Multiple antiviral components from an extract of Datura stramonium

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Graduate Student Theses, Dissertations, &
Graduate School
Professional Papers
1995
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Albert Charles Grobe
The University of Montana
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Grobe, Albert Charles, "Multiple antiviral components from an extract of Datura stramonium" (1995).
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Multiple Antiviral Components from an
Extract of Datura stramonium
by
Albert Charles Grobe
s.
B. , University of Montana 1990
Presented in Partial Fulfillment of the Requirements
for the Degree of
Master of Science
University of Montana
1995
Approved by
Committee Chairman
Dean, Graduate School
Date
UMI Number: EP37614
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Grobe, Albert Charles, M. S., May 1995 Microbiology
Multiple Antiviral Components from an Extract of
Dettura stramonium»
Committee Chairman: Dan C. DeBo
The objective of this project was to further characterize
the compound(s) from Datura stramonium leaves responsible
for antiviral activity. A variety of protein purification
techniques were explored in the isolation of the active
material. The purification steps finally established as
useful were (in this order) : 60-80% eunmonium sulfate
precipitation, dialysis, heat^treatment at 60*C, and
chromatography on DEAE-ce 1 lu lose followed by Sephadex G-200.
The first peak eluted (following the void volume peak) from
the G-200 column contained the majority of the antiviral
activity. This peak was further characterized by SDS-PAGE,
tested for RNase activity, and digested with trypsin. All
antiviral fractions contained a protein of -17,000 MW and
the antiviral activity was destroyed by trypsin. The
initial fractions from this peak also contained PKase
activity, while the remainder of the fractions were only
antiviral. The heat-treated extract was also fractionated
using centrifugal filters having lOOK, 30K, and lOK
molecular weight cut-offs. These fractions were
characterized by SDS-PAGE, and tested for antiviral and
RNase activity. The +10,000 (10,000-30,000 MW), +30,000
(30,000-100,000 MW), and +100,000 (above 100,000 MW)
molecular weight fractions were antiviral, while only the
+30,000 and +100,000 fractions showed RNase activity. Data
suggest the antiviral material is present as a -87,000 MW
complex which is not precipitated by heat. The data suggest
the antiviral complex is composed of proteins of -17,000 MW
and -12,000 MW, an antiviral protein of unknown size, and an
RNase-active protein. Future studies of the material
present in the complex should help to elucidate the nature
of the components present.
11
Acknowledgements
First, I thank my professor, Dr. Dan DeBorde for his
infinite patience with me and my project. Without his help,
support, and understanding this project might have never
been finished. His lab has supported this project for the
last five years with time, space, and supplies .
My sincere thanks to Drs. North and Chess in for their
invaluable support and for serving on my committee. My
thanks to both for the use of their equipment, lab space,
and time.
Thanks to Brad Mitchell, Neal Goodwin, and Dr. Mike
Minnick for their technical expertise and assistance. Your
help with this project is greatly appreciated.
To all my friends, who even though they usually had no
idea what I was talking about 'this time', gave me the
support to get through the good and bad times. You will not
be forgotten.
And last, but certainly not least, to my family. To my
mother, father, and sisters for their patience and
understanding when I told them it would be 'done soon'.
Thanksi
iii
Table of Contents
Page
Abstract................................. *.............. *ii
Acknowledgements........................................ iii
Table of Contents........................................ iv
List of T a b l e s ........................ vi
List of Figures........................................ vii
I. Intr oduct ion.........................................1
A. Antiviral Proteins from Plants................ 2
1) Induced Antiviral Proteins................2
2> Endogenous Antiviral Proteins.......... «.4
B. Purpose of Study............................... 8
XI • Materials and Methods..............................10
A. Buffers........................................ 10
B. Growth of Plants.............................. 10
C. Protein Content «......................... 10
D» Purification and Titration of TMV............ 11
E. Preparation of the Datura Extract........ 12
F. Inhibition Assay.............................. 13
G. Heat Treatment of Datura Extract............. 14
H. Molecular Filtration.......................... 15
I. Column Preparation............................ 15
1) DEAE-ce 1 lu lose.................... 15
2) CM-cellulose............................. 16
3) Sephadex G-200........................... 16
J. Column Chromatography of Extract............. 17
1> DEAE-Cellulose........................... 17
2) CM-cellulose............................. 18
3 ) Sephadex G-200........................... 18
I. SDS-PAGE....................................... 19
1) SDS-PAGE Reagents and Buffers........... 19
2) SDS-PAGE Gel Preparation................ 20
3 ) SDS-PAGE Procedure.......................21
K. Staining.......................................22
1) Coomassie Brilliant Blue Stain for
Proteins.................................22
2) Silver Staining for Proteins............ 22
L. Molecular Weight Determination............... 23
1) By SDS-PAGE.............................. 23
2) By Sephadex G-200........................23
M. RNase Activity in Agarose Gels............... 24
N. Inhibition of RNase Activity................. 25
P. Protease Treatment of Column Fractions.......25
III. Results..........*...................................27
A Titration of TMV................................ 27
B. Heat Treatment of Datura Extract...............27
C. Molecular Filtration............................ 32
D. SDS-PAGE of Molecular Filtration Fractions....35
IV
E. RNase Activity of Molecular Filtration
Fractions......... *........................... 38
F. DEAE-cellulose..................................38
G. CM-cellulose....................................47
H. Sephadex G-200..................................52
I. SDS-PAGE of Column Fractions...................53
J. RNase Activity in Agarose Gels................ 64
K. Inhibition of RNase Activity...................64
L. Protease Treatment of Column Fractions........71
M. Native Molecular Weight........................71
IV. Discussion........................................... 79
Future Studies...................................... 82
V. Literature Cited................... ;................ 84
List of Tables
Page
Table 1: Titration of Tobacco Mosaic Virus........... 28
Table 2: Heat Treatment of Datura Extract............ 30
Table 3: Antiviral Activity of Molecular
Filtration Fractions......................... 33
Table 4: Antiviral Activity of DEAE-Cellulose
Column Fractions *...... ;............... 43
Table 5: Antiviral Activity of CM-cellulose
Fraction Pools................................50
Table 6: Antiviral Activity at 6-200 Sephadex
Fractions (Pooled)........................... 56
Table 7: Antiviral Activity of Individual
G-200 Sephadex Column Fractions..............58
Table 8: Trypsin Treatment of Co limn Fractions...... 72
Table 9: Calibration of Sephadex G-200 Column and
Determination of Native Molecular Weight••••76
VI