A serological comparison of potato virus X protein prior and subsequent to partial enzymatic
hydrolysis
by Gary Allen Secor
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Botany
Montana State University
© Copyright by Gary Allen Secor (1971)
Abstract:
Potato virus X protein in the native and depolymerized states were serologically compared prior and
subsequent to partial enzymatic hydrolysis. SDS gel electrophoresis and Sephadex chromatography
were used to detect changes in molecular weight of the protein. Reciprocal cross-absorption and
Ouchterlony double diffusion tests were conducted to detect changes in antigenic specificity of the
protein. It was concluded that the PVX protein molecules prior and subsequent to hydrolysis are
serologically identical, although a 15% difference in molecular weight exists. The region(s) of the
polypeptide chain lost must constitute an immunosilent portion(s) of the molecule. Degraded viral
protein is more susceptible to hydrolysis by trypsin and chymotrypsin than is native protein. In presenting this thesis in partial fulfillment of the require­
ments for an advanced degree at Montana State University, I agree that
the Library shall make it freely available for inspection.
I further
agree that permission for extensive copying of this thesis for scholar
Iy purposes may be granted by my major professor, or, in his absence,
by the Director of Libraries.
It is understood that any copying or
publication of this thesis for financial gain^shall not be allowed
without my written permission.
Signature
ture /
I
Date
Al
T y V iA M f jz l
\ __ /
z V
f T V ,
A SEROLOGICAL COMPARISON OF POTATO VIRUS X PROTEIN
PRIOR AND SUBSEQUENT TO PARTIAL ENZYMATIC HYDROLYSIS
by
GARY ALLEN SECOR
A t h e s is s u b m itte d to th e Graduate F a c u lt y i n p a r t i a l
f u l f i l l m e n t o f th e re q u ire m e n ts f o r th e degree
of
MASTER OF SCIENCE
in
Botany
Approved:
Head, M ajor Department
'Chairman, Examining Committee
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1971
iii
ACKNOWLEDGMENTS.
I would l i k e to acknowledge the, a s s is ta n c e o f th e f o l l o w i n g :
F i r s t and fo re m o s t, Dr. J. F. Shepard, who as my m a jo r a d v i s o r ,
gave me guidance and encouragement, was p a t i e n t w i t h my e r r o r s and
ta u g h t me much th ro u g h o u t t h i s s tu d y .
Dr. I .
W. C a r r o l l and Dr. J. W. J u t i l a who n o t o n ly acted as
the re m a in in g members o f my co m m ittee, b u t a ls o o f f e r e d guidance,
a d v ic e and encouragement.
The s t a f f and g radu ate s tu d e n ts f o r t h e i r h e lp f u ln e s s and
i n v i g o r a t i n g d is c u s s io n s .
Miss Becky M a r t in f o r t e c h n ic a l a s s is ta n c e .
Mrs. W a lte r Secor f o r t y p in g th e f i n a l copy o f t h i s m a n u s c rip t.
The Department o f Botany and M ic r o b io lo g y f o r a re se a rch
a s s i s t a n t s h i p to i n i t i a l l y
begin my s t u d i e s , and th e Montana Potato
Improvement A s s o c ia t io n f o r f i n a n c i a l a s s is ta n c e which a llo w e d me to
c o n tin u e my e d u c a tio n .
iv
TABLE OF CONTENTS
Page
.
ii
ACKNOWLEDGMENTS . . : ...................................
. iii
TABLE OF CONTENTS . . . . . . . . . . . .
. iv
LIST OF FIGURES . . . . . . . . . . . . .
. vi
ABSTRACT......................
o
. vii
.
INTRODUCTION. . . . . . . . . . . . . .
.
I
MATERIALS AND METHODS . . . . . . . . . .
.
7
.
7
P r e p a ra tio n o f degraded PVX p r o t e i n .
.
8
A c ryla m id e gel e l e c t r o p h o r e s is
.
8
V ir u s p u r i f i c a t i o n
. . . . . . . . .
Sephadex chrom atography.
. . .
. . ....
9
.
A n tise ru m p r o d u c tio n . . . . . . . .
.
10
S e r o logy . . . . . . o . . . . . . .
.
10
Enzymatic, d i g e s t i o n .
. • 11
13
RESULTS . . . .
E f f e c t . o f p u r i f i c a t i o n on th e PVX s u b u n it .
. . ..........................
13
I
P re p a ra tio n o f p u r i f i e d PVX-IP and PVX-QN
. . . . . . . . .
S e r o lo g ic a l r e l a t i o n s h i p between PVX-IP and PVX-ON . . . . .
20
22
P re p a ra tio n and s e r o lo g ic a l re la te d n e s s o f PVX-IP and PVX-ON
degraded p f o t e i h
. . . . . . . . . . . . . . . . . . . . . . .
23
E f f e c t o f t r y p s i n on degraded p r o t e i n .
. . . . . . . .
E f f e c t o f c h y m o tryp sin on PVX and PVX degraded p r o t e in
DISCUSSION
SUMMARY
U l/il X I
e
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B
e
e
e
e
e
e
e
e
e
e
e
e
e
e
o
e
e
e
e
e
e
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e
e
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B
e
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LITERATURE CITED
. .
vi
LIST OF FIGURES
Page
F ig u re I
F ig u re 2
F ig u re 3
F ig u re 4
F ig u re 5
F ig u re 6
F ig u re
I
Graph used f o r m o le c u la r w e ig h t d e t e r ­
m in a tio n u t i l i z i n g SDS s p l i t gel p o l y ­
a c ry la m id e e le c t r o p h o r e s is . . . . . . . . . . .
14
R e p re s e n ta tiv e SDS s p l i t p o ly a c ry la m id e
g e ls o f known p r o t e i n standards used
f o r c o n s t r u c t i o n o f m o le c u la r w e ig h t
d e t e r m in a t io n graph. . . . . . . . . . . . . .
15
SDS s p l i t p o ly a c r y la m id e g e ls showing
a PVX-M p r e p a r a t io n and a PVX-M p r e ­
p a r a t io n subsequent t o t r y p s i n d ig e s ­
tio n . e e o . . o e . . o . . . . .
.
.
.
.
o
IG
SDS p o ly a c r y la m id e s p l i t g e ls demonstra­
t i n g PVX-IP degraded p r o t e in and PVX-ON
degraded p r o t e in p r e p a r a tio n s compared
t o c h y m o try p s inogen. . . . . . . . . . . . . .
18
Graph used f o r m o le c u la r w e ig h t d e t e r ­
m in a tio n using Sephadex G-200 column
chrom atography . . . . . . . . . . . . . . . .
21
SDS p r e p a r a t iv e p o ly a c r y la m id e e l e c t r o ­
p h o r e t i c gel i l l u s t r a t i n g s e p a ra tio n
p o s s ib le between PVX-IP degraded p r o t e in
and PVX-ON degraded p r o t e i n . . . . . . . . . .
25
O u ch te rlo n y double d i f f u s i o n t e s t i l l u s ­
t r a t i n g a n t ig e n i c i d e n t i t y between PVX-IP
and PVX-ON degraded p r o t e i n . . . . . . . . . .
27
vii
ABSTRACT
P o ta to v i r u s X p r o t e i n in th e n a t iv e and dep olym erized s ta te s
were s e r o l o g i c a l l y compared p r i o r and subsequent t o p a r t i a l enzymatic
h y d ro ly s is .
SDS gel e le c t r o p h o r e s is and Sephadex chromatography were
used t o d e t e c t changes i n m o le c u la r w e ig h t o f th e p r o t e i n .
R e c ip ro ­
cal c r o s s - a b s o r p t io n and O u c h te rlo n y double d i f f u s i o n t e s t s were
conducted t o d e t e c t changes in a n t ig e n i c s p e c i f i c i t y o f the p r o t e i n .
I t was concluded t h a t th e PVX p r o t e in m olecules p r i o r and subsequent
t o h y d r o l y s is are s e r o l o g i c a l l y i d e n t i c a l , a lth o u g h a 15% d i f f e r e n c e
i n m o le c u la r w e ig h t e x i s t s .
The r e g i o n (s ) o f the p o ly p e p tid e chain
l o s t must c o n s t i t u t e an im m u n o sile n t p o r t i o n ( s ) o f th e m o le c u le .
Degraded v i r a l p r o t e i n i s more s u s c e p t ib le t o h y d r o l y s is by t r y p s i n
and chymot r y p s i n than i s n a t iv e p r o t e i n .
INTRODUCTION
The im portance o f m a in t a in in g n a t iv e v i r u s p a r t i c l e s f o l l o w i n g
p u r ific a tio n
i s o b v i o u s , f o r to s tu d y these i n f e c t i o u s . a g e n t s , they
must be c h e m ic a lly and s t r u c t u r a l l y c o m p le te , as w e ll &s b i o l o g i ­
c a lly fu n c tio n a l.
re s u lts .
b io lo g ic a l
O the rw ise e x p e rim e n ta tio n may r e s u l t i n e r r a t i c
For example, e r r o r s in i d e n t i f i c a t i o n , c o m p o s itio n ,
f u n c t io n and r e l a t i o n s h i p s w i t h o t h e r v ir u s e s may r e s u l t
i f a p o r t i o n o f th e s t r u c t u r a l m olecu le i s changed o r l o s t upon
p u rific a tio n .
I t has been dem onstrated w i t h some p l a n t v ir u s e s
t h a t p u r i f i c a t i o n may r e s u l t i n th e p r o d u c tio n o f in c o m p le te v i r i o n s .
Koenig e t al
(12) r e c e n t l y r e p o r te d t h a t th e m o le c u la r w e ig h t o f the
p o ta to v i r u s X (PVX) p r o t e i n s u b u n it may be a f f e c t e d by th e method
i n which th e v i r u s i s p u r i f i e d from to bacco.
When i n f e c t i v e homo­
genates a re in c u b a te d o v e r n ig h t a t room te m p e ra tu re , th e a u th o rs
suggested th e presence o f p r o t e o l y t i c enzymes i n tobacco sap which
are capable o f c o n v e r t in g th e normal p r o t e i n s u b u n it w i t h a m o le c u la r
w e ig h t o f 29,800 to one w i t h a m o le c u la r w e ig h t o f 24,000.
w i t h tobacco mosaic v i r u s
S tu d ie s
(TMV) by Rees and S h o rt (20) have shown
t h a t p u r i f i c a t i o n o f t h i s v i r u s from beans r e s u l t s in p a r t i c l e s
which are s e r o l o g i c a l l y d i s t i n c t from those p u r i f i e d from to b a c c o ,
presumably due to th e a c t io n o f c a rb o xyp e p tid a se s on v i r u s p a r t i c l e s .
O ther s t u d ie s w i t h v ir u s e s have shown t h a t a r t i f i c i a l
d i g e s t io n may m arke dly a f f e c t c e r t a i n v i r a l
p r o p e rtie s .
enzymatic
Koenig e t al
2
(12) dem onstrated t h a t t r y p s i n tre a tm e n t o f p u r i f i e d PVX d u p lic a te s
th e s u b u n it m o le c u la r w e ig h t lo s s t h a t occurs upon in c u b a t io n o f
i n f e c t i v e homogenates.
In a d d i t i o n , th e e l e c t r o p h o r e t i c m o b i l i t y
o f th e t r y p s i n t r e a t e d PVX was d i s t i n c t from t h a t o f th e u n tre a te d
PVX.
However, d e t a i l e d s e r o lo g ic a l s t u d ie s were n o t conducted.
Bawden and P i r i e
( I ) have shown . t h a t pepsin d ig e s t io n o f PVX r e s u l t s
in a lo s s o f i n f e c t i v i t y and s e r o lo g ic a l a c t i v i t y .
S tu d ie s w ith
TMV have been most e n l ig h t e n in g f o r c o r r e l a t i n g p r o t e in a l t e r a t i o n
i n p a r t i c l e s and changes in a n t ig e n i c s p e c i f i c i t y .
H a r r is and
K n ig h t (7) have shown t h a t t re a tm e n t o f TMV w i t h c a rb o x yp e p tid a se
r e s u l t s in th e removal o f th e t h r e e C -te rm in a l amino a c id s .
R e s u lta n t TMV p a r t i c l e s are s e r o l o g i c a l l y d i s t i n c t from undigested
v ir io n s .
S u r p r i s i n g l y , th e removal o f tw e lv e a d d i t i o n a l amino
a c id s from th e C -te rm in a l end o f th e s u b u n its had, no f u r t h e r e f f e c t .
on a n t i g e n i c s p e c i f i c i t y
(10).
Sengbusch and Wittmann (23) have
a ls o dem onstrated th e s u s c e p t i b i l i t y o f TMV to s e r o lo g ic a l change
f o l l o w i n g a d i f f e r e n t form o f a l t e r a t i o n in p rim a ry s t r u c t u r e .
A
s in g l e amino a c id s u b s t i t u t i o n in th e TMV p o ly p e p tid e c h a in may
r e s u l t i n a m olecu le s e r o l o g i c a l l y d i s t i n c t from w i l d ty p e p r o t e i n ,
p ro v id e d t h a t th e s u b s t i t u t i o n occu rs a t th e prope r l o c a t i o n .
N i b l e t t and Semancik (15) dem onstrated t h a t th e e l e c t r o ­
p h o r e t i c components o f cowpea mosaic v i r u s and o f bean pod m o t t le
3
v i r u s would m ig r a te a t a d i f f e r e n t r a t e f o l l o w i n g d i g e s t i o n by c e r ­
t a i n enzymes.
Chidlow and Tremaine (3) r e p o r te d th e a c t io n o f v a r io u s enzymes
on cowpea c h l o r o t i c m o t t le v i r u s and concluded t h a t t r y p s i n d ig e s t io n
caused disasse m b ly o f th e v i r u s and a s u b u n it m o le c u la r w e ig h t lo s s
o f 20%.
Reassembled t r y p s i n d ig e s te d v i r u s was s e r o l o g i c a l l y com­
pared to i n t a c t v i r u s and found to be n o n - i d e n t i c a l .
By c o n t r a s t ,
ca rb o x y p e p tid a s e and ch ym o tryp sin removed 0.5% and 10% o f the
p r o t e i n r e s p e c t i v e l y , caused no d i s s o c i a t i o n o f th e v i r u s , and the
s e r o lo g ic a l
p r o p e r t ie s o f th e v i r u s were u n a ffe c te d .
P e tte rs s o n (18) s tu d ie d enzym atic d ig e s t io n o f ade novirus
hexons and th e e f f e c t t h i s d i g e s t io n had on a n t ig e n i c s p e c i f i c i t y
o f th e hexons.
He found t h a t t r y p s i n d ig e s te d 5-10% o f th e hexon
b u t no change i n s e r o lo g ic a l p r o p e r t ie s accompanied th e lo s s .
However, when s u b t i l i s i n , papain o r ch ym o tryp sin were u sed, a
g r e a t deal o f th e hexon was d ig e s t e d , le a v in g o n ly th e hexon " c o r e " ,
which was s e r o l o g i c a l l y d i s t i n c t from i n t a c t hexons.
SDS p o ly a c r y la m id e gel e le c t r o p h o r e s is has become a r e l i a b l e
te c h n iq u e f o r th d s tu d y o f p r o t e in m o le c u le s , e s p e c i a l l y as a to o l
f o r d e t e r m in a t io n o f m o le c u la r w e ig h ts .
O r i g i n a t i o n o f a c ry la m id e
gel e le c t r o p h o r e s is by O rn s te in and Davis (4 , 16) was f o llo w e d by
th e use o f SDS i n th e system by S h apiro e t a I
(2 5 ) .
Weber arid
4
Osborn (32) c o n firm e d a c c u ra te m o le c u la r - w e ig h t d e t e r m in a t io n using
SDS gel e l e c t r o p h o r e s is .
The a c t io n o f SDS as a d e n a tu ra n t o f p r o ­
t e i n s was e x p la in e d by Reynolds and Tan ford (21) and P i t t - R i v e r s
and Ambesi Impiombato ( 1 9 ) , who r e p o r te d t h a t as SDS den a tu re s a ,
p r o t e i n , i t a tta c h e s t o th e p r o t e in and th e r e s u l t a n t amalgams
v a r y d i r e c t l y in t h e i r m o le c u la r r a d iu s w i t h m o le c u la r w e ig h t.
C o nsequ ently, an a c c u ra te r e l a t i o n s h i p between Stoke
1S
r a d iu s and
m o le c u la r w e ig h t - e x is t s which can be used f o r m o le c u la r w e ig h t
d e t e r m in a t io n v ia SDS p o ly a c r y la m id e gel e l e c t r o p h o r e s is .
Several
papers have co n firm e d th e accuracy o f m o le c u la r w e ig h t d e te r m in a tio n
using SDS gel e l e c t r o p h o r e s is
(13, 24, 3 4 ).
Dunker and Rueckert (5)
in tr o d u c e d s p l i t gel e le c t r o p h o r e s is which a llo w e d th e t e s t m olecule
t o be e le c tr o p h o r e s e d in th e same gel as a known m o le c u le , f u r t h e r
in c r e a s in g th e r e l i a b i l i t y o f m o le c u la r w e ig h t d e t e r m in a t io n w ith
SDS gel e l e c t r o p h o r e s is .
L ik e w is e , SDS column chromatography has been used f o r the
s tu d y o f p r o t e in s
(9 ).
Fish e t aI
(6) have e lu c id a t e d th e a c t io n
o f SDS as a d e n a tu ra n t o f p r o t e in s and t h e i r use in gel chroma­
to g r a p h y , and found t h a t a s t a t i c r e l a t i o n s h i p e x i s t s between
S t o k e 's r a d iu s ,and m o le c u la r w e ig h t.
Page1 and Godin (17) and
S h a lla and Shepard (24) have a c c u r a t e l y determ ined m o le c u la r ,weights
o f p r o t e i n m olecules using Sephadex G-200.
5
I t i s th e purpose o f t h i s paper t o dem onstrate th e enzym atic
d ig e s t io n o f PVX and o f i t s
degraded p r o t e in by both n a t u r a l and
a r t i f i c i a l methods and t o r e p o r t a s e r o lo g ic a l comparison o f the
two r e s u l t a n t forms o f PVX and o f PVX degraded p r o t e i n .
In h is -
re v ie w on some m o le c u la r aspe cts o f a n t i g e n i c i t y , Sela (22) p o in ts
t o th e r o l e o f c o n fo rm a tio n in a n t i g e n i c s p e c i f i c i t y .
He c i t e s
seve ra l cases -in which n a t iv e and denatured p r o t e in s are n o t c r o s s ­
r e a c t i v e w i t h each o t h e r s ' a n tib o d y p o p u la tio n s due t o th e r o l e o f
secondary and t e r t i a r y s t r u c t u r e i n d e te r m in in g immunopotency.
a case i n p o i n t i s PVX.
Such
S tu d ie s by -Shepard and S h a lla (2 4 , 28)
have shown t h a t as PVX i s d e p o ly m e riz e d , th e re le a s e d p r o t e in sub­
u n i t s undergo u n f o ld in g and e n t e r i n t o a new c o n fo rm a tio n a l
s ta te .
T h is change in c o n fo rm a tio n o f th e p r o t e i n i s accompanied by a
change i n th e a n t ig e n i c s p e c i f i c i t y o f th e p r o t e i n so d r a s t i c t h a t
PVX and the. degraded p r o t e in are o n ly s l i g h t l y r e l a t e d as a n tig e n s .
I t i s t h e r e f o r e necessary to compare th e PVX forms s e p a r a t e ly from
th e PVX degraded p r o t e i n forms s in c e th e two are a n t i g e n ic a l I y
d is tin c t.
Because PVX i s a good model v i r u s f o r s tu d y , s in c e n a tu r a l
enzym atic d i g e s t io n does o c c u r , i t was o f i n t e r e s t to s tu d y the
e f f e c t on th e im m unological p r o p e r t ie s o f th e v i r a l
p a r t i a l p o ly p e p tid e c h a in lo s s would have.
p ro te in th a t
SDS s p l i t gel p o l y ­
6
a c ry la m id e e l e c t r o p h o r e s is and SDS Sephadex G-200 column chroma­
to g ra p h y were used as an assay system f o r p rim a ry s t r u c t u r a l changes.
O u c h te rlo n y double d i f f u s i o n and r e c ip r o c a l c r o s s - a b s o r p t io n
s t u d ie s were used f o r , d e t e c t io n o f changes in a n t ig e n i c s p e c i f i c i t y .
MATERIALS AND METHODS
V ir u s p u r i f i c a t i o n .
The i s o l a t e o f p o ta to v i r u s X (PVX)
was th e same as t h a t used in p re v io u s s t u d ie s in t h i s l a b o r a t o r y
(29).
N ic o t ia n a tabacum L. v a r .
in c re a s e h o s t.
'W h ite B u r le y ' was th e sys te m ic
P u r i f i c a t i o n o f th e v i r u s f o llo w e d th e procedures
o f Koenig and Bercks (11) and W e tte r ( 3 2 ) .
In g e n e r a l, t h i s
in c lu d e d hom ogenization o f f r e s h s y s t e m i c a l l y in f e c t e d t i s s u e in
0.05 M sodium c i t r a t e b u f f e r pH 6 .0 c o n t a i n in g 0.2% sodium s u l f i t e
and 0.2% a s c o r b ic a c i d , c h lo r o fo r m e m u l s i f i c a t i o n , and se v e ra l
c y c le s o f d i f f e r e n t i a l
u ltr a c e n trifu g a tio n .
Three a l t e r n a t i v e schemes were used i n th e b a s ic p u r i f i c a t i o n
p ro c e d u re .
Im m e d ia te ly p u r i f i e d PVX (PVX-IP) was prepa red by
e m u ls i f y i n g i n f e c t i v e homogenates from f r e s h l y h a rvested le aves
w i t h c h lo r o fo r m w i t h i n t h r e e m inutes a f t e r hom ogenization (1 2 ).
A l t e r n a t i v e l y , PVX was p u r i f i e d by f i l t e r i n g
i n f e c t i v e homogenates
th ro u g h c h e e s e c lo th and a llo w in g t h i s crude j u i c e to in c u b a te
o v e r n i g h t a t roorii te m p e ra tu re b e fo re f u r t h e r p u r i f i c a t i o n .
p r e p a r a t io n s were d e s ig n a te d as PVX-ON ( 1 2 ) .
p r e p a r in g PVX was a ls o used.
These
A t h i r d method o f
Thesd p r e p a r a t io n s ,
(PVX-M), c o n s is te d '
o f crude homogenates which had been a llo w e d t o remain a t room
te m p e ra tu re o r a t 4 ° C fro m f i f t e e n m inutes to seve ra l hours befo re
c h lo r o fo r m c l a r i f i c a t i o n and f u r t h e r p u r i f i c a t i o n , as d e s c r ib e d .
P u r i t y o f th e v i r a l
p r e p a r a t io n s was a s c e r ta in e d by ( i )
8
c l a r i t y o f th e f i n a l
high speed p e l l e t ( i i ) r e a c t i v i t y o f v i r a l
p r e p a r a t io n s in r a d i a l
im m u n o d iffu s io n p la t e s c o n t a in in g a n tis e ru m
prepared a g a in s t h e a lt h y h o s t m a te r ia l
(28)
( iii)
d e g ra d a tio n p ro d u c ts in sodium dodecyl s u l f a t e
gel e le c t r o p h o r e s is system s, and ( i i i i )
homogeneity o f
(SDS) p o ly a c r y la m id e
r e a c t i v i t y o f h e a lt h y h o s t -
m a t e r ia l w i t h a n tis e ru m provoked by th e v i r a l
p r e p a r a t io n s .
C o n c e n tra tio n s o f v i r a l and degraded v i r a l
p r o t e in p r e p a ra tio n s
were .determined s p e c t r o p h o to m e t r ic a l l y as per Shepard and Secor (2 9 ).
P re p a ra tio n o f degraded PVX p r o t e i n . —
Degraded PVX p r o t e in
was o b ta in e d by d e p o ly m e r iz a tio n o f th e s u b u n its w i t h e i t h e r
p y r i d i n e o r SDS.
fin a l
P y r id in e was added t o v i r a l
p r e p a ra tio n s a t a
c o n c e n tr a t io n o f 30% and then removed by d i a l y s i s o v e r n ig h t
a g a in s t a l a r g e volume o f 0.005 M sodium c i t r a t e pH 8 . 0 .
For SDS
d e g r a d a tio n , SDS and 2-mercaptQethandl we^e each added t o th e v ir u s
s o lu tio n a t a f in a l
hours.
c o n c e n tr a t io n o f 1%, in cu b a te d a t 37°C f o r two
The r e s u l t a n t v i r a l
p r o t e in was used f o r e i t h e r s e r o lo g ic a l
s tu d ie s o r f o r a p p l i c a t i o n on e l e c t r o p h o r e t i c g e ls .
A c ryla m id e gel e l e c t r o p h o r e s i s . - -
SDS s p l i t gel p o ly a c r y la m id e ,
e le c t r o p h o r e s is was used f o r d e t e r m in a t io n o f m o le c u la r w e ig h t and
as an assay o f homogeneity ( 5 ) .
PVX p r o t e i n was prepared f o r
e le c t r o p h o r e s is by SDS in c u b a t io n o f v i r u s
(24) and e le c tro p h o re s e d
on SDS s p l i t g e ls having a f i n a l c o n c e n tr a t io n o f 10% a c ry la m id e
9
p lu s 1% SDS (24)'.
Gels were s ta in e d w it h 0.025% -Coomassie b r i l l i a n t
b lu e (Colab Chicago H e ig h t s , 111.) and d e sta in e d w i t h 7.5% a c e t ic
a c id p lu s 5.0% methanol
(24).
( 2 ) , o r s p e c i f i c a l l y s ta in e d w i t h a n tib o d y
M o le c u la r w e ig h ts o f degraded v i r a l
p r o t e i n were e stim a te d by
comparing t h e i r m o b i l i t i e s t o th e m o b i l i t i e s o f known p r o t e in s
prepared in th e same manner.
The f o l l o w i n g standard p r o t e in s were
used: bovine serum album in (BSA MW=67,000), ovalbum in (oval MW=
4 5 , 0 0 0 ) , c h y m o try p s inogen (chymo MW=25,000), tobacco mosaic v i r u s
U - I s t r a i n monomers (TMV monomer MW=17,300) and r ib o n u c le a s e
( RNAse MW=13,700).
TMV (from Dr. T. A. S h a ll a. U n i v e r s i t y o f
C a l i f o r n i a , D a vis) monomers were prepared by in c u b a t io n o f TMV a t
37°C f o r two hours in 1% SDS p lu s 1% m e rc a p to e th a n o l.
P r e p a r a tiv e SDS gel e le c t r o p h o r e s is was a ls o conducted i n a
s i m i l a r manner, b u t i n l a r g e r g la s s tubes (12cm X 20mm).
Three to
f i v e mg o f p r o t e i n i n 4 0 0 ju l o f b u f f e r were e le c tro p h o re s e d per tube
a t 40-50 m i l lia m p s f o r 12-16 hours.
The PVX degraded p r o t e i n bands
were lo c a te d w i t h a n t ib o d y , a c c o rd in g to th e method d e s c rib e d by
S h a lla and Shepard (2 4 )..
Sephadex chrom ato graphy. - -
Sephadex G-200 e q u i l i b r a t e d w ith
0.05 M T r is - H C l pH 7 .2 c o n t a in in g 1% SDS was used f o r c o n f ir m a t io n
o f m o le c u la r w e ig h t d e te r m in a tio n s o f degraded v i r a l
p ro te in .
M o le c u la r w e ig h ts were e s tim a te d by comparing the m o b i l i t i e s o f
10
known p r o t e i n s , s i m i l a r to th e method o f S h a lla and Shepard (2 4 ).
The p r o t e i n s ta n d a r d s ; BSA, o v a l , chymo and cytochrome C ( c y to C
MW=13,7 0 0 ), were prepared by in c u b a t io n o f
tha n o l f o r two hours a t 37°C.
1%
SDS p lu s
1%
m ercaptoe-
The f r a c t i o n s , u s u a ll y 2 .2 m l, were
c o l l e c t e d w i t h an a u to m a tic f r a c t i o n c o l l e c t o r and m o n ito re d a t
280nm w i t h a Beckman DB s p e c tro p h o to m e te r.
A n tise ru m p r o d u c t i o n . - -
A n tis e r a to whole PVX and t o PVX-M
degraded p r o t e i n a n t is e r a were prepared by e m u ls if y in g 2 mg o f
a p p r o p r ia t e a n tig e n w i t h an equal volume o f F reund 's in c o m p le te
a d ju v a n t and i n j e c t i n g a r a b b i t in t r a m u s c u l a r l y e very seven days
o v e r a fo u r-w e e k p e r io d .
A n t is e r a a g a in s t PVX-IP and PVX-ON degraded p r o t e i n were
provoked by i n j e c t i n g degraded PVX p r o t e i n p u r i f i e d by. th e p r e ­
p a r a t iv e SDS gel system.
degraded v i r a l
Four w eekly i n j e c t i o n s o f 2-3 mg o f
p r o t e i n were a d m in is te r e d o v e r a one month p e r io d .
B le e d in g s were conducted 2-6 weeks f o l l o w i n g th e i n i t i a l
in je c ­
t i o n by c u t t i n g th e m arg in a l ear v e in o f th e r a b b i t and c o l l e c t i n g
40-50 ml o f b lo o d .
The a n tis e ru m was a llo w e d to se p a ra te from the
c l o t t e d red c e lls . , c o l l e c t e d , c e n t r if u g e d a t 10,000 £ f o r 10 m inutes
and s to re d fro z e n a t O0C.
S e r o lo g y . - -
A n t is e r a were t i t e r e d by e i t h e r tube p r e c i p i t i n
o r double d i f f u s i o n methods.
The tube p r e c i p i t i n procedure was
11
used t o e v a lu a te whole v i r u s a n t is e r a a c t i v i t y .
A n tise ru m was
s e r i a l l y d i l u t e d w i t h 0.05 M T r is - H C l pH 7 .0 c o n t a in in g 0.85%
s a lin e .
An equal volume o f v i r u s a t 0 .2 mg/ml was added and the
m ix t u r e was in cu b a te d o v e r n ig h t a t 4°C.
Double d i f f u s i o n t e s t s were
c a r r i e d o u t i n 0.9% io n a g a r d is s o lv e d in 0.05 M T r is - H C l pH 7 .0
c o n t a in in g 0.85% s a l i n e .
For a n tis e ru m t i t e r d e t e r m in a t io n to
degraded p r o t e i n , two f o l d d i l u t i o n s o f serum were made, placed in
th e c e n t r a l a n tis e ru m w e ll and degraded p r o t e i n a t 0 . 1 - 0 . 2 mg/ml
placed i n th e p e r ip h e r a l a n tig e n w e l l s .
R eactions were a llo w e d to
develop o v e r n i g h t a t room te m p e ra tu re b e fo re - e v a lu a t io n .
For
e s t a b l i s h i n g s e r o lo g ic a l r e l a t i o n s h i p s , degraded p r o t e in concen­
t r a t i o n s ranged from 0 , 1 - 0 . 2 mg/ml.
Enzymatic d i g e s t i o n . —
T r y p s ih (3X c r y s t a l l i z e d W o rth in g to n
F re e h o ld , N . J . ) and chymot r y p s i n
(3X c r y s t a l l i z e d Sigma S t. L o u is ,
Mo.) were used f o r d i g e s t io n e x p e rim e n ts .
Unless o th e r w is e s p e c i f i e d
d i g e s t i o n w i t h t r y p s i n and c h y m o tryp sin was c a r r i e d o u t a r a t i o o f ,
I . Aig enzyme per I mg o f s u b s t r a te o v e r n i g h t a t room te m p e ra tu re in
0.05 M N a - c i t r a t e b u f f e r pH 8 . 0 .
F o llo w in g in c u b a t io n o f e i t h e r
whole v i r u s o r degraded p r o t e in w i t h c h y m o try p s in , th e enzyme was
i n h i b i t e d w i t h L - l - t o s y l a m i d e - 2 - p h e n y le t h y l c h lo ro m e th y l
ketone
HCl (TPCK Sigma S t . L o u is , Mo.) using a m o d ifie d procedure o f Shaw
e t al
( 2 7 ) . TPCK was d is s o lv e d i n a b s o lu te methanol and added to
12
th e virus-enzym e m ix t u r e a t a r a t i o o f th r e e p a r ts TPCK t o one p a r t
enzyme and in c u b a te d f o r two hours a t room te m p e ra tu re .
Aqueous
N - ^ - P - t o s y l - L - I y s i n e c h lo ro m e th y l ketone (TICK S igm a-S t. L o u is , Mo.)
was used to i n h i b i t t r y p s i n a f t e r in c u b a t io n and was a ls o used a t a
r a t i o o f t h r e e to one (27, 2 8 ).
The tim e r e q u ir e d f o r complete i n h i b i t i o n o f t r y p s i n w i t h TLCK
and chymot r y p s i n w i t h TPCK was d e te rm in e d .
Hide powder azure
( C a lbiochem Los A n g e le s, C a l i f . ) , an in s o l u b l e chromogenic s u b t r a t e ,
was used t o assay th e e f f e c t i v e n e s s o f i n h i b i t i o n o f enzym atic
a c tiv ity .
F ive mg o f hide powder azure were mixed w i t h 2 ml o f
0.005 M Na- c i t r a t e b u f f e r pH 8 .0 in se ve ra l tu b e s .
One hundred
Ug o f t r y p s i n p lu s 300 jjg o f TLCK were mixed and 100 jjg o f chymot r y p s i n p lu s SOOug o f TPCK were m ixed, and these m ix tu r e s were
added to th e tubes c o n t a in in g th e h id e powder azure a t h o u r ly
in te rv a ls .
S o l u b i l i z a t i o n o f dye due to enzym atic d i g e s t io n was
s p e c t r o p h o t o m e t r i c a ll y analyzed a t 595 nm w i t h a Beckman DB
sp e c tro p h o to m e te r a f t e r a l lo w in g th e h id e powder azure p lu s enzymei n h i b i t o r m ix tu re s t o in c u b a te f o r v a r io u s tim es a t 37°C.
RESULTS
E f f e c t o f - p u r i f i c a t i o n on th e PVX . s u b u n i t . —
Koenig e t al
(12) r e c e n t l y r e p o r te d the e f f e c t t h a t method o f p u r i f i c a t i o n may
have upon th e m o le c u la r w e ig h t o f th e PVX s u b u n it .
The r e p o r t
s t a te d t h a t PVX s u b u n it co u ld possess a m o le c u la r w e ig h t o f e i t h e r
24,000 o r 29,800.
In th e p re s e n t s t u d y , experim ents were conducted
t o dete rm in e w hether s i m i l a r r e s u l t s c o u ld be produced using the
s t r a i n o f PVX r o u t i n e l y used here and under o u r c o n d it io n s .
P r e l im in a r y e x a m in a tio n o f PVX was conducted w i t h p r e p a r a t io n s
which v a r ie d in tim e between hom ogenization and c h lo r o fo r m e m u ls i­
f i c a t i o n from f i f t e e n m inutes to se ve ra l hours (PVX-M).
PVX-M
p r d t e i n was prepared by in c u b a t in g v i r u s w i t h sodium dodecyl s u l f a t e
(SDS).
M o le c u la r w e ig h ts o f th e s u b u n its were e s tim a te d from an
SDS s p l i t gel e l e c t r o p h o r e s is m o le c u la r w e ig h t curve ( F ig . I )
c o n t r u c te d from data w i t h known p r o t e i n standards ( F ig . 2 ) .
In itia l
expe rim e nts in d ic a t e d t h a t v i r u s p a r t i c l e s in PVX-M
p r e p a r a t io n s were composed o f m ix tu re s o f p r o t e in s u b u n it s , i . e . ,
both i n t a c t and p a r t i a l I y degraded p o ly p e p tid e c h a in s .
These
p r e p a r a t io n s f r e q u e n t l y c o n ta in e d s u b u n its o f both m o le c u la r
w e ig h ts i n a p p ro x im a te ly equal r a t i o s , as determ ined by i n t e n s i t y
and s iz e o f th e s t a in e d p o r t io n s o f th e SDS s p l i t g e ls
( F ig . 3 ) .
The m o le c u la r w e ig h ts o f th e two s iz e s o f s u b u n its was c a lc u la t e d
t o be 29,000 f o r th e l a r g e r and 24,500 f o r the s m a lle r .
14
MOL. WTx IO" 4
OVAL
PVX I P
MONOMER
CHYMO
FVX ON MONOMER
T MV MONOMER
RNAse
.2
.3
.4
.5
6
.7
.8
.9
O
1.1
12
13
14 15
MOBILITY
F ig u re I .
E s tim a tio n o f m o le c u la r w e ig h ts u t i l i z i n g th e SDS s p l i t
gel e l e c t r o p h o r e s is system.
M o b i l i t y equals th e r a t i o
o f the d is ta n c e the p r o t e in m ig ra te s to th e d is ta n c e
chym otrypsinogen m ig r a te s .
Both graphs p l o t t e d by
method o f l e a s t squares.
15
F ig u re 2.
SDS s p l i t g e ls o f f o u r o f the standards used to c o n s t r u c t
th e m o le c u la r w e ig h t e s t im a t io n curve shown i n f i g u r e I .
C=chym otrypsinogen, R = rib o n u c le a s e , O=ovalb um in, B=bovine
serum album in.
16
F ig u re 3.
SDS s p l i t p o ly a c ry la m id e g e ls comparing th e m o b i l i t i e s o f
PVX degraded p r o t e i n and c h y m o try p s inogen ( C ) . On the l e f t
i s a t y p i c a l gel showing th e r e s u l t s o f a PVX-M p r e p a ra tio n
d e m o n s tra tin g the o ccurrence o f both the 29,000 m o le c u la r
w e ig h t s u b u n it (upper band) and the 24,500 m o le c u la r w e ig h t
s u b u n it ( lo w e r band) in the same p r e p a r a t io n .
On the r i g h t
i s e l e c t r o p h o r e t i c a n a ly s is o f th e same p r e p a r a t io n a f t e r
in c u b a t io n o f the v i r u s w i t h t r y p s i n , d e m o n s tra tin g com­
p l e t e c o n v e rs io n to th e 24,500 m o le c u la r w e ig h t s u b u n it.
17 Crude homogenate m a n ip u la tio n was t e s te d as a means o f p r o ­
ducin g v i r a l p r e p a r a t io n s composed o f ,subunits o f e i t h e r , b u t n o t
b o th , m o le c u la r w e ig h ts .
Crude homogenates were e m u ls i f i e d w ith
c h lo r o fo r m im m e d ia te ly . a f t e r hbm ogenization and, a f t e r f u r t h e r
p u rific a tio n ,
PVX-IP was ahalyzed f o r s u b u n it m o le c u la r w e ig h t w it h
SDS s p l i t gel e l e c t r o p h o r e s is .
Assays o f PVX-IP p r e p a r a tio n s
suggested a homogeneous s u b u n it m o le c u la r w e ig h t o f 29,000-300
( F ig .
I).
Some PVX s o l u t i o n s were p u r i f i e d a f t e r a llo w in g th e crude
homogenate t o in c u b a te o v e r n ig h t a t room tem p era ture and subse quen tly
p u r i f i e d as d e s c rib e d f o r PVX-ON. • These v i r u s suspensions were a ls o
analyzed f o r s u b u n it m o le c u la r w e ig h t by a c ry la m id e gel e le c tr o p h o ­
r e s i s , and y ie ld e d p r i m a r i l y s u b u n its having a m o le c u la r w e ig h t o f
24,500-500 ( F ig . I ) .
F ig u re 4 i l l u s t r a t e s , t y p i c a l p a t t e r n s in SDS
s p l i t g e ls o f PVX-IP and PVX1
-ON degraded p r o t e in compared w i t h the
chym otrypsi,nogen s ta n d a rd .
The consiste n cy, o f s u b u n it m o le c u la r Weight was i n v e s t i g a t e d in
v i r a l p r e p a r a t io n s o b ta in e d by- PVX-IP and PVX-ON p u r i f i c a t i o n .
R e s u lts from tw e n ty expe rim e nts in d ic a t e d t h a t PVX-IP p u r i f i c a t i o n
c o n s i s t e n t l y produced v i r u s p a r t i c l e s composed o f th e 29,000 molec­
u l a r w e ig h t s u b u n its e x c l u s i v e l y , s in c e o n ly these s t r u c t u r e u n it s
were d e t e c t a b le i n e l e c t r o p h o r e t i c g e ls ..
18
F ig u re 4.
T y p ic a l SDS s p l i t g e ls showing p a tte r n s o b ta in e d a f t e r
e l e c t r o p h o r e t i c a n a ly s is o f PVX-IP and PVX-ON p r o t e in
using c h y m o try p s inogen as th e com parative m arker p r o t e in
( C ) . On th e l e f t i s th e gel o f PVX-IP degraded p r o t e in
and on th e r i g h t i s the gel o f PVX-ON degraded p r o t e i n .
Degraded p r o t e i n o b ta in e d by SDS in c u b a t io n .
19
The minimum amount o f p r o t e i n d e t e c t a b le in SDS s p l i t g e ls was
d e te rm in e d .
S e n s i t i v i t y o f th e assay procedure f o r d e t e c t in g the
s u b u n its i n a v i r u s p r e p a r a t io n depended upon th e minimum amount o f
p r o t e i n v i s i b l e a f t e r Coomassie b r i l l i a n t b lu e s t a i n i n g .
SDS
degraded PVX p r o t e i n was s e r i a l l y d i l u t e d in th e s ta n d a rd e l e c t r o ­
p h o r e t i c b u f f e r , a p p lie d a t th e to p o f th e g e l s , and e le c tr o p h o r e s e d .
The minimum d e t e c t a b le c o n c e n tr a t io n o f p r o t e in a f t e r s t a i n i n g was
found t o be 30 jug/m l i n th e a p p lie d p r e p a r a t io n .
However, PVX-ON p r e p a r a t io n s which co n ta in e d o n ly th e 24,500
m o le c u la r w e ig h ts s u b u n its were more d i f f i c u l t t o o b t a i n .
P u rifie d
PVX-ON suspensions were i n c o n s i s t e n t , i . e . , sometimes r e s u l t i n g in
homogeneous s o l u t i o n s o f th e 24,500 s u b u n it s , b u t more f r e q u e n t l y
r e s u l t i n g in s o l u t i o n s c o n t a in in g both th e 29,000 and th e 24,500
s u b u n it s .
P r e p a ra tio n s o f PVX-IP and PVX-ON were a ls o s e r o l o g i c a l l y
assayed f o r homogeneity.
PVX was degraded w it h SDS and th e degraded
p r o t e i n e le c tr o p h o r e s e d on sta n d a rd s iz e d SDS a c ry la m id e g e ls .
F o llo w in g e l e c t r o p h o r e s is f o r t h r e e h o u rs , the g e ls were surrounded
w i t h PVX-M degraded p r o t e i n a n tis e ru m im pregnated i n agar acco rd in g
t o S h a lla and Shepard ( 2 4 ) .
F r e s h ly prepared PVX-IP and PVX-ON
assayed i n t h i s manner d is p la y e d a s i n g l e component r e a c t i v e w ith
a n tib o d y on th e c irc u m fe re n c e o f th e g e l .
The s e n s i t i v i t y o f the
20
s e r o lo g ic a l assay method f o r minimum d e t e c t a b le a n tig e n c o n c e n tr a ­
t i o n was a s c e r t a in e d .
PVX degraded p r o t e i n was s e r i a l l y d i l u t e d
10 f o l d from 10 mg/ml t o 0.0001 m g/m l, e le c tr o p h o r e s e d in acryla m id e
g e l s , and s t a in e d w i t h a c o n s ta n t le v e l o f a n tib o d y .
The r e s u l t s
in d ic a t e d t h a t th e a n tib o d y assay-method was s e n s i t i v e t o 30 xig/m l.
Sephadex gel chrom atography o f reduced PVX degraded p r o t e in in
th e presence o f SDS was t e s te d as an a d d i t i o n a l method o f d e t e r ­
m in in g degraded v i r a l
p r o t e i n m p le c u la r w e ig h ts and o f c o n f ir m in g
those c a l c u la t e d from SDS s p l i t gel e l e c t r o p h o r e s is d a t a .
SDS de­
graded PVX-IP and PVX-ON p r e p a r a t io n s 'With a s t a r t i n g volume o f
2-3 ml and a c o n c e n t r a t io n o f 10-15 mg/ml were chromatographed on
Sephadex G-200 e q u i l i b r a t e d w i t h 0.05 M T r i s b u f f e r pH 7 .0 con­
t a i n i n g 1.0% SDS.
E l u t i o n p r o f i l e s compared w i t h s i m i l a r l y
t r e a t e d sta n d a rd s in d ic a t e d m o le c u la r w e ig h ts f o r PVX-IP and
PVX-ON s t r u c t u r e u n i t s o f 28,500 and 24,000 r e s p e c t i v e l y : ( F ig . 5)..
The v a lu e s o b ta in e d f o r th e m o le c u la r ,w e ig h ts o f PVX-IP and
PVX-0N s u b u n i t s - o f 29,000 and 24,500 by SDS s p l i t gel e l e c t r o ­
p h o re s is and 28,500 and 24,000 by SDS Sephadex chromatography
agree c l o s e l y w i t h K o e n ig 's f i n d i n g s o f 29,800 and 2 4 ,0 0 0 , and
c o n fir m s th e v a r i a b i l i t y in the PVX p r o t e i n m o le c u la r w e ig h t
w i t h mode o f v i r a l
p u rific a tio n .
P r e p a ra tio n o f p u r i f i e d PVX-IP and PVX-ON. - -
D e gradation o f
21
MOL. WT X IO " 4
OVAL
PVX IP MONOMER
CHYMO1
VXON MONOMER
CYTO C
.1 .2.
.3
.4
.5
.6 .7 .8
.9
IO LI
12 1.3
14 1.5
MOBILITY
F ig u re 5.
Graph c o n s tr u c te d f o r e s t im a t io n o f m o le c u la r w e ig h ts by
SDS Sephadex G-200 column chrom atography. M o b i l i t y equals
th e r a t i o o f th e e l u t i o n volume (m l) o f th e p r o t e in to the
e l u t i o n volume o f cytochrome C,
22
PVX-IP r e s u lt e d in s u b u n it s o l u t i o n s homogeneous in th e a c ry la m id e
gel assay system.
s c r ib e d r o u t i n e l y .
Hence, PVX-IP was prepared in th e manner de­
However, s in c e p r o d u c tio n o f PVXt-ON by crude
homogenate in c u b a t io n was i n c o n s i s t e n t , ' a more r e l i a b l e method o f
o b t a i n in g v i r u s p r e p a r a t io n s composed o f onTy th e lo w e r m o le c u la r .
w e ig h t s u b u n its was in v e s t i g a t e d .
Since Koenig e t al
(12) re p o rte d
t h a t in c u b a t io n o f PVX W ith t r y p s i n d u p lic a t e d th e m o le c u la r w e ig h t
c o n v e rs io n t h a t o c c u rre d w i t h crude homogenate in c u b a t io n , enzy­
m a tic d ig e s t io n was i n v e s t i g a t e d as an a l t e r n a t i v e means o f p r e ­
p a r in g PVX-ON. ■ I n c u b a tio n o f PVX w i t h t r y p s i n a t a s u b s t r a t e to
enzyme r a t i o o f 1000:1 f o r 1a p p ro x im a te ly tw e lv e hours r e s u lt e d in
I
■
;
th e c o n s i s t e n t c o n v e rs io n to v i r u s composed o f o n ly d e t e c t a b le
s u b u n its w i t h a m o le c u la r Weight o f 24,500 ( F ig . 3 ) .
D is s o c ia t io n
O
o f th e v i r u s subsequent to t r y p s i n d i g e s t i o n as r e p o r te d f o r cowpea
m o t t le v i r u s by Chidlow arid,Tremaine (3) was n o t o b se rve d ,
determ ined by r e t e n t i o n o f stream b i r e f r i n g e n c e .
as
T r y p s in d ig e s te d
PVX was t h e r e f o r e used to, prepare PVX-ON except as n o te d .
S e r o lo g ic a l r e l a t i o n s h i p between PVX-IP and PVX-ON. - it
A fte r
had been co n firm e d t h a t two m o le c u la r w e ig h ts o f PVX p r o t e in
could be prepared homogeneously, th e s e r o lo g ic a l r e la te d n e s s o f
th e two forms was d eterm ined .
and a n t is e r a t o th e two v i r a l
PVX-IP and PVX-ON were p u r i f i e d
forms were prepa red .
A fte r s ix
23
weeks, th e homologous t i t e r o f PVX-ON a n tis e ru m reached 1 :8192 , and
th e homologous t i t e r o f PVX-IP a n tis e ru m was 1:4096.
R e cip ro ca l
c r o s s - a b s o r p t io n t e s t s were conducted t o determ ine i f any s e r o lo g ic a l
d i f f e r e n c e s e x is t e d between these two forms o f PVX.
The r e s u l t s
showed no r e s id u a l a c t i v i t y t o homologous a n tig e n a f t e r removal o f
th e immune complex fdrmed by o v e r n ig h t a b s o rp tio n w i t h h e te ro lo g o u s
a n tig e n a t 4 mg v i r u s per ml o f a n tis e ru m .
C o n tro l t e s t s conducted
by a b s o r p tio n w i t h homologous a n tig e n and subsequent t i t e r i n g t o
homologous a n tig e n dem onstrated t h a t a b s o rp tio n was com plete a t the
l e v e l s used.
P o r tio n s o f th e a n tig e n s used in the c r o s s - a b s o r p t io n
s tu d ie s had been determ ined to be homogenous w i t h r e s p e c t t o s u b u n it
m o le c u la r w e ig h t p r i o r to use in th e r e c ip r o c a l c r o s s - a b s o r p t io n
s t u d ie s .
I t was concluded from the se s tu d ie s .th a t PVX-IP and PVX^ON
were s e r o l o g i c a l l y i d e n t i c a l d e s p it e th e f a c t th e y were composed o f
p r o t e i n s u b u n its o f d i s t i n c t m o le c u la r w e ig h ts .
P r e p a ra tio n and s e r o lo g ic a l r e la te d n e s s o f PVX-IP and PVX-ON .
degraded p r o t e i n . - - ■E x p e rim e n ta tio n had shown t h a t th e n a t iv e
p r o t e i n o f PVX-IP and PVX-ON were a n t i g e n i c a l l y i d e n t i c a l .
However,
s in c e d e p o ly m e r iz a tio n o f PVX s u b u n its r e s u l t i n g i n a change in
a n t ig e n i c s p e c i f i c i t y from th e n a t iv e form o f th e s u b u n it (24, 30)
and th e e f f i c a c y o f using denatured p r o t e i n to dete rm in e s e r o lo g ic a l
r e la te d n e s s (31) have been d e m on strate d, i t was necessary t o se­
24
r o l o g i c a l l y compare th e denatured p r o t e i n s u b u n its o f PVX.
PVX-IP
and PVX-ON were p u r i f i e d and th e degraded p r o t e in o f each species
prepared by SDS in c u b a t io n .
As a f u r t h e r p r e c a u tio n a g a in s t
contam in ants n o t d e t e c t a b le w i t h e i t h e r th e ,c h e m ic a l o r th e se­
r o lo g ic a l
homogeneity assay f o l l o w i n g e l e c t r o p h o r e t i c a n a l y s i s , an
a d d i t i o n a l p u r i f i c a t i o n ste p was employed.
A cryla m ide gel e l e c t r o ­
p h o re s is o f SDS degraded p r o t e in o f PVX-IP o r PVX-ON was conducted
on a p r e p a r a t iv e s c a le t o f u r t h e r p u r i f y th e p r o t e in to be used f o r
im m u n iz a tio n .
A n tig e n bands were i d e n t i f i e d w it h a n tib o d y and, as
shown i n f i g u r e 6 , t h i s method was capable o f s e p a ra tin g th e 29,000
from th e 24,500 m o le c u la r w e ig h t s t r u c t u r a l
p r o t e in s .
When used to
prepare e i t h e r P V X -IP 'o r PVX-ON degraded p r o t e in f o r im m u n iz a tio n ,
a s i n g l e band in the gel. was observed.
SDS s p l i t g e ls were e l e c t r o -
phoresed f o r each p r e p a r a t io n to f u r t h e r c o n fir m th e homogeneity and
m o le c u la r w e ig h t o f t h e , p r o t e i n .
F o llo w in g p r e p a r a t iv e gel e l e c t r o ­
p h o r e s is , t h a t p o r t io n o f the a c ry la m id e gel c o n t a in in g th e degraded
v ir a l
p r o t e i n was s l i c e d from th e g e l , smashed i n t o a s l u r r y w ith
2 ml o f 0.005 M N a - C it r a t e pH 8 .0 b u f f e r and d ia ly z e d 6-8 hours
a g a in s t th e same b u f f e r to remove unbound SDS.
A n tis e r a to PVX-IP
and PVX-ON degraded p r o t e i n were prepared by i n j e c t i n g t h i s r e s u l t a n t
s lu rry .
A n tis e r a to PVX-IP and PVX-ON degraded p r o t e i n both reached
a t i t e r o f 1:8 to SDS prepared homologous a n tig e n s i x weeks, a f t e r
25
F ig u re 6.
SDS p r e p a r a t iv e e l e c t r o p h o r e t i c gel o f PVX-IP and PVX-ON
degraded p r o t e in a f t e r i d e n t i f i c a t i o n o f th e p r o t e in bands
w i t h a n t ib o d y .
The gel i l l u s t r a t e s s e p a r a tio n p o s s ib le
between the two species o f degraded p r o t e i n , and the
u s e fu ln e s s o f t h i s method f o r use in p re p a rin g degraded
PVX p r o t e in f o r im m u n iz a tio n .
The upper band re p re se n ts
l o c a t i o n o f PVX-IP degraded p r o t e in and th e lo w e r band
re p re s e n ts lo c a t i o n o f th e PVX-ON degraded p r o t e i n .
26
th e i n i t i a l
in je c tio n .
conducted t o d e t e c t i f
O u c h te rlo n y double d i f f u s i o n t e s t s were
any s e r o lo g ic a l d if f e r e n c e s e x i s t e d between
these two forms o f s u b u n it s .
s e r o lo g ic a l
1%
SDS.
PVX degraded p r o t e in was prepared f o r
t e s t i n g by d e p o ly m e r iz a tio n w i t h e i t h e r
30%
p y r id i n e o r
P r o t e in prepared w i t h SDS was analyzed f o r homogeneity
b e fo r e - use i n double d i f f u s i o n .
P y r id in e has been p r e v io u s ly shown
t o y i e l d m a in ly dim ers when used t o dep o lym e rize PVX ( 2 4 ) .
Double
d i f f u s i o n t e s t s conducted w i t h SDS prepared a n tig e n in d ic a t e d s e ro ­
lo g ic a l
i d e n t i t y between th e two, fohns o f PVX s u b u n its when te s te d
by th e s tandard method f o r d e t e c t in g d i f f e r e n c e s in a n t ig e n i c
s p e c i f i c i t y v i a double d i f f u s i o n
( F ig . 7 ) , using a n t is e r a to e i t h e r
PVX-IP o r PVX-ON degraded p r o t e i n .
Double d i f f u s i o n assays were
s i m i l a r l y conducted using PVX-IP and PVX-ON degraded p r o t e i n p r e pared e l e c t r o p h o r e t i c a l l y .
The degraded v i r a l
.
p r o t e in was e l e c t r o -
phoresed on sta n d a rd s iz e SDA a c ry la m id e g e ls and th e p r o t e i n in
th e g e ls i d e n t i f i e d th ro u g h th e use o f a n t ib o d y .
th e gel, c o n t a in in g th e v i r a l
That p o r t io n o f
s u b u n its was e x c is e d , these d is c s
o r g a n iz e d i n t o th e d e s ire d s e r o lo g ic a l p a t t e r n , and surrounded
w i t h sta n d a rd double d i f f u s i o n a g a r.
A c e n t r a l w e ll was then r e ­
moved and charged w i t h e i t h e r PVX-IP o r PVX-ON degraded p r o t e in
a n tis e ru m and th e r e a c t io n s a llo w e d to deve lop .
These r e s u l t s a ls o
in d ic a t e d t h a t th e two forms o f PVX s t r u c t u r a l p r o t e i n are se-
27
F ig u re 7.
O u c h te rlo n y double d i f f u s i o n t e s t comparing PVX-IP and
PVX-ON degraded p r o t e i n re a c te d a g a in s t PVX-IP degraded
p r o t e i n a n tis e ru m .
I=PVX-IP degraded p r o t e i n and
O=PVX-ON degraded p r o t e i n .
N o tic e the c o n t i n u i t y o f the
p r e c i p i t i n l i n e s and th e absence o f s p u r s , i n d i c a t i n g
s e r o lo g ic a l i d e n t i t y o f th e a n tig e n s .
C o n c e n tra tio n s o f
both a n tig e n s o l u t i o n s equ als a p p ro x im a te ly 0 .2 mg/ml.
28
ro lo g ic a lly id e n tic a l.
Because i t was p o s s ib le t h a t SDS breakdown o f PVX r e s u lt e d in
th e p r o d u c tio n o f monomeric s o l u t i o n s o f s u b u n its which would n o t
show a s in g l e d e te rm in a n t d i f f e r e n c e between th e PVX-IP and PVX-ON
degraded p r o t e i n i n double d i f f u s i o n t e s t s , p y r i d i n e degraded PVX-IP
and PVX-ON dimers were compared f o r s e r o lo g ic a l r e la t e d n e s s .
P y ri­
d in e d e g ra d a tio n o f each species was perform ed and th e two forms
compared i n double d i f f u s i o n using both PVX-IP and PVX-ON degraded
p ro te in a n t is e r a .
The r e s u l t s again in d ic a t e d s e r o lo g ic a l
between th e two forms o f v i r a l
id e n tity
p ro te in .
Since t r y p s i n prepared PVX-ON was used i n th e e xp e rim e n t in
most cases, i t was o f i n t e r e s t to compare th e a n t ig e n i c s p e c i f i c i ­
t i e s o f t r y p s i n prepared PVX-ON and PVX-ON p u r i f i e d f o l l o w i n g
in c u b a t io n o f crude homogenates.
PVX-ON was p u r i f i e d by both methods
and the, degraded p r o t e i n o b ta in e d by SDS in c u b a t io n .
Standard double
d i f f u s i o n t e s t s were conducted to compare the two forms o f PVX-ON
p r o t e in a g a in s t PVX-ON degraded p r o t e i n a n tis e ru m .
The r e s u l t s o f
these t e s t s dem onstrated t h a t th e method o f p re p a rin g PVX-ON had no
e f f e c t upon th e a n t i g e n i c s p e c i f i c i t y o f PVX-ON degraded p r o t e in .
E f f e c t . o f t r y p s i n on degraded p r o t e i n . —
PVX d is s o c ia t e d
p r o t e i n has been shown t o be. co n ve rte d i n t o a d i f f e r e n t conform a­
tio n a l
s t a t e than when assembled i n t o whole v i r u s p a r t i c l e s
(2 4 ).
'
29
Tests were conducted to. dete rm in e whether t h i s d e n a tu re d , but
c h e m ic a lly i d e n t i c a l
s u b u n it would be, more s e n s i t i v e t o t r y p s i n
d ig e s t io n than th e n a t iv e o r assembled p r o t e i n , and hence be ,se­
r o l o g i c a l l y d i s t i n g u i s h a b l e from u n d ig e ste d p r o t e i n .
s e r i a l l y d i l u t e d i n t e p f o . ld in crem ents from an i n i t i a l
o f 10 m g /m l,to 0.001 mg/ml.
p y r i d i n e degraded v i r a l
te m p e ra tu re .
T r y p s in was
c o n c e n tr a t io n
To each c o n c e n tr a t io n was added I mg o f
p r q t e i n and in c u b a te d two hours, a t room
Each m ix t u r e was, then i n h i b i t e d by TICK.
The tim e r e q u ir e d f o r i n h i b i t i o n o f t r y p s i n by TLCK was d e t e r ­
mined u sin g th e h id e powder,azure system.
The absorbance o f the
t r y p s i n i n h i b i t i o n t e s t p r e p a r a t io n decreased from 0.61 o p t i c a l
d e n s it y u n i t s t o 0.015 o p t i c a l d e n s it y u n i t s a f t e r one .,hour in c u ­
b a t io n . : V i r a l
p r o t e i n - t r y p s i n p r e p a r a t io n s were i n h i b i t e d w i t h TLCK
f o r two hours t o in s u r e com plete , i n h i b i t i o n .
M ik i & K n ig h t (14) have r e p o r te d t h g t PVX p r o t e i n , degraded
w i t h g u a n id in e h y d r o c h lo r id e , r e s u lt e d i n 18 p e p tid e s when d ig e s te d
w i t h t r y p s i n a t a r a t i o o f PVX p r o t e i n t o t r y p s i n o f 1 0 :1 .
The
s t u d ie s conducted in t h i s expe rim e nt demonstrated t h a t PVX degraded
p r o t e i n was s u f f i c i e n t l y d ig e s te d a t l e v e l s o f p r o t e i n to t r y p s i n
o f 1 :1 0 , 1:1 and 10:1 t o have no r e a c t i v i t y rem a in in g when te s te d
a g a in s t PVX-IP o r PVX-ON degraded p r o t e i n a ntise ru m i n double
d iffu s io n .
However, a t l e v e l s o f v i r a l
p r o t e in to t r y p s i n o f
30
100:1 and 100 0:1 , the degraded p r o t e i n would r e a c t w i t h degraded
v ira l
p r o t e i n a n tis e ru m , b u t w i t h a r e a c t io n o f n o n - i d e n t i t y when
compared t o u n d ig e ste d v i r a l
p ro te in .
E f f e c t o f c h y m o try p s in on PVX-and PVX degraded p r o t e i n . —
The e f f e c t o f c h y m o try p s in on PVX and PVX degraded p r o t e i n was a ls o
te s te d .
Koenig e t al
(12) have r e p o r te d t h a t c h y m o tryp sin had no
e f f e c t upon assembled PVX s u b u n it s .
T h is was t e s te d i n o u r la b o r a ­
t o r y by in c u b a t in g PVX-IP w i t h c h y m o try p s in and using TPCK t o t e r m i ­
nate th e r e a c t i o n .
A l iq u o t s o f PVX were in cu b a te d w i t h chymo­
t r y p s i n a t a r a t i o o f 100 0:1 , as conducted w i t h t r y p s i n d i g e s t i o n
expe rim e nts o f PVX, and a f t e r i n h i b i t i o n o f the enzyme, th e molecu­
l a r w e ig h t o f th e v i r a l
s u b u n its f o l l o w i n g SDS d e g ra d a tio n was
determ ined w i t h th e SDS s p l i t gel system.
The tim e r e q u ir e d f o r
i n h i b i t i o n o f c h y m o try p s in w i t h TPCK was d e te rm in e d .
Using th e hide
powder azure system a g a in , the absorbance o f the c h y m o try p s in i n ­
h i b i t i o n t e s t p r e p a r a t io n decreased from 0.55 o p t i c a l d e n s it y u n i t s
t o 0.01 o p t i c a l d e n s it y u n i t s a f t e r one h ou r.
t i o n , th e TPCK i n h i b i t e d v i r a l
As an e x t r a precau­
p r o t e i n - c h y m o tr y p s in m ix tu r e s were
in c u b a te d f o r two hours t o in s u r e com plete i n h i b i t i o n .
The r e s u l t s
o b ta in e d in d ic a t e d t h a t c h y m o try p s in had no e f f e c t upon th e m o le c u la r
w e ig h t o f th e s u b u n it s , f o r th e a c ry la m id e gel a n a ly s is demonstrated
o n ly one s t a in a b le band, r e p r e s e n t a t iv e o f a m o le c u la r w e ig h t o f
31
29,000.
Chymotrypsin in cu b a te d PVX-IP was degraded w i t h SDS and
s e r o l o g i c a l l y compared t o SDS degraded u n tre a te d PVX-IP i n double
d iffu s io n .
These r e s u l t s in d ic a t e d t h a t th e s e r o lo g ic a l
p r o p e r t ie s
o f PVX-IP were l i k e w i s e u n a ffe c te d by c h y m o tryp sin in c u b a t io n .
Experiments were conducted t o dete rm in e th e e f f e c t t h a t chymo­
t r y p s i n would have upon PVX degraded p r o t e i n .
Chymotrypsin was
s e r i a l l y d i l u t e d from 10 mg/ml t o 0.001 mg/ml and to each concen­
t r a t i o n was added I mg o f PVX degraded p r o t e in which had been p re ­
pared w i t h p y r i d i n e .
The m ix t u r e was in c u b a te d f o r two hours a t
room te m p e ra tu re and th e enzyme i n h i b i t e d by adding TPCK and a llo w in g
to in c u b a te f o r two h o u rs.
v ir a l
These s t u d ie s showed t h a t a t l e v e l s o f
p r o t e i n to enzyme o f 1 :1 0 , 1 :1 , 10,:I and 1 0 0 :1 , th e PVX de­
graded p r o t e i n r e a c t i v i t y w i t h degraded p r o t e in a n tis e ru m was
c o m p le te ly e lim in a t e d in double d i f f u s i o n t e s t s .
v ira l
But a t l e v e l s o f
p r o t e i n t o enzyme r a t i o s o f 100 0:1 , the v i r a l
p r o t e i n would
r e a c t w i t h PVX degraded p r o t e in a n tis e ru m i n double d i f f u s i o n , but
th e r e a c t io n was one o f n o n - i d e n t i t y .
DISCUSSION
Presumably an enzyme or, enzymes in tobacco sap are r e s p o n s ib le
f o r th e c o n v e rs io n o f th e m o le c u la r w e ig h t o f the PVX s u b u n it from
29,000 to 24,500 under c e r t a i n c o n d i t i o n s .
This m o le c u la r w e ig h t
c o n v e rs io n can be d u p lic a t e d by in c u b a t in g PVX w i t h t r y p s i n .
These
two m o le c u la r w e ig h t forms o f p r o t e i n , whether e x i s t e n t as assembled
o r dep olym erized p r o t e i n , were compared s e r o l o g i c a l l y f o r any d i f f e r ­
ence i n a n t ig e n i c s p e c i f i c i t y which may o c cu r f o l l o w i n g a lo s s o f
n e a r l y 15% o f th e p o ly p e p tid e c h a in .
P u r i f i e d whole PVX p a r t i c l e s
composed e x c l u s i v e l y o f s u b u n its o f e i t h e r m o le c u la r w e ig h t were
compared by r e c ip r o c a l cro ss a b s o r p tio n s t u d ie s .
PVX degraded
p r o t e in o f both m o le c u la r w e ig h ts was o b ta in e d by both p y r i d i n e and
SDS d e g ra d a tio n .
In a l l
cases, th e two forms o f assembled o r de­
graded PVX p r o t e i n , i . e . , p r o t e i n c o n s i s t i n g o f e i t h e r 29,000 o r
24,500 m o le c u la r w e ig h t, were found to be s e r o l o g i c a l l y i d e n t i c a l .
T h is la c k o f a n t i g e n i c s p e c i f i c i t y change w it h PVX p r o t e in is
i n c o n t r a s t t o TMV expe rim e nts whereby one amino a c id s u b s t i t u t i o n
i n o r th e lo s s o f as few as th r e e amino a c id s from th e p r o t e in may
cause a d e t e c t a b le s e r o lo g ic a l change.
However, th e r e s u l t s are in
accordance w i t h the expe rim e nts w i t h cowpea c h l o r o t i c m o t t le v ir u s
i n which a 10% lo s s o f p r o t e in caused no s e r o lo g ic a l change, b u t a
20% lo s s was accompanied by a s e r o lo g ic a l change.
In a d d i t i o n , a
lo s s o f 5-10% o f th e m o le c u la r w e ig h t o f ade novirus hexons caused no
33 '
change in a n t i g e n i c s p e c i f i c i t y b u t a g r e a t e r l o s s ,
( e x a c t data n o t
a v a i l a b l e ) , caused a change in a n t i g e n i c s p e c i f i c i t y .
I t seems
e v id e n t from t h i s data t h a t p o ly p e p tid e ch a in lo s s and a n t ig e n i c
s p e c i f i c i t y change cannot be n e c e s s a r ily c o r r e la t e d o r p r e d ic t e d .
The 15% m o le c u la r w e ig h t lo s s has no in f lu e n c e upon a n t ig e n ic
s p e c i f i c i t y o f th e PVX m o le c u le , anc) mode o f p u r i f i c a t i o n a p p a r e n tly
does n o t re n d e r s e r o lo g ic a l
i d e n t i f i c a t i o n s tu d ie s i n v a l i d .
im p o r t a n t when c o n s id e r in g s e r o lo g ic a l d e t e c t io n o f PVX.
This i s
A n tig e n ic
s p e c i f i c i t y seems t o r e s id e in 85% o r le s s o f th e m o le c u le , meaning
t h a t v a r i a t i o n can o c c u r in th e re m a in in g 15% o f the m olecu le w i t h ­
o u t a f f e c t i n g th e a b i l i t y t o be d e te c te d by s e r o lo g ic a l methods.
I f th e v a r i a t i o n in s t r a i n s o f v i r u s r e s id e n t in th e m a t e r ia l te s te d
occurs i n th e p o r t i o n o f th e p e p tid e l o s t upon enzym atic d i g e s t i o n ,
i d e n t i f i c a t i o n o f PVX would n o t be s e r i o u s l y im p a ire d .
Because o f
the la c k o f any a n t i g e n i c d i s p a r i t y between th e two form s o f PVX
p r o t e i n , th e r e g io n o r r e g i o n s , (perhaps both t e r m in i are d i g e s t e d ) ,
o f th e p o ly p e p tid e ch a in l o s t , i . e . ,
15% o f th e m o le c u la r w e ig h t,
must c o n s t i t u t e an im m u n o sile n t re g io n o r re g io n s o f th e m olecule
c o n t a in in g none o f th e th r e e o r f o u r a n tib o d y com bining s i t e s per
monomer as r e p o r te d by Shepard and S h a lla (3 0 ).
However, s in c e
broad spectrum o r s p e c i f i c proteases may occu r in some p l a n t t i s ­
sues, f o r example bean c a rb o x y p e p tid a s e ( 2 0 ) , mode o f p u r i f i c a t i o n
34
may in f l u e n c e th e s t r u c t u r e o f c e r t a i n v i r i o n s 1p u r i f i e d and hence
th e a n t ig e n i c s p e c i f i c i t y s u f f i c i e n t l y t h a t even i d e n t i f i c a t i o n by
im m u n o d iffu s io n c o u ld be q u e s tio n a b le .
The phenomenon o f enzymatic
d i g e s t io n o f v i r i o n s d u r in g r o u t i n e p u r i f i c a t i o n may be q u i t e un­
p r e d i c t a b l e and c o u ld have c o n c e iv a b ly o c c u rre d w i t h some v i r u s ( e s )
and gone unreco gnize d.
D ig e s tio n o f v i r a l
p r o t e in by some c e l l u l a r
enzymes, may o c cu r i n s t a n t l y upon r u p t u r e o f c e l l s and /or, t h e i r
o r g a n e lle s and le a d .to c o n fu s io n i n i n t e r p r e t a t i o n o f d a ta .
The r e s u l t s have shown t h a t degraded p r o t e in o f PVX i s more
s u s c e p t ib le t o d i g e s t i o n by t r y p s i n and ch ym o tryp sin than assembed
p ro te in .
The g r e a t e r s u s c e p t i b i l i t y o f th e degraded PVX p r o t e in
s u b u n its to enzym atic d i g e s t io n may b e , a t t r i b u t a b l e to th e hypoth­
e s is t h a t th e depolym erized s u b u n its have undergone a change in
c o n fo rm a tio n which a llo w s f o r th e in c re a s e d d i g e s t i o n , s i m i l a r
to th e phenomenon found by H u l t i n and S j o q v i s t (8) w i t h r a t l i v e r
ribosom es.
In t h e i r r e p o r t , th e y proposed t h a t under c e r t a i n w e l l -
d e fin e d c o n d i t i o n s , th e ribosomes w i l l
undergo a change in c o n f o r ­
m ation and a " lo o p " on th e ribosom e, which was f o r m e r ly r e s i s t a n t
to c h y m o try p s in d i g e s t i o n , becomes s u s c e p t ib le to chym o tryp sin
d ig e s tio n .
The m o le c u la r w e ig h ts o f 29,000 and 24,500 f o r th e two forms
o f th e PVX s u b u n its agree c l o s e l y w i t h th e r e s u l t s o f Koenig e t
35
al
(12).
The small d is c re p a n c y i s p o s s i b l y a t t r i b u t a b l e to e l e c t r o ­
p h o r e t i c p ro c e d u ra l d i f f e r e n c e s .
The use o f s p l i t g e ls u t i l i z i n g
SDS p o ly a c r y la m id e e le c t r o p h o r e s is i s p o t e n t i a l l y th e most accu ra te
method c u r r e n t l y i n use o f d e te r m in in g m o le c u la r w e ig h ts o f v i r a l
p r o t e i n in t h i s range .
The m o le c u la r w e ig h ts r e p o r te d a ls o agree
c l o s e l y w i t h th e r e p o r t o f th e m o le c u la r w e ig h t o f PVX s u b u n its
r e p o r te d by S h a lla and Shepard (24) o f 25,000.
Even though t h e i r
r e s u l t s o f a n t ig e n i c d i s p a r i t y between PVX and i t s degraded p r o t e in
were achieved th ro u g h th e use o f th e lo w e r m o le c u la r w e ig h t o f the
two forms o f PVX p r o t e i n r e p o r te d in t h i s p u b l i c a t i o n , t h e i r r e s u l t s
can be e x t r a p o la t e d a c c u r a t e l y f o r th e com plete s u b u n it s in c e the
l a r g e r and th e s m a lle r are a n t i g e n i c a l l y i d e n t i c a l .
F i n a l l y , th e PVX system may be a model system f o r e x a c t ly
l o c a t i n g and i d e n t i f y i n g th e a n t ig e n i c d e te rm in a n ts o f a m o le cu le .
I t i s c l e a r t h a t 15% o f th e PVX m o le cu le i s n o t r e s p o n s ib le f o r
a n tig e n ic ity .
Through th e use o f s e l e c t i v e cleavage te c h n iq u e s ,
e i t h e r enzym atic o r n o n -e n z y m a tic , m o d i f i c a t i o n o f PVX p r o t e in may
r e s u l t in i d e n t i f i c a t i o n o f immunogenic re g io n s o f th e PVX p o l y ­
p e p tid e c h a in .
SUMMARY
P o ta to v i r u s X (PVX) has been shown to lo s e a p o r t i o n o f i t s
p o ly p e p tid e ch a in d u r in g p u r i f i c a t i o n o r by t r y p s i n d i g e s t i o n .
By
in c u b a t in g crude i n f e c t i v e homogenates o v e r n ig h t b e fo re p u r i f y i n g
th e v i r u s , o r by in c u b a t in g p u r i f i e d v i r u s w i t h t r y p s i n , a s u b u n it
m o le c u la r w e ig h t change from 29,000 t o 24,500 w i l l o c c u r .
The change
in m o le c u la r w e ig h t was co n firm e d by sodium dodecyl s u l f a t e
(SDS)
s p l i t gel p o ly a c r y la m id e e l e c t r o p h o r e s is and Sephadex 0-200 column
chrom atography.
The e f f e c t o f th e 15% m o le c u la r w e ig h t lo s s due t o enzym atic
h y d r o l y s is upon th e a n t i g e n i c s p e c i f i c i t y o f PVX and o f PVX degraded
p r o t e i n was i n v e s t i g a t e d .
w e igh ts, o f th e v i r a l
I t was necessary to compare both m o le c u la r
p r o t e i n w h ile i n e i t h e r the n a t iv e (assembled)
s t a t e o r th e degraded (d e p o ly m e riz e d ) s t a t e , f o r p r e v io u s s tu d ie s
have shown t h a t PVX and i t s degraded p r o t e in are o n ly s l i g h t l y
r e la t e d as a n t ig e n s .
A n t is e r a were made t o p u r i f i e d PVX composed o f e i t h e r , b u t n o t
b o th , s t r u c t u r a l u n i t m o le c u la r w e ig h ts .
A p r e p a r a t iv e SDS gel
e le c t r o p h o r e s is system was used to prep a re degraded p r o t e i n o f
e i t h e r m o le c u la r w e ig h t f o r use in th e p ro d u c tio n o f a n t i s e r a to
PVX degraded p r o t e i n .
R e ciprocal c r o s s - a b s o r p t io n t e s t s and
O u c h te rlo n y double d i f f u s i o n t e s t s were employed t o d e t e c t d i f f e r ­
ences i n a n t ig e n i c s p e c i f i c i t y which m ig h t accompany th e p o ly p e p tid e
37
c h a in lo s s .
Experim ental r e s u l t s i n d i c a t e ^ t h a t no change i n a n t i ­
g e n ic s p e c i f i c i t y was found t o accompany the 15% m o le c u la r w e ig h t
lo s s i n PVX s t r u c t u r a l
p r o t e in f o l l o w i n g enzym atic h y d r o l y s i s .
T r y p s in d i g e s t io n o f PVX degraded p r o t e in was more complete
than d i g e s t i o n o f n a t iv e v i r u s .
Depending upon th e c o n c e n tr a t io n
o f t r y p s i n , PVX degraded p r o t e in was e i t h e r im m u n o lo g ic a lly non­
r e a c t i v e o r re a c te d w i t h an O u c h te rlo n y p r e c i p i t i n l i n e o f non­
i d e n t i t y w i t h u n tr e a te d PVX degraded p r o t e i n .
Chymotrypsin had no
marked in f l u e n c e upon e i t h e r th e m o le c u la r w e ig h t o r th e a n t ig e n i c
s p e c i f i c i t y o f PVX.
However, PVX degraded p r o t e in was a ls o suscep­
t i b l e to d ig e s t io n by t h i s enzyme.
I t was h ypo the sized t h a t
c o n fo r m a tio n a l changes i n the depolym erized PVX p r o t e i n a llo w s f o r
th e g r e a t e r s u s c e p t i b i l i t y to t r y p s i n and c h y m o try p s in .
I t was concluded t h a t t r y p s i n d ig e s t io n o r in c u b a t io n o f
i n f e c t i v e homogenates o f PVX caused a s u b u n it m o le c u la r w e ig h t change
from the normal o f 29,000 to 24,500 , and t h a t t h i s 15% m o le c u la r
w e ig h t lo s s had no in f l u e n c e upon th e a n t ig e n i c s p e c i f i c i t y o f
e i t h e r n a t iv e o r degraded PVX p r o t e i n ,
F u rth e rm o re , th e r e g io n ( s )
l o s t upon h y d r o l y s is c o n s t i t u t e s an im m u n o sile n t p o r t i o n o f the
m o le c u le .
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