Separation and Analysis of Honeybee Venom ComponentsLevi BlazerLiz DenningLaura RhodesJuniata CollegeResearch Advisors: Dr. Lorraine Mulfinger and Dr. Michael Boyle

Honeybee Venom Components

Melittin: Our Primary InterestComprises 50% of raw honeybee venomHas antibacterial and lytic propertiesMelittin tetramer (4 protein chains)

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 beadsOur column optimized for melittin

Separation According to SizeSmall molecules enter the pores of the beads and flow through the column more slowlyLarge molecules will not be able to enter the beads and will flow through more quickly

Sephedex Gel ChromatographyUV MONITORCOLUMNFRACTIONS

Honeybee Venom Sample: 20mg/mLMelittin???????PhospholipaseHyaluronidase

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Lyophilization-Freeze Drying ProcessPurpose: ability to reconstitute peptide into varied solvents as necessary for certain experimentation

Lyophilization ProcessLowering the temperature and pressure draws out solvent vapor leaving behind frozen faction sample

Solvent removed via sublimationSolid phase Gas phase

Analysis of Column Fractions

Gel Electrophoresis

SELDI-TOF Mass Spectrometry

Purpose of Gel Electrophoresis Determine purity of column separationCompare with whole bee venomIdentify protein components of whole venomFigure 1: Shows electrophoresis components

Polyacrylamide Gel ElectrophoresisSamples placed in 20% sucrose solution

Bands separated by chargeStained in Rapid ReagentFigure: Shows PAGE gel results. (In lane 6: Whole Bee Venom, in lane 5: Melittin Standard, and in lane 1-4: Melittin Fractions)

Polyacrylamide Gel ResultsTop Gel:Lane 1: Whole Bee VenomLane 2: Melittin StandardLane 3-6: Melittin FractionsBottom Gel:Lane 1: Whole Bee VenomLane 2: Melittin StandardLane 3-6: Phospholipase A FractionsMelittin FractionsPhospholipase A Fractions 1 2 3 4 5 61 2 3 4 5 6

AcknowledgementsDr. Lorraine Mulfinger Assistant professor,Juniata College Dept. of Chemistry

Dr. Marielena McGuireField Scientist, Mid-Atlantic Region,Ciphergen Biosystems, Inc.Dr. Michael BoyleVon Lebig chair in Biomedical Sciences, Juniata College Dept. of Biology

Dr. Tom Lyons FisherProfessor of ChemistryJuniata College Dept. of Chemistry

ReferencesAltmann 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) 217-23Mulfinger 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