lawrence AJ, Pearce FL, Vernon CA. 1984. The purification and characterization of hyaluronidase from the venom of the honey bee, Apis Mellifera. Eur J Biochem 139(2) 217-23 Mulfinger LS. 1989. Synergestic activity of honey bee venom with antibiotics. The Pennsylvania State University. Staay FJ, Fanelli RJ, Blokland A, Schmidt BH. 1999. Behavioral effects of apamin, selective inhibitor of the SKCA channel in mice and rats. Neurosci. Biobehav. Rev. 23 1087-1110

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