Separation and Analysis of Honeybee Venom Components

Levi BlazerLiz DenningLaura RhodesJuniata CollegeResearch Advisors: Dr. Lorraine Mulfinger and Dr. Michael Boyle

Honeybee Venom Components

Component Molecular Weight (Da)

Hyaluronidase 41,000

Phospholipase 15,700-19,700

Melittin 2,847

Apamine 2,027

Melittin: Our Primary Interest

Comprises 50% of raw honeybee venom

Has antibacterial and lytic properties

Melittin 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 beads Our column optimized for melittin

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

Sephedex Gel Chromatography

UV MONITOR

COLUMN

FRACTIONS

Honeybee Venom Sample: 20mg/mL

0

100

200

-25 25 75 125 175 225 275 325 375

Time

Rel

ativ

e A

bsor

banc

e

Melittin

???????Phospholipase

Hyaluronidase

Lyophilization-Freeze Drying Process

•Purpose: ability to reconstitute peptide into varied solvents as necessary for certain experimentation

Lyophilization Process

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

Solvent removed via sublimation

– Solid phase Gas phase

Analysis of Column Fractions

Gel Electrophoresis

SELDI-TOF Mass Spectrometry

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

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)

Polyacrylamide Gel Results

• 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

Phospholipase A Fractions

1 2 3 4 5 6

1 2 3 4 5 6

Acknowledgements

Dr. Lorraine MulfingerAssistant 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 Chemistry

Juniata College Dept. of Chemistry

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