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

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

FRACTION
S
UV
MONITOR

COLUMN

Honeybee Venom Sample:

20mg/mL
Melittin

Relative Absorbance

200

Phospholipase
100

???????

Hyaluronidase

0
-25

25

75

125

175
Time

225

275

325

375

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

Figure: Shows PAGE gel results. (In lane 6: Whole Bee Venom, in lane 5: Melittin
Standard, and in lane 1-4: Melittin Fractions)

Bands

separated by charge
Stained in Rapid Reagent

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

1 2 3 4 5 6

Melittin Fractions

1 2

34 5 6

Phospholipase A Fractions

Acknowledgements

Dr. Lorraine Mulfinger

Assistant 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

References
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The purification and characterization of hyaluronidase from the
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217-23
Mulfinger LS. 1989. Synergestic activity of honey bee venom with
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Staay FJ, Fanelli RJ, Blokland A, Schmidt BH. 1999. Behavioral
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