STUDIES OK METHYLATION REACTIONS IN PLANTS:
THE ORIGIN OF TEE METHYL CARBON OF
NICOTINE FORMED BY NICOTIANA RUSTICA

By
S te w a rt A n g lin Brown

A THESIS

S u b m itte d t o t h e S ch o o l o f G ra d u a te S tu d ie s o f M ichigan
S t a t e C o lle g e o f A g r ic u ltu r e and A p p lied S c ie n c e
in . 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 t h e d e g re e o f
DOCTOR OF PHILOSOPHY
D epartm ent o f C h em istry
1951

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ACKNOWLEDGMENT
The w r i t e r w ish e s t o e x p re s s h i s a p p r e c i a t i o n t o
Dr# R ic h a rd TJ# Byerrum , whose a d v ic e and w i l l i n g
c o o p e r a tio n h a v e g r e a t l y f a c i l i t a t e d t h e c o m p le tio n
o f t h i s p ro b lem , and t o s e v e r a l o t h e r members o f t h e
D ep artm en t o f C h em istry who h av e g iv e n a d v ic e fro m
tim e t o tim e# S p e c ia l g r a t i t u d e i s a l s o d u e t o Dr# E#
H# L u c a s, P r o f e s s o r of H o r t i c u l t u r e , w hose a c t i v e
a s s is ta n c e in c u ltiv a tin g th e p la n ts used in th e in ­
v e s t i g a t i o n s h a s b e e n a n in v a l u a b le c o n t r ib u t i o n t o
t h e s u c c e s s o f t h i s work#
■ F in a lly , t h e w r i t e r w ish e s t o th a n k t h e Eastm an
Kodak Company o f R o c h e s te r, New Y ork, and t h e D ep art­
ment o f C h em istry o f M iohigan S t a t e C o lle g e f o r t h e i r
g e n e r o s i t y i n p r o v id in g f e llo w s h ip s u n d e r w hich t h i s
work was c a r r i e d o n .
**********
******
****
**

*

TABLE OF CONTENTS

INTRODUCTION...............................................
EXPERIMENTAL........................ *.................................................................

PAGE
1
4

S y n th e s is o f R a d io a c tiv e P r e c u r s o r s ..................• * ..♦
4
P r e p a r a t i o n o f t h e Tobacco P l a n t s
6
M e th y la tio n E x p e rim en ts w ith D ,L -M e th io n in e .. . • .
8
U ptake o f m e th io n in e fro m s o l u t i o n ..................
8
I s o l a t i o n and p u r i f i c a t i o n o f n i c o t i n e . • • • .
9
A d m in is tr a tio n of C -^ -m e th y l D ,L -m e th io n in e 10
M e th y la tio n E x p erim en ts w ith F o r m a t e . 14
U ptake o f fo rm a te from s o l u t i o n ....................
16
A d m in is tr a tio n o f
fo rm a te sodium s a l t . .
18
*

DISCUSSION.............................................................................

20

SUMMARY.............................................................................

26

REFERENCES................................................................................................

27

APPENDIX

29

...............

INTRODUCTION
The i n v e s t i g a t i o n d e s c r ib e d h e r e was u n d e r ta k e n w ith t h e d u a l
o b j e c t i v e o f e l u c i d a t i n g some a s p e c ts o f tw o d i s t i n c t problem s#

The

f i r s t o f t h e s e in v o lv e d t h e p o s s ib le r o l e o f t r a n s m e t h y la t io n i n
p l a n t m e ta b o lism , an d t h e second co n ce rn e d pathw ays i n th e b io s y n th e ­
s i s of a lk a lo id s *
I n t h e c o u rs e o f t h e p a s t d ecad e a c o n s id e r a b le body o f e v id e n c e
h a s b e e n a c c u m u la te d t o e s t a b l i s h t h e im p o rta n c e o f tr a n s m e th y la ti o n
i n t h e a n im a l o rg an ism ( l ) *

I n a d d i t i o n , i t h a s b e e n shown t h a t t h e

mould N eu ro sp o ra c r a s s a c a n s y n th e s iz e c h o lin e b y s te p w is e m e th y la tio n
o f e th a n o la m in e , a lth o u g h t h e s o u rc e of th e m e th y l g ro u p s i s unknown
(2 )#

B u t, w h ile i t s e x i s t e n c e h a s b e e n p o s tu l a t e d ( 3 , 4 , 5) a lm o st

n o th in g h a s b e e n done t o e s t a b l i s h b y d i r e c t e x p e r im e n ta tio n w h e th e r
o r n o t th e r e a c t i o n o c c u rs i n t h e h ig h e r p la n t#

I n t h e l a t t e r con­

n e c t i o n , t h e o n ly e v id e n c e h a s b e e n o b ta in e d by means o f i n v i t r o
e x p e rim en ts#

B a rre n sc h e e n and v o n V aly i-N ag y ( 6 ) a d m in is te r e d m e th i­

o n in e t o ground w heat germ s and found t h a t t h e s y n th e s is o f c r e a t i n e
i n c r e a s e d s i x - t o e i g h t - f o l d i n th e p re s e n c e o f g ly co cy am in e.
At t h e commencement o f t h e p r e s e n t s tu d y no o n e , t o t h e w r ite r * s
know ledge, had a tte m p te d to d e m o n s tra te tr a n s m e th y la tio n i n t h e i n t a c t
p la n t#

E a r l i e r t h i s y e a r su ch an a tte m p t was r e p o r te d by Kirkwood

M arion ( 7 ) i n a s tu d y o f t h e f o rm a tio n o f t h e a l k a l o i d h o rd e n in e i n
s p ro u te d b a r le y #

They f e d C^4-m e th y l c h o lin e w hich h a s b e e n shown t o

a c t , a t l e a s t i n d i r e c t l y , a s a m eth y l d o n o r i n t h e a n im a l (8 )*

The

h o rd e n in e i s o l a t e d d u rin g t h i s e x p e rim e n t p o s s e s s e d n e g l i g i b l e r a d i o ­
a c t i v i t y , and t h e a u th o r s c o n c lu d e d t h a t t h e N -m ethyl g ro u p s o f
h o rd e n in e do n o t a r i s e fro m t h e c h o lin e - m e th io n in e system *
The compound ch o sen f o r s tu d y i n o u r w ork was n i c o t i n e , one o f
t h e to b a c c o a l k a l o i d s , w hich c o n ta in s a n N -m ethyl group*

T h is com­

pound was s e l e c t e d b e c a u s e o f t h e r e l a t i v e e a s e o f grow ing to b a c c o
w hich
p l a n t s , t h e f a c i l i t y w ith An i c o t i n e c an b e i s o l a t e d and p u r i f i e d , and
i t s c o m p a ra tiv e ly w ell-k n o w n c h e m istiy *

F u rth e rm o re , i t a f f o r d e d a n

o p p o r tu n ity o f c o n t r i b u t i n g t o th e second o f t h e two problem s r e f e r r e d
t o a b o v e , t h a t o f a l k a l o i d b io s y n th e s is *

I n t h i s f i e l d a n a p p r e c ia b l e

amount o f w ork h a s b ee n p u b lis h e d on t h e p h y s io lo g i c a l a s p e c ts ( 9 , 1 0 ) ,
but l i t t l e

i s known c o n c e rn in g th e p r e c u r s o r s o f t h e a l k a l o i d s o r t h e

ch em ica l pathw ays in v o lv e d i n a l k a l o i d fo rm a tio n *

In re g a rd to p re ­

c u r s o r s i t a p p e a r s t h a t t h e o n ly s i g n i f i c a n t w ork w ith n i c o t i n e h a s
b een t h a t o f K le in and L in s e r (1 1 ) and o f Dawson ( 1 2 ) , who h av e ob­
t a i n e d e v id e n c e t h a t p r o l i n e an d p y r r o lid o n e c a r b o x y lic a c id a c t a s
p r e c u r s o r s i n to b a c c o .

T h is e v id e n c e was o b ta in e d by in tr o d u c in g t h e s e

compounds i n t o e x c is e d s h o o ts and n o tin g an i n c r e a s e i n t h e amount o f
n i c o t i n e form ed i n t h e c o u r s e o f t h e a d m i n i s t r a t i o n p e rio d *

On t h e

b a s i s o f l a t e r e x p e rim e n ts , how ever, Dawson (9 ) h a s w ith d raw n h i s
o r i g i n a l c o n t e n t i o n t h a t p r o l i n e and p y r r o lid o n e c a r b o x y lic a c id a r e
n i c o t i n e p r e c u rs o rs *
I n v ie w o f t h e e x i s t i n g s t a t e o f know ledge i n t h e a b o v e-m en tio n ed
f i e l d s , i t a p p e a re d p ro m isin g t o s tu d y t h e p o s s i b l e o r i g i n o f t h e

N -m ethyl g ro u p of n i c o t i n e by t r a n s m e t h y l a t i o n , u s in g c a r b o n - 14 a s a
ra d io a c tiv e tra c e r#

By f e e d in g t o to b a c c o p l a n t s m e t h y l - l a b e l l e d

compounds t h a t h av e b e e n shown t o a c t a s m eth y l d o n o rs i n t h e a n im a l,
f o llo w e d by i s o l a t i o n and d e g r a d a tio n o f t h e n i c o t i n e s y n th e s iz e d , i t
was hoped to o b ta in in f o r m a tio n c o n c e rn in g t h e r o l e o f tr a n s m e th y la ­
t i o n i n a l k a l o i d b io g e n e s is #

EXPERIMENTAL
S y n th e s is o f R a d io a c tiT e P r e c u r s o r s
The r a d i o a c t i v e t r a c e r s u s e d i n t h i s -work w ere C^4 -m e th y l D ,Lm e th io n in e and C^4 - f o r m ic a c i d sodium s a l t *

The l a t t e r was p u rc h a s e d

fro m Oak R id g e u n d e r a l l o c a t i o n fro m t h e U n ite d S t a t e s A tom ic E n erg y
Commission*

The m e th io n in e was s y n th e s iz e d from C^4 ~m ethyl i o d i d e

w h ich was o b ta in e d fro m T r a c e r l a b , I n c * , a ls o u n d e r a l l o c a t i o n fro m
t h e Atom ic E nergy Commission*
The r e a c t i o n s em ployed i n t h e s y n th e s is o f C14 -m e th y l U*Lm e th io n in e a r e su n m arized below :

(1 )

CHg-CHg-CH-COOH
S
HBL
£
+ C^H^-CHqCI
CH^-CHg-pH-COOH
UHg

UH3 ( 1 )
Sfa

D, L -S-b o n zy lh o m o cy stein e
""

D,L-homocy s t i n e
(2 )

CgHfi-CH^-S-Caj-CHa-pH-COOH
NH2

CfiHK-CH^-S-CHp-CHj>-pH-COOH + C14sE^I
fa 2

KH3 ( l )
C14H3-S-CH2-CHg-CH-C00H
la
*
m z
D, L-m eth i o n in e

The s y n th e s is o f S -b en zy lh o m o cy stein ew as c a r r i e d o u t a s d e s c r ib e d
by du V igneaud an d P a t t e r s o n (13)*

The m e th y la tio n s te p was p erfo rm ed

a c c o rd in g t o t h e method o f M e lv ille , R ac h ele and K e l l e r ( 1 4 ) , b u t
b e c a u s e t h e C^4-m e th y l io d id e was o b ta in e d i n a g l a s s am poule a m o d ifi­
c a t i o n o f t h e a p p a r a tu s was n e c e s s a ry *

—4—

T his i s shown i n F ig u r e I .

FIGURE I

The equipm ent was d e s ig n e d so t h a t a p o r t i o n o f t h e m e th y l io d id e
c o u ld b e r e t a i n e d i n a c lo s e d tu b e f o r u s e i n a n o th e r s y n t h e s i s .

The

t r a p H and t h e r e a c t i o n v e s s e l J shown i n th e F ig u r e a r e e s s e n t i a l l y
t h e same a s t h o s e u s e d by M e lv ille e t a l .

T rap H c o n ta in e d a n a p p ro ­

p r i a t e q u a n t i t y o f i n a c t i v e m e th y l i o d id e , c a l c u l a t e d t o g iv e a y i e l d
o f 500 mg. o f m e th io n in e , when com bined w ith t h e a c t i v e compound.
The a p p a r a tu s was sw ept w ith n i t r o g e n g a s , w hich was d r i e d by f i r s t
p a s s in g i t th ro u g h a s o d a -lim e to w e r and th e n th ro u g h a t r a p o f con­
s t r u c t i o n s i m i l a r t o H, c o o le d i n a s o l i d c a rb o n d io x id e - m eth y l
c e llo s o lv e b a th .

The ammonia, w hich s e rv e d a s t h e r e a c t i o n medium,

was d r i e d b y p a s s a g e o f th e g as th ro u g h t h e soda-lim © to w e r b e f o r e
c o n d e n s a tio n i n v e s s e l J .

UVhen i t was d e s ir e d t o in tr o d u c e t h e m eth y l

i o d i d e , t h e r e a c t i o n v e s s e l J an d t r a p H w ere c o o le d i n CO2 - c e l l o s o l v e
b a t h s , s to p - c o c k B was s e t a s i l l u s t r a t e d i n t h e F ig u r e , and t h e d ry
n i t r o g e n was a d m itte d a t A*

The g la s s - e n c l o s e d plummet E was th e n

drawn by a m agnet i n t o t h e lo n g tu b e and a llo w e d t o f a l l , b re a k in g t h e
s e a le d c a p i l l a r y D o f t h e g l a s s am poule F c o n ta in in g t h e a c t i v e m eth y l
io d id e .

The l a t t e r was v a p o r iz e d s lo w ly by h e a tin g t h e am poule t o

a b o u t 50° i n a w a te r b a t h , and sw ept th ro u g h t h e a p p a r a tu s i n a s tr e a m
o f n itro g e n .

Some o f t h e v a p o u r condensed i n t r a p H and t h e r e s t was

c a r r i e d i n t o t h e r e a c t i o n v e s s e l*

TWhen t h e r e q u ir e d volum e o f 'th e

a c t i v e m e th y l io d id e h ad been sw ept o u t o f t h e am poule, s to p - c o c k C was
c lo s e d an d s to p - c o c k B r e v e r s e d , c o n f in in g t h e re m a in d e r o f t h e m eth y l
io d id e .

The n i t r o g e n was th e n a d m itte d a t G, an d t h e h e a t t r a n s f e r r e d

5

t o t r a p H.

The re m a in in g s te p s i n th e r e a c t i o n and p u r i f i c a t i o n w ere

c a r r i e d o u t a s d e s c r ib e d by M e lv ille and co-w o rk ers#
P r e p a r a t i o n o f t h e Tobacco P la n ts
The to b a c c o u s e d i n t h e s e s t u d i e s was IT ic o tia n a r u s t i c a L#, v a r .
h u m ilis , a h ig h -n ic o tin e s tr a in #

The seeds'*’ w ere p la n te d i n f l a t s i n

t h e g re e n h o u se an d t r a n s p l a n t e d a f t e r a b o u t t h r e e weeks i n t o s m a ll
p o t s , w here th o y w e re grown u n t i l th e y had a t t a i n e d a h e i g h t o f a t
l e a s t s i x in c h e s #

D uring t h i s i n t e r v a l t h e p l a n t s w ere o c c a s io n a lly

su p p lem en ted w i t h com m ercial p la n t fo o d m ix tu re a s r e q u ir e d .

The tim e

n e c e s s a a y f o r t h e p l a n t s t o a t t a i n t h e d e s ir e d s i z e was fro m two to
t h r e e m o n th s, t h e v a r i a t i o n p resu m ab ly b e in g due t o s e a s o n a l e f f e c t s *
The p l a n t s w ere p re p a re d f o r t h e h y d ro p o n ic a d m in is t r a t io n o f th e
r a d i o a c t i v e m a t e r i a l s a s f o ll o w s .

The e a r t h was c a r e f u l l y rem oved

fro m t h e r o o t s , f i r s t by s h a k in g an d th e n by w ash in g u n d e r a s tr e a m o f
w a te r.

As many a s p o s s i b l e of t h e a d h e rin g s o i l p a r t i c l e s w ere removed

i n t h i s w ay.

The r o o ts w ere th e n im m ersed i n a 0.01% s o l u t i o n o f

W yandotte d e t e r g e n t g e rm ic id e Uo. 1528** f o r a t l e a s t one h o u r , w ith
o c c a s io n a l a g i t a t i o n , t o re d u c e t h e b a c t e r i a l p o p u la tio n .

F o llo w in g a

b r i e f r i n s e u n d e r t a p w a t e r , t h e r o o ts o f each p l a n t w ere im m ersed i n
50 m l. o f a n in o r g a n ic n u t r i e n t s o l u t i o n i n a 125 m l. E rlen m ey er f l a s k .

* The seed s w ere o b ta in e d th ro u g h t h e c o u r te s y o f Dr# N* A# MacRae
o f t h e C an ad ia n D epartm ent o f A g r i c u l t u r e , C e n tr a l E x p e rim e n ta l
Farm , O ttaw a.
2
T h is m a t e r i a l was o b ta in e d from t h e W yandotte C hem icals C o rp .,
W y an d o tte, M ic h ., th ro u g h th e M ich ig an S t a t e C o lle g e D ep artm en t of
H o rtic u ltu re .

The n u t r i e n t s o l u t i o n was p r e p a re d by d i l u t i n g t h e s to c k s o l u t i o n ,
t h e c o m p o sitio n o f w hich i s shewn i n T a b le I , I s 3*

The n u t r i e n t s o lu ­

t i o n a l s o c o n ta in e d a n a p p r o p r i a t e q u a n t i t y o f th e C ^ - l a b e l l e d
compound b e in g i n v e s t i g a t e d , a s -w ill b e d e s c r ib e d l a t e r .

TABLE I
COMPOSITION OF THE NUTRIENT SOLUTION

W ater

11.

Magnesium s u lp h a te s MgSO^s

C alciu m n i t r a t e s Ca(N0g)2

1 g.

Ammonium s u lp h a te s (NH^)gS0^;250 mg.

P o ta s s iu m c h l o r id e s KC1
F e r r ic c h lo rid e

250 mg.
2 mg.

250 mg.

P o ta s s iu m d ih y d ro g e n
p h o s p h a te : KHgPO^s

250 mg.

D uring t h e a d m i n i s t r a t i o n o f t h e i s o to p e s i t was n e c e s s a r y t o
grow t h e p l a n t s i n a fume h ood, t o g u a rd a g a i n s t an y h e a l t h h a z a rd
<
1i

a r i s i n g fro m t h e p o s s i b l e l i b e r a t i o n of C Og th ro u g h r e s p i r a t i o n .
A s o u rc e o f l i g h t was s u p p lie d by tw o 3 6 - in c h , 3 0 -w a tt f lu o r e s c e n t
tu b e s and a 1 0 0 -w a tt in c a n d e s c e n t b u lb , p la c e d a b o u t 14 In c h e s ab o v e
t h e to p s o f t h e p l a n t s .

The l i g h t i n t e n s i t y a t t h e l e v e l o f t h e u p p e r

le a v e s was fo u n d t o b e i n t h e ra n g e o f 200 to 250 f o o t - c a n d l e s .

The

l i g h t was l e f t on a p p ro x im a te ly 12 h o u rs o u t o f 24 w h ile t h e p l a n t s
w ere g ro w in g , and d i s t i l l e d w a te r was added a s r e q u i r e d .

M e th y la tio n E x p e rim e n ts w ith D, L -M eth io n in e
I n a tte m p tin g t o e s t a b l i s h t h e o r i g i n o f t h e n i c o t i n e m e th y l
c a rb o n by t r a n s m e t h y l a t i o n i t a p p e a re d l o g i c a l f i r s t to t i y m e th y ll a b e l l e d m e th io n in e a s a s o u rc e o f th e m e th y l g ro u p .

T h is compound

h a s lo n g b e e n e s t a b l i s h e d a s a m eth y l d o n o r i n th e a n im a l ( l ) » and
i t h a s b e e n shown by d o u b le l a b e l l i n g urith C^4 and d e u te riu m t h a t t h e
m e th y l g ro u p o f m e th io n in e i s t r a n s f e r r e d a s a n e n t i t y , w ith o u t i n t e r ­
m e d ia te o x i d a tio n an d r e d u c tio n ; i n o t h e r w o rd s, a t r u e tr a n s m e th y l­
a tio n (1 5 ).
U ptake of m e th io n in e fro m s o l u t i o n .

B e fo re a d m in is te r in g t h e

r a d i o a c t i v e m e th io n in e t o t h e p l a n t s , i t was n e c e s s a r y t o a s c e r t a i n
t h e b e s t m ethod o f in c o r p o r a ti n g t h e compound i n t o t h e p l a n t .

It

a p p e a r e d , on a n a p r i o r i b a s i s , t h a t t h e m ost a d v an tag eo u s m ethod, i f
f e a s i b l e , w ould b e t o a llo w t h e p la n t t o ab so rb th e m e th io n in e fro m
aq u eo u s s o l u t i o n th ro u g h t h e r o o t s , inasm uch a s n i c o t i n e s y n th e s is h a s
b e e n shown t o ta k e p la c e i n t h e r o o ts ( 1 6 ) .

F i r s t , how ever, a s u i t a b l e

a n a l y t i c a l p r o c e d u re h ad t o b e found i n o r d e r t o d e te rm in e (1 ) t h e
r a t e o f u p ta k e by t h e p l a n t , and (2 ) w h e th e r th e m e th io n in e was de­
s tr o y e d by m icro o rg a n ism s i n t h e medium b e f o r e t h e r e was o p p o r tu n ity
f o r i t s a b s o r p t i o n by t h e p l a n t .

The m ethod o f a n a l y s is ch o sen was

t h a t o f M cCarthy and S u l l i v a n ( 1 7 ) , a c o lo r im e t r ic m ethod b a s e d on t h e
r e a c t i o n o f m e th io n in e w ith n i t r o p r u s s i d e to g iv e a re d c o lo u r .
To t e s t t h e u p ta k e o f m e th io n in e t h r e e m illig ra m s o f t h e amino
a c i d was a d m in is te r e d i n t h e n u t r i e n t s o l u t i o n t o each o f s ix p l a n t s .

A t t h e end o f t w e n t y - f o u r h o u rs t h e p l a n t s w ere rem oved fro m t h e s o lu ­
t i o n and t h e r o o t s w ere r i n s e d i n t o t h e r e s i d u a l medium, w hich was
t h e n a n a ly z e d f o r m e th io n in e .

I t was fo u n d t h a t i n t h e tw e n ty - f o u r

h o u r p e r io d 67 t o 80^ o f t h e m e th io n in e was a b s o rb e d .

No e v id e n c e o f

b a c t e r i a l grow th i n t h e medium was o b se rv e d d u rin g t h i s tim e , b u t a s
a c h ec k a n e x p e rim e n t was c a r r i e d o u t i n w hich t h e medium was in o c u ­
l a t e d by t h e a d d i t i o n o f a few r o o t f ra g m e n ts .

A f t e r tw e n ty - f o u r h o u rs

t h e a n a l y s is showed no d e t e c t a b l e l o s s o f m e th io n in e a s com pared t o
u n in o c u la te d c o n t r o l s .

Even a f t e r t h e sev en day a d m i n i s t r a t i o n p e r io d

s u b s e q u e n tly a d o p te d t h e n u t r i e n t s o lu t i o n s d id n o t e x h i b i t t h e t u r ­
b i d i t y and s lim y c o n s is te n c y o r odour c h a r a c t e r i s t i c o f e x te n s iv e
b a c t e r i a l g ro w th .
I s o l a t i o n and p u r i f i c a t i o n o f n i c o t i n e .

The p ro c e d u re u s e d

th ro u g h o u t t h i s work f o r t h e i s o l a t i o n an d p u r i f i c a t i o n o f n i c o t i n e was
a s fo llo w s s

The p l a n t s t o b e w orked u p w ere removed from t h e n u t r i e n t

s o l u t i o n and t h e r o o ts w ere r in s e d w ith d i s t i l l e d w a te r , t h e e x c e ss
b e in g b l o t t e d o f f w ith c h e e s e c lo th .

The p la n t s w ere th e n c u t i n t o

s m a ll p i e c e s w ith s c i s s o r s and d r ie d u n d e r h e a t lam ps a s r a p i d l y a s
p o s s ib le .

Toward th e end o f t h e d x y in g p e r io d th e te m p e r a tu re was m ain­

t a i n e d a t 100° C. f o r a b o u t an h o u r.

T/Vhen t h e m a t e r i a l had b e e n d r ie d

i t was ground f i n e l y i n a m o rta r , m ixed w ith ab o u t o n e - te n th o f i t s
w e ig h t o f c a lc iu m h y d ro x id e , and s t e a m - d i s t i l l e d .

The d i s t i l l a t e was

c o n c e n tr a te d , and p u r i f i c a t i o n o f th e a l k a l o i d was a c c o m p lish e d by two
s u c c e s s iv e a z e o t r o p i c d i s t i l l a t i o n s from a l k a l i n e medium a s d e s c r ib e d

by S m ith (1 8 )*

T h is was f o llo w e d by rem oval o f th e w a te r fro m t h e

a c i d d i s t i l l a t e u n d e r red u c ed p r e s s u r e , th e n i c o t i n e c r y s t a l l i z i n g
o u t a s t h e h y d r o c h lo r id e .

T h is was d is s o lv e d i n m eth an o l p lu s a l i t t l e

w a t e r , an d a s a t u r a t e d m e th a n o lic s o l u t i o n o f p i c r i c a c id was added i n
ex cess.

A f t e r t h e s o l u t i o n h ad s to o d f o r a s h o r t tim e t h e p r e c i p i t a t e

o f n i c o t i n e d i p i c r a t e was f i l t e r e d o f f , w ashed w ith m e th a n o l, and r e ­
c r y s t a l l i z e d fro m h o t w ater*

(M* P# 2 2 4 -5 ° C .; re c o rd e d v a lu e 224° C.

( 1 9 ).)
A dm inl s t r a t j on o f C^ - m e t h y l D, L-met h io n in e *

I n p r e lim in a r y

e x p e rim e n ts w ith t h e a d m i n i s t r a t i o n o f r a d io - m e th io n in e t h e to b a c c o
p l a n t s w ere a llo w e d t o grow i n c o n ta c t w ith t h e is o to p e f o r a p e r io d
c

o f tw e n ty - f o u r h o u rs*

Two m illig ra m s o f m e th io n in e , c o n ta in in g 10

c o u n ts p e r m in u te ^ , was g iv e n to each p l a n t .

On w orking u p t h e p l a n t

m a t e r i a l , how ever, o n ly v e r y low a c t i v i t y (a b o u t 25 c .p .m . o v e r b a c k ­
ground a t i n f i n i t e t h i c k n e s s ) c o u ld b e d e m o n stra te d i n t h e n i c o t i n e
p ic ra te .

The tim e i n c o n ta c t w ith t h e is o to p e w as, t h e r e f o r e , e x te n d e d

t o se v e n d a y s , u s in g t h e same q u a n t i t y o f a d m in is te r e d m e th io n in e .

The

n i c o t i n e p i c r a t e i s o l a t e d a f t e r t h i s lo n g e r i n t e r v a l was found t o
p o s s e s s c o n s id e r a b ly g r e a t e r a c t i v i t y , h a v in g a maximum s p e c i f i c a c t i v i t y ,
c a l c u l a t e d f o r z e ro sam ple t h i c k n e s s ^ , o f t h e o r d e r o f 4 ,0 0 0 c .p .m . p e r
m illim o le *

One r e c r y s t a l l i z a t i o n was found s u f f i c i e n t t o b r in g t h e

3 T h is was d e te rm in e d by c o u n tin g a n i n f i n i t e l y t h i n l a y e r . A ll
c o u n ts w e re made on a N u c le a r In s tru m e n t & Chem ical C orp. endwindow c o u n te r on t h e to p s h e l f o f th e c o u n te r a s se m b ly . The
o v e r a l l e f f i c i e n c y was 7%.
^ F o r c a l c u l a t i o n s , s e e A ppendix I .

10-

a c t i v i t y t o a c o n s ta n t v a l u e .

The se v e n -d a y a d m i n i s t r a t i o n p e r io d

w as u s e d i n a l l s u b s e q u e n t e x p e rim e n ts .
I n o r d e r t o e s t a b l i s h w h e th e r o r n o t t h e r a d i o a c t i v i t y i n th e
n i c o t i n e was l o c a l i z e d i n t h e m e th y l c a rb o n i t was n e c e s s a r y to c le a v e
t h e m e th y l g ro u p from t h e n i c o t i n e m o le c u le , and o b t a in i t i n t h e fo rm
o f a s o l i d compound s u i t a b l e f o r c o u n tin g .

The f i r s t m ethod t r i e d f o r

t h e rem o v al o f t h e m e th y l group was t h a t o f L a ib l i n ( 2 0 ) , who r e p o r te d
h a v in g i s o l a t e d m eth y lam in e a f t e r c o m p le te o x i d a tio n o f n i c o t i n e by
a l k a l i n e p e rm a n g a n a te .

S e v e ra l a tte m p ts t o d u p l i c a t e t h i s r e s u l t ,

u s in g o n e gram o f n i c o t i n e , p ro v ed u n s u c c e s s f u l.

As th e q u a n t i t i e s

a v a i l a b l e i n t h i s work w e re much s m a l l e r , i t was n o t c o n s id e re d o f
p r a c t i c a l v a lu e t o c o n tin u e w ith t h i s m ethod, an d i t was ab an d o n ed .
G r e a te r s u c c e s s was e n c o u n te re d u s in g a p ro c e d u re o r i g i n a l l y d e s ­
c r ib e d b y H e rz ig and Meyer (2 1 ) and l a t e r a d a p te d by P r e g l (2 2 ) a s a
g e n e r a l m icro -m eth o d f o r m e th y l- and e th y l-im in o d e te r m in a tio n s .

T h is

te c h n iq u e in v o lv e s h e a t i n g n i c o t i n e w ith h y d r io d ic a c id t o form t h e
t e r n a r y ammonium s a l t and th e n s p l i t t i n g o f f m eth y l io d id e a t a
h i g h e r te m p e r a tu r e .

I n t h e P r e g l m ethod th e m eth y l io d id e i s sw ept

th ro u g h a l c o h o l i c s i l v e r n i t r a t e and th e r e s u l t i n g p r e c i p i t a t e of s i l v e r
io d id e i s w e ig h e d , b u t i n t h e p r e s e n t work i t was n e c e s s a r y , o f c o u rs e ,
t o r e c o v e r t h e m e th y l r a d i c a l .

Simmonds and co -w o rk ers ( 2 3 ) , who d e -

m e th y la te d t i s s u e p r o t e i n s w ith h y d r io d ic a c i d , r e a c te d m e th y l io d id e
w ith trim e th y la m in e t o form tetram ethylam m onium i o d id e , a s o l i d .

T h is

p r i n c i p l e was m o d ifie d i n o u r work by t h e s u b s t i t u t i o n o f t r i e t h y l a m i n e ,

-1 1 -

IO

CD

111

o:
3
O
IL.

■which we fo u n d t o r e a c t q u a n t i t a t i v e l y w ith m eth y l io d id e t o fo rm
m e th y l trie th y la m m o n iu m i o d i d e , and w hich h a s t h e a d v a n ta g e o f much
lo w e r v o l a t i l i t y a s com pared t o trim e th y la m in e *
The d e m e th y la tio n a p p a r a tu s u s e d , a m o d ifie d form o f t h a t d e s ­
c r ib e d b y P r e g l * a s shown i n F ig u r e I I #

The m a t e r i a l t o b e d a m e th y la te d

( t h e e q u i v a l e n t o f 50 mg# o f n i c o t i n e was u s e d ) i s p la c e d i n t h e r e ­
a c t i o n v e s s e l A, t o g e t h e r w ith 45 mg* o f ammonium i o d i d e , two d ro p s o f
5% g o ld c h l o r i d e s o l u t i o n and 3 ml# o f h y d r io d ic a c id ( s p e c i f i c g r a v i t y
1 * 7 ).

The g a s-w a sh in g v e s s e l B c o n ta in s 1*5 m l. o f t h e C dS O ^N s^^O g

s o l u t i o n d e s c r ib e d b y P r e g l, t o rem ove io d in e and h y d ro g en io d i d e .

The

r e c e i v e r C c o n ta in s a 5% e t h a n o l i c s o l u t i o n o f t r i e t h y l a m i n e , and i s
c o o le d i n a CC ^-m ethyl c e l l o s o l v e b ath #
I t was n o t fo u n d p r a c t i c a l t o u s e t h e n i c o t i n e p i c r a t e a s such f o r
d e m e th y la tio n , a s d i f f i c u l t y was e n c o u n te re d i n g e t t i n g i t i n t o s o lu ­
tio n *

The p i c r a t e w a s, t h e r e f o r e , d is s o lv e d i n d i l u t e sodium h y d ro x id e

and th e n i c o t i n e was re c o v e re d by a z e o tr o p ic d i s t i l l a t i o n a s b e fo re *
The a c i d d i s t i l l a t e was t h e n c o n c e n tr a te d , t h e c o n c e n tr a tio n b e in g com­
p l e t e d i n t h e r e a c t i o n f l a s k A, w hich was d is c o n n e c te d fro m t h e r e ­
m a in d e r o f t h e d e m e th y la tio n a p p a ra tu s *
A f t e r t h e a d d i t i o n o f th e o t h e r r e a c t a n t s a s d e s c r ib e d ab o v e, t h e
f l a s k A was re c o n n e c te d and h e a te d

i n t h e c u p r ic o x id e b a th *

A slo w

® H e a tin g w ith h y d r io d ic a c id c a u s e s a g r a d u a l e tc h in g o f t h e g l a s s ,
e v e n t u a l l y r e n d e r in g i t f r a g i l e . I t i s n o t a d v is a b le t o u s e t h e
v e s s e l m ore th a n s i x o r sev en tim e s#

12-

s tr e a m o f n i t r o g e n -was p a s s e d i n th ro u g h t h e s id e - a r m D, an d t h e b a th
te m p e r a tu r e was r a i s e d t o 200° C. i n 20 to 25 m in u tes*

By t h i s tim e

some co n d en sed h y d r io d ic a c i d h ad c o l l e c t e d i n t h e TJ-tube tr a p *

The

te m p e r a tu r e was th e n r a i s e d s lo w ly t o 3 5 0 -6 0 ° C*, and h e ld t h e r e f o r
45 m in u te s*

The h e a t was rem oved and t h e sw eeping c o n tin u e d u n t i l

t h e r e a c t i o n f l a s k h ad co o led *

The r e c e iv i n g tu b e was th e n t a k e n o f f

a n d t h e d e l i v e r y t i p r in s e d w ith a s m a ll amount o f e th a n o l*

The

a l c o h o l i c s o l u t i o n was sh a k e n , co rk ed t i g h t l y , and a llo w e d t o s ta n d
o v e r - n i g h t a t room te m p e ra tu re *

I n t h e m orning m ost o f th e a lc o h o l was

rem oved by h e a t i n g , and t h e l a s t o f t h e s o lv e n t and t h e ex cess am ine
w e re e v a p o ra te d i n a vacuum d e s i c c a t o r .

A w h ite c r y s t a l l i n e r e s id u e

o f m e th y l trie th y la m m o n iu m i o d id e was re c o v e re d i n 55 -7 0 ^ o f t h e o r e t i c a l
y i e l d , b a s e d on n i c o t i n e *

(.Anal* C a lc d . f o r

I , 52*20.

Pounds

I , 5 2 .3 1 .)
T h is q u a te r n a r y compound was c o u n te d u n d e r th e same c o n d itio n s a s
t h e n i c o t i n e p i c r a t e and fo u n d to p o s s e s s a c t i v i t y .

The maximum

s p e c i f i c a c t i v i t y was c a l c u l a t e d on a m illim o le b a s i s u s in g t h e fo rm u la
shown i n A ppendix I .

The f a c t o r fb ’ u s e d was t h e same a s t h a t o b ta in e d

fro m t h e p i c r a t e c u rv e ; t h i s p ro c e d u re in tr o d u c e s some e r r o r b u t t h e
ira g n itu d e o f t h e e r r o r p ro b a b ly i s low ( 2 4 ) .

The maximum s p e c i f i c

a c t i v i t i e s o f t h e n i c o t i n e p i c r a t e and m eth y l trieth y lam m o n iu m io d id e
sam p les o b ta in e d i n t h r e e e x p e rim e n ta l ru n s a r e shown i n T a b le I I .

13-

TABLE I I
LOCATION OF RADIOACTIVITY IN THE NICOTINE MOLECULE AFTER
c 14- m ethionine a d m in istra tio n
Maximum s p e c i f i c a c t i v i t y
( c o u n ts p e r m in u te p e r m illim o le )
E x p e rim e n t No.
N ic o tin e D i p i c r a t e

Q u a te rn a ry I o d id e

1 . (39 p l a n t s )

4 .2 8 x 103

4 .4 2 x 103

2 . (40 p l a n t s )

4 .2 4 x 103

3 .9 2 x 103

. 3 . (39 p l a n t s )

7 .5 3 x 103

7 .6 0 x 103

I t c an b e s e e n fro m t h e s e f ig u r e s t h a t t h e re c o v e ry o f t h e n i c ­
o t i n e a c t i v i t y a s t h e m eth y l gro u p o f t h e q u a te rn a ry io d id e ran g ed
fro m a b o u t 93 t o 103%, i n d i c a t i n g t h a t , w ith i n e x p e rim e n ta l e r r o r , a l l
t h e a c t i v i t y i s l o c a l i z e d i n t h e m eth y l carb o n of t h e n i c o t i n e .

I t is

n o t c l e a r w hat c a u se d t h e r e l a t i v e l y h ig h v a lu e s o b ta in e d i n E xperim ent
3 , . a s t h e t r e a tm e n t was k e p t a s u n ifo rm a s p o s s i b l e i n a l l th e
e x p e r im e n ts .

The e x p la n a tio n may l i e , a g a in , i n s e a s o n a l i n f l u e n c e s ,

a s t h e p l a n t s w ere grown a t d i f f e r e n t tim e s of t h e y e a r .
M e th y la tio n E x p erim en ts w ith F orm ate
On t h e b a s i s o f r e c e n t e x p e rim e n ts fo rm a te , o r some o n e -c a rb o n
compound w hich h a s b ee n d e s ig n a te d a s " fo rm a te ” , a p p e a rs to h av e an
im p o r ta n t r o l e i n m e th y la tio n r e a c t i o n s ,

du V igneaud and h i s c o -w o rk e rs ,

i n e x p e rim a n ts w ith r a t s u s in g C1 4 , have r e c e n t ly shown t h a t fo rm a te can
a c t a s a p r e c u r s o r o f t h e m eth y l c a rb o n o f c h o lin e ( 2 5 ) .

-1 4 -

I n v ie w o f t h e

f in d i n g s o f Sakami (2 6 ) and o f W e issb ach , Elwyn and S p rin s o n ( 2 7 ) , i t
i s p r o b a b le t h a t t h e c o n v e rs io n ta k e s p la c e by way o f s e r i n e , a s shown
s c h e m a tic a lly below :
HC*OOH 4

-C00H

HOC*Hr,-CH-COOH
fe ,

"F o rm ate"

G ly c in e

—

S e r in e

(H gW -C H g-^H gO H )_________

E th a n o la m in e

£( C ^ ^ N - C H g - C ^ g 6O H jI OH

C h o lin e

I t i s p r o b a b le t h a t o th e r i n t e r m e d i a te s , a s y e t unknown, a l s o e n te r
i n t o t h i s tr a n s f o r m a t i o n .

C h o lin e can t r a n s f e r i t s m eth y l c a rb o n ,

th ro u g h b e t a i n e , t o m e th io n in e ( 8 ) .

On th e o th e r h and, m e th io n in e can

s u p p ly a m eth y l c a rb o n t o s a r c o s in e ( 2 8 ) , and M ackenzie (2 8 ) h a s shown
t h a t t h e m e th y l g ro u p o f s a r c o s in e can b e o x id iz e d t o fo rm a te by r a t
l i v e r h o m o g en ates.

Most o f t h i s fo rm a te i s s u b s e q u e n tly o x id iz e d

f u r t h e r t o c a rb o n d io x id e , b u t i t i s c o n c e iv a b le t h a t p a r t o f i t e n te r s
t h e fo rm a te p o o l o f t h e o rg an ism and i s re c o n v e rte d in t o th e m ethyl
c a rb o n o f m e th io n in e by th e r e a c t i o n s shown ab o v e , i f c o n d itio n s e x i s t
u n d e r w hich s y n th e s i s of t h e m e th y l group i s ta k in g p l a c e .
U nder n o rm al c o n d itio n s i n t h e p l a n t t h e l a b i l e m eth y l groups m ust
b e s y n th e s iz e d fro m s im p le r compounds, a s an exogenous s o u rc e o f l a b i l e
m e th y ls i s u n a v a i l a b l e .

I f th e s i t u a t i o n i s an alo g o u s t o t h a t in t h e

a n im a l, fo rm a te a d m in is te r e d t o a p la n t w ould b e in c o r p o r a te d i n p a r t
i n t o l a b i l e m eth y l g ro u p s and i n t o v a r io u s m e th y la te d com pounds.

15-

That

s u c h i s t h e c a s e h a s b een shown by Kirkwood and M arion ( 7 ) , who f e d
C^4 - f o r m a te t o s p r o u tin g b a r l e y and i s o l a t e d c h o lin e and h o rd e n in e
c o n ta in in g

i n t h e m eth y l groups#

F o rm a te may b e in v o lv e d i n m e th y la tio n r e a c t i o n s i n two w ays:
( 1 ) by c o n v e rs io n f i r s t t o l a b i l e m eth y l g ro u p s, w hich a r e t h e n t r a n s ­
f e r r e d a s s u c h , o r (2 ) by a c t i n g as a m ore d i r e c t p r e c u r s o r , p o s s ib ly
a r i s i n g fro m ’l a b i l e ' 1 m e th y l gro u p s by o x id a tio n #

I t was f e l t t h a t

i t m ig h t b e p o s s i b l e t o com pare t h e fo rm a tio n o f t h e n i c o t i n e m eth y l
c a rb o n fro m f o rm a te w ith t h a t from m e th io n in e , and th u s o b ta in some
i n d i c a t i o n a s t o w hich i s t h e more d i r e c t p re c u rs o r#

T here a r e o b v io u s­

l y s e v e r a l c o m p lic a tio n s i n any such e x p e rim e n t; i t i s n e c e s s a r y t o a s ­
sume t h a t t h e r a t e s o f u p ta k e a r e ab o u t t h e sam e, t h a t t h e s i z e o f t h e
p o o ls i n t h e p l a n t rem ains c o n s ta n t, and t h a t t h e r e i s no v a r i a t i o n i n
th e r a t i o o f n i c o t i n e form ed d u rin g t h e p e r io d o f t h e ex p erim en t to
t h a t p r e v io u s ly s to r e d i n th e p la n t#

I t can b e se e n from T a b le I I t h a t

t h e r e i s i n r e a l i t y a v a r i a t i o n i n one o r more o f th e s e f a c t o r s , t h e
s p e c i f i c a c t i v i t y o f t h e n i c o t i n e i n E xperim ent 3 b e in g ab o u t 75# h ig h e r
th a n t h a t i n t h e o th e r two ex p erim en ts*

I t f o llo w s , t h e r e f o r e , t h a t

an y ch an g e i n t h e a c t i v i t y of t h e n i c o t i n e s u b se q u e n t t o t h e a d m in is ­
t r a t i o n o f f o r m a te , a s com pared to t h a t fo llo w in g t h e a d m i n i s t r a t i o n o f
m e th io n in e , w ould h av e to be of t h e o r d e r of s e v e r a l hundred p e r c e n t
b e f o r e any i n f e r e n c e s a s t o pathw ays c o u ld b e drawn#
U ptake o f fo rm a te fro m s o l u t i o n *

As i n t h e e x p e rim e n ts w ith

m e th io n in e , i t was d e s i r a b l e to g a in some id e a o f t h e r a t e o f u p ta k e

-1 6 -

o f f o rm a te fro m s o l u t i o n by t h e p la n ts #

U n f o r tu n a te ly , how ever, no

m icrom ethod f o r f o rm a te co u ld b e found w hich was co n d u c iv e t o r e s u l t s
a s a c c u r a t e a s w ere o b ta i n a b l e i n t h e m e th io n in e ex p erim en ts#

The

p ro c e d u re f i n a l l y a d o p te d was t h a t o f G ra n t ( 2 9 ) , i n w hich m e rc u ric
c h l o r i d e i s re d u c e d b y fo rm a te to m ercurous c h lo r id e ; t h e l a t t e r i s
i n t u r n r e a c t e d w ith a p h o sp hom olybdic- p h o s p h o tu n g s tic a c id t o
p ro d u c e a b lu e c o l o u r .

I n a p p ly in g t h i s p ro c e d u re to fo rm a te i n t h e

p l a n t n u t r i e n t s o l u t i o n s i t was found t h a t t h e ammonium s a l t i n th e
medium c a u s e d p r e c i p i t a t i o n on t h e a d d i t i o n o f t h e m e rc u ric c h l o r id e
r e a g e n t; h en c e i t was n e c e s s a r y t o remove t h e ammonia by f i r s t m aking
t h e s o l u t i o n a l k a l i n e and b o i l i n g .

An a d d i t i o n a l c o m p lic a tio n was th e

p r o g r e s s i v e d ev elo p m en t o f t u r b i d i t y a f t e r fo rm a tio n o f t h e b lu e c o lo u r ,
f o llo w e d b y t h e s e p a r a t i o n of a g e la tin o u s p r e c i p i t a t e ; even t h e m ost
e x h a u s tiv e w ash in g o f t h e m ercurous c h lo r id e f a i l e d t o p r e v e n t t h i s #
N e v e r t h e l e s s , i t was fo u n d p o s s i b l e t o o b ta in a t l e a s t s e m i- q u a n tita ­
t i v e a n a ly s e s on fo rm a te i n t h e n u t r i e n t medium#

Over a f o r t y - e i g h t

h o u r p e r io d to b a c c o p l a n t s w ere found t o a b so rb fo rm a te from a s o l u t i o n
o f one m illig r a m o f t h e sodium s a l t i n 50 ml# o f t h e n u t r i e n t medium; a
q u a n t i t y o f th e o r d e r o f h a l f t h e t o t a l p r e s e n t was ta k e n up i n t h i s
tim e , t h e amount v a r y in g somewhat w ith p l a n t siz e #

No l o s s by b a c t e r i a l

a c t i o n , i n f l a s k s in o c u la te d w ith p l a n t ro o t f ra g m e n ts , co u ld be ob­
s e rv e d i n th e i n t e r v a l .

I n su b se q u e n t e x p e rim e n ts, a s i n th e c a s e o f

t h o s e w ith m e th io n in e , no s ig n s o f e x te n s iv e b a c t e r i a l grow th w ere
o b se rv e d d u r in g a se v en -d ay a d m i n is tr a tio n p e r io d , and a m ic ro s c o p ic
e x a m in a tio n o f th e f i l t r a t e from t h e r e s i d u a l medium a t t h e end o f t h i s

-1 7 -

tim e c o n firm e d "th is o b s e rv a tio n *

To check f u r t h e r th e u p ta k e of

fo rm a te by t h e p l a n t s a d e te r m in a tio n was made d u rin g one o f th e ru n s
by d r y in g and c o u n tin g an a l i q u o t o f t h e n u t r i e n t s o lu tio n *

In le s s

th a n t h r e e days fro m t h e s t a r t o f t h e r u n , o n ly ab o u t one p e r c e n t of
t h e a d m in is te r e d r a d i o a c t i v i t y c o u ld b e a c c o u n te d f o r i n t h i s way*
Thus by t h i s tim e , a t m o st, v i r t u a l l y a l l o f t h e fo rm a te had b een ab ­
s o rb e d b y t h e p la n t*
14
Admini s t r a t i on o f C - f o r m a te sodium s a l t *

I n a d m in is te r in g r a d i o ­

a c t i v e fo rm a te t o t h e to b a c c o p l a n t s i t was d e s i r a b l e , f o r p u rp o se s o f
c o m p a riso n , t o m a in ta in t h e c o n d itio n s o f tr e a tm e n t a s s i m i l a r t o th o s e
o f t h e m e th io n in e e x p e rim e n ts a s p o s s ib le *

I t was n o t f e a s i b l e , o f

c o u r s e , t o m a in ta in c o n s ta n t t h e p re v io u s tr e a tm e n t o f t h e p l a n t s , b e ­
c a u s e o f s e a s o n a l v a r i a t i o n s i n l i g h t , te m p e r a tu r e , and o th e r f a c to r s *
D i l u t i o n o f a sam p le o f C ^ - f o r m a t e was c a r r i e d o u t so t h a t 0*91 mg*
o f t h e sodium s a l t , c o n ta in in g 10

C

c o u n ts p e r m in u te was g iv e n to each

p l a n t ; t h e m o la r q u a n t i t y and th e t o t a l c o u n ts p e r m in u te w ere th u s t h e
same a s t h a t o f t h e m eth io n in e*

The p l a n t s w ere m a in ta in e d u n d e r t h e

same grow ing c o n d itio n s a s d e s c rib e d p r e v io u s ly f o r th e s e v e n -d a y p e rio d *
At t h e end o f t h i s tim e th q y w ere worked up a s b e f o r e , and n i c o t i n e was
a g a in i s o l a t e d a s t h e d i p i c r a t e .

A f te r c o u n tin g , t h e n i c o t i n e was r e ­

c o v e re d and d e m e th y la te d a s i n th e m e th io n in e e x p e rim e n ts and t h e r e s u l t ­
in g q u a te r n a ry io d id e was counted*
i n two ru n s o f p l a n t s .

-1 8 -

T a b le I I I shows t h e v a lu e s o b ta in e d

TABLE I I I
LOCATION OF RADIOACTIVITY IN THE NICOTINE MDIECULE AFTER
FORMATE ADMINISTRATION

Maximum s p e c i f i c a c t i v i t y
( c o u n ts p e r m in u te p e r m illim o le )
E x p e rim e n t No#
N ic o tin e D ip ic r a te

Q u a te rn a ry I o d id e

1 . (25 p l a n t s )

9 .6 x 102

9 .3 x 102

2 . (29 p l a n t s )

4 .4 x 102

4 .1 x 102

I t i s e v id e n t fro m t h e s e r e s u l t s t h a t t h e r e i s some in c o r p o r a t io n
o f t h e fo rm a te c a rb o n i n t o n i c o t i n e , and t h a t m ost i f n o t a l l o f t h e
a c t i v i t y i s i n t h e m e th y l carbon#

Hie low count i n t h i s c a s e i n t r o ­

d u ces r e l a t i v e l y l a r g e r e r r o r s , and i t i s n o t p o s s i b l e t o compare t h e
s p e c i f i c a c t i v i t i e s o f th e n i c o t i n e and t h e q u a te rn a ry io d id e w ith t h e
d e g r e e o f a c c u r a c y o b ta in a b le i n t h e m e th io n in e experim ents#
A co m p a riso n o f t h e v a lu e s re c o rd e d in T a b le I I I w ith th o s e of
T a b le I I r e v e a l s t h a t t h e fo rm er a r e c o n s id e ra b ly low er#

The lo w e s t

s p e c i f i c a c t i v i t y o b ta in e d f o r th e m eth y l carb o n i n t h e m e th io n in e
e x p e rim e n ts ( 1 1 , 2) i s o v e r f o u r tim e s a s h ig h a s t h e h ig h e r o b ta in e d
a f t e r fo rm a te a d m i n i s t r a t i o n ( 1 1 1 ,1 ) , -whereas a t t h e o th e r extrem e t h e
v a l u e I I f 3 i s n e a r l y n i n e t e e n tim e s i n ex cess o f 1 1 1 ,2 .

I t ap p ears,

t h e r e f o r e , t h a t when C ^ - f o r m a te i s f e d to N# r u s t i c a t h e s p e c i f i c
a c t i v i t y o f th e n i c o t i n e m eth y l c a rb o n i s o l a t e d i s a b o u t one o r d e r o f
m a g n itu d e lo w er th a n t h a t -which r e s u l t s a f t e r f e e d in g C14-m e th y l
m e th io n in e , u n d e r c o n d itio n s m a in ta in e d a s n e a r ly a s p o s s i b l e c o n s ta n t#

-1 9 -

DISCUSSION
The r e s u l t s o f t h e s e i n v e s t i g a t i o n s show t h a t t h e m eth y l carb o n
o f m e th io n in e c a n a c t a s a p r e c u r s o r o f t h e m eth y l carb o n o f n i c o t i n e
i n v i v o , and t h a t t o an a p p r e c ia b ly l e s s e r e x te n t fo rm a te can a l s o a c t
as a p r e c u r s o r *

The d a ta p r e s e n te d c o n s t i t u t e th e f i r s t d i r e c t chem i­

c a l e v id e n c e t h a t t r a n s m e t h y la ti o n ta k e s p la c e i n h ig h e r p la n ts *

The

lo w e r s p e c i f i c a c t i v i t i e s o b ta in e d u s in g fo rm a te s e rv e a s an i n d ic a ­
t i o n t h a t t h e m e th io n in e m eth y l g roup i s n o t o x id iz e d t o fo rm a te and
th e n re d u c e d d u rin g t h e t r a n s f e r o f t h e m eth y l carb o n to n i c o t i n e .

If

such a n o x id a tio n and r e d u c tio n does o c c u r , t h e a d m in is t r a tio n o f
fo rm a te s h o u ld h av e r e s u l t e d i n a s p e c i f i c a c t i v i t y f o r t h e n i c o t i n e
m eth y l a t l e a s t a s h ig h a s t h a t o b ta in e d fo llo w in g th e f e e d in g of
m e th io n in e *

I t can b e i n f e r r e d from o u r r e s u l t s t h a t t h e f u n c tio n o f

fo rm a te i n t r a n s m e t h y l a t i o n i s , a l t e r n a t i v e l y , t h a t o f a p r e c u r s o r i n
t h e b i o s y n t h e s i s o f l a b i l e m eth y l groups such as t h a t o f m e th io n in e .
I t i s p r o b a b le , a s p o in te d o u t e a r lie r ,* t h a t fo rm a te i s c o n v e rte d to
th e l a b i l e m eth y l by way o f s e r i n e i n some m anner a s y e t in c o m p le te ly
u n d e r s to o d .
The f i n a l p ro o f t h a t t h e m e th io n in e m eth y l group i s t r a n s f e r r e d as
an e n t i t y t o n i c o t i n e m ust a w a it t h e c o m p le tio n o f e x p e rim e n ts in v o lv ­
in g d o u b l e - l a b e l l i n g w ith c arb o n -1 4 and d e u te riu m .

T hese e x p e rim e n ts

a r e now b e in g co n d u cted i n t h i s l a b o r a t o r y , and th e r e s u l t s w i l l b e
fo rth c o m in g a t a l a t e r d a t e .

-2 0 -

I t i s somewhat d i f f i c u l t t o r e c o n c il e th e r e s u l t s o f o u r e x p e r i­
m en ts w ith th o s e o f Kirkwood and M arion ( 7 ) , who o b ta in e d c o n s id e r a b le
i n c o r p o r a t i o n o f fo rm a te c arb o n i n t o t h e m eth y l g ro u p s o f h o rd e n in e b u t
o n ly n e g l i g i b l e i n c o r p o r a t i o n from t h e m eth y l carb o n o f c h o li n e .

How­

e v e r Wing ( 3 0 ) , i n p r e lim in a r y e x p e rim e n ts i n t h i s l a b o r a t o r y , h a s
fo u n d e v id e n c e t h a t c h o lin e i s s u b je c t t o q u i t e r a p id d e s t r u c t i o n hy
b a c t e r i a l a c t i o n i n t h e p l a n t n u t r i e n t s o l u t i o n , and a s th e above
a u th o r s do n o t r e f e r t o an y p r e c a u tio n s t a k e n to r e s t r i c t b a c t e r i a l
g ro w th i t i s p o s s i b l e t h a t much o f t h e c h o lin e was d e s tro y e d b e f o r e i t
c o u ld b e a b s o rb e d by th e b a r l e y .

I t would a p p e a r t h a t f u r t h e r i n v e s t i ­

g a t i o n i s i n d i c a t e d , p o s s i b l y in v o lv in g a d i r e c t com parison of t h e
e f f e c t s o f c h o lin e and m e th io n in e u n d e r s t e r i l e c o n d iti o n s , i n t h e same
s p e c ie s ,
Dawson ( 4 ) h a s shown by means o f g r a f t i n g ex p e rim e n ts t h a t i n s p e c ie s
w h ich p ro d u c e n o m i c o t i n e , su ch a s N, g l u t i n o s a an d H. g la u c a , t h i s
a l k a l o i d i s p ro d u c ed n o t i n t h e r o o t s , b u t i n t h e le a v e s , by d em eth y lat i o n o f n i c o t i n e t r a n s l o c a t e d from t h e r o o t s .

On t h e b a s is o f th e s e

f i n d i n g s h e h a s ad v an ced th e p l a u s i b l e h y p o th e s is t h a t a tra n s m e th y la ­
t i o n r e a c t i o n t a k e s p la c e i n t h e l e a f , i n which n i c o t i n e s e rv e s a s t h e
i

m e th y l d o n o r and some a s y e t unknown compound o r compounds as t h e
a c c e p to r.

To e x p la in t h e f a c t t h a t many s p e c ie s , e . g . H. tabacum and

N. r u s t i c a , c o n ta in l i t t l e

o r no n o m ic o t in e h e s u g g e s ts t h a t such

s p e c ie s h a v e l o s t t h e en zy m atic sy stem r e s p o n s ib le f o r t h e tr a n s m e th y la t i on*

-2 1 -

The d a t a p r e s e n te d i n t h i s p a p e r , how ever, c o n s t i t u t e s tr o n g e v i ­
d e n c e t h a t , i n N. r u s t i c a a t l e a s t , a tra n s m e t h y la t in g sy ste m i s
o p e r a t i v e d u r in g t h e p rim a ry s y n th e s is o f n ic o tin e #

As i t i s p ro b a b le

t h a t t h e enzyme sy ste m s in v o lv e d i n th e s e two tr a n s m e th y la tio n r e a c t i o n s
w ould b e v e iy c l o s e l y r e l a t e d i f n o t a c t u a l l y i d e n t i c a l , i t a p p e a rs t o
t h e w r i t e r u n l i k e l y t h a t o n e sy stem w ould b e l o s t i n t h e c o u r s e o f
n a t u r a l s e l e c t i o n and t h e o th e r r e ta in e d #

F or t h i s re a s o n i t seems de­

s i r a b l e to a d v a n c e a n a l t e r n a t i v e h y p o th e s is , w hich i s b a se d on th e
a s s u m p tio n t h a t n o m i c o t i n e , once form ed i n t h e l e a f , can b e rean eth y la t e d a t t h e e x p en se o f m e th io n in e , o r some o th e r l a b i l e m eth y l d o n o r.
T h is i s i l l u s t r a t e d d ia g ra m m a tic a lly b elo w .

I f tr a n s m e th y la tio n in v o lv ­

in g n i c o t i n e a s a m eth y l d onor

R -H

HOMOCYSTEINE (

NORNICOTINE

METHIONINE (

^R -CR

ta k e s p l a c e i n t h e l e a f , i t i s r e a s o n a b le to p o s t u l a t e a second t r a n s ­
m e th y la tio n a t t h i s s i t e i n w hich n o m i c o tin e a c t s as a m eth y l a c c e p to r .
A lth o u g h t h e i n v e s t i g a t i o n s o f Dawson (1 6 ) i n d i c a t e t h a t n i c o t i n e can
b e com plefcely s y n th e s iz e d o n ly i n th e r o o t th e y do n o t, a s James (1 0 )
h a s p o in te d o u t , e x c lu d e t h e p o s s i b i l i t y t h a t t h e l e a v e s , t o o , may form
n i c o t i n e i f p ro v id e d w ith s u i t a b l e p r e c u rs o rs #

-2 2 -

Even a s i d e from t h i s

c o n s i d e r a t i o n , t h e r e i s no e v id e n c e t o s u g g e s t t h a t t h e r o o t i s th e
o n ly o rg a n -where m e th y la tio n can o c c u r .

I f th e r e a c t i o n s i n t h e l e a f

a r e a s r e p r e s e n te d i n t h e d ia g ra m i t i s no lo n g e r n e c e s s a r y t o p o s tu ­
l a t e t h e l o s s o f r e a c t i o n B i n a s p e c ie s such a s IT. r u s t i c a j i t i s
n e c e s s a iy o n ly t h a t t h e r a t i o o f r e a c t i o n r a t e A to r e a c t i o n r a t e B
d i f f e r fro m o n e s p e c i e s t o a n o t h e r .

I n N. r u s t i c a , f o r exam ple, i f B

w ere a slo w r e a c t i o n and A r e l a t i v e l y r a p i d , t h e n o r n ic o tin e form ed w ould
so o n b e r e m e th y la te d th ro u g h t h e m e th io n in e sy stem and h e n c e w ould n o t
a c c u m u la te .

C o n v e rs e ly , i n no m i c o tin e - p ro d u c in g s p e c i e s , t h e accum ula­

t i o n o f n o r n ic o ti n e c o u ld b e e x p la in e d i f A w ere a v e ry slo w r e a c t i o n
an d B a m ore r a p id o n e .

I n t h i s c a s e n o m i c o t i n e would b e form ed f a s t e r

th a n i t c o u ld b e re m e th y la te d an d w ould te n d to a c c u m u la te i n t h e l e a f .
The s i t u a t i o n o b ta in in g i n IT. g la u c a in tr o d u c e s f u r t h e r com plexi­
tie s .

I t h a s b een fo u n d t h a t c e r t a i n s t r a i n s o f t h i s s p e c ie s s t o r e

some n i c o t i n e a s w e ll a s n o m i c o t i n e , w h ereas o th e r s do n o t p o s s e s s
n i c o t i n e i n d e t e c t a b l e am ounts ( 4 ) .

Dawson (4 ) h as e x p la in e d t h i s d i f ­

f e r e n c e on t h e b a s i s o f l o s s o f t h e tr a n s m e th y la tin g m echanism i n th e
fo rm e r c a s e .

"While t h i s a p p e a rs t o be t h e s im p le s t e x p la n a tio n , i t

co u ld a l s o be e x p la in e d by assum ing t h a t i n th e s t r a i n s w hich a c c u m u la te
no n i c o t i n e r e a c t i o n B i s s u f f i c i e n t l y r a p id t o d e m e th y la te a l l t h e
n i c o t i n e a r i s i n g fro m p rim a ry s y n th e s is p lu s t h e s m a ll amount b e in g sup­
p l i e d th ro u g h r e a c t i o n A.

We b e l i e v e , a s does Dawson ( 9 ) , t h a t t h e

l i m i t i n g f a c t o r i n r e a c t i o n A i s most l i k e l y t o be t h e su p p ly o f t h e
h y p o t h e t i c a l m eth y l a c c e p t o r , w hich i s presum ably s u p p lie d th ro u g h t r a n s ­
lo c a tio n .

The a c c u m u la tio n o f n i c o t i n e in o th e r s t r a i n s o f N. g la u c a

-23

c o u ld , ‘th e r e fo r © , b e e x p la in e d by a r e d u c tio n i n th e r a t e of r e a c t i o n
B c a u s e d by a m ore l i m i t e d su p p ly o f t h e a c c e p to r .

L eaves of N. g la u c a

grown on p l a n t s g r a f t e d t o t h e h i g h - n i c o t i n e p ro d u c in g W. ta b a cum have
b e e n o b s e rv e d t o be c a p a b le o f c o n s id e r a b ly g r e a t e r r a t e s o f n o m i c o t i n e
s y n th e s is ( 4 ) .

We h a v e o n ly t o assum e h e r e t h a t a g r e a t e r s u p p ly o f

t h e m e th y l a c c e p to r i s p ro d u ced c o n c u r r e n tly w ith t h e n i c o t i n e i n th e
r o o t s and t r a n s p o r t e d to t h e enzyme sy ste m i n th e l e a v e s .

T h is i s n o t

i n c o n s i s t e n t w ith t h e slow r a t e of r e a c t i o n B p r e v io u s ly p o s tu l a t e d
f o r N. t a b a cum, a s o n ly t h e r a t e r e l a t i v e t o r e a c t i o n A was th e n b e in g
c o n s id e re d *

The l i m i t i n g f a c t o r i n r e a c t i o n A may w e ll b e t h e a v a i l ­

a b i l i t y o f m e th io n in e o r some r e l a t e d compound a s a m eth y l d o n o r i n
th e le a f .
I t s h o u ld b e em phasized t h a t th e above scheme i s a lm o st e n t i r e l y
h y p o th e tic a l.

We c o n s id e r i t p r o b a b le t h a t t h e d i f f e r e n c e s i n n i c o t i n e

and n o m i c o t i n e c o n c e n tr a tio n s h etw een s p e c ie s and s t r a i n s a r e d u e t o
v a r i a t i o n s i n r e a c t i o n r a t e s , and u l t i m a t e l y to th e a v a i l a b i l i t y o f t h e
v a r i o u s r e a c t a n t s , b u t Dawson*s h y p o th e s is rem ains a p o s s i b i l i t y and
c a n n o t by any means b e r u le d o u t on t h e s tr e n g t h o f e x i s t i n g e v id e n c e .
I t i s a p p a r e n t, o f c o u r s e , t h a t much m ore e x p e rim e n ta l work w i l l b e
r e q u ir e d b e f o r e any f i n a l c o n c lu s io n can be re a c h e d .

The f i r s t th i n g

t o b e d e te rm in e d sh o u ld b e w h e th e r o r n o t t h e m eth y l group o f n i c o t i n e
i s , in r e a lity , la b ile .

T h is in fo r m a tio n sh o u ld b e o b ta in a b le by s tu d y ­

in g t h e f a t e o f C -^ -m e th y l n i c o t i n e in tro d u c e d i n t o an i n t a c t p l a n t .
I f n i c o t i n e can b e shown i n t h i s way t o a c t a s a m eth y l d o n o r, t h e f i r s t

-2 4 -

p o s i t i v e e v id e n c e f o r a b i o l o g i c a l f u n c tio n of t h e a l k a l o i d s w i l l h av e
b e e n o b ta in e d *

An e x te n s io n o f th e s e t r a c e r te c h n iq u e s m ig h t a ls o

e l u c i d a t e t h e o t h e r i n t e r - r e l a t i o n s h i p s w hich e x i s t among th e to b a c c o
a lk a lo id s .

-2 5 -

SUMMARY
1* U i c o t i n e i s o l a t e d fro m U ic o tia n a r u s t i c a L ., v a r* h u m il is ,
p r e v i o u s l y fe d m e th io n in e c o n ta in in g carb o n -1 4 i n t h e m eth y l
g ro u p , h a s b e e n shown t o p o s s e s s r a d i o a c t i v i t y .

By means o f de­

g r a d a t i o n e x p e rim e n ts t h i s a c t i v i t y h a s b e e n fo u n d to b e l o c a l ­
iz e d i n t h e m e th y l c a rb o n .
2 . A f te r a d m i n i s t r a t i o n o f C ^ - f o r m a te t o t h i s s p e c ie s , n i c o t i n e of
a p p r e c i a b l y lo w e r s p e c i f i c a c t i v i t y h a s b een o b ta in e d .

M ost, i f

n o t a l l o f t h e a c t i v i t y h a s a l s o b e e n shown t o r e s id e i n t h e
m e th y l c a rb o n .
3 . Chi th e b a s i s o f t h e s e r e s u l t s i t i s p o s tu la te d t h a t fo rm a te i s
f i r s t c o n v e rte d i n t o l a b i l e m eth y l g ro u p s , su ch a s t h a t o f
m e th io n in e , and t h a t t h e s e a r e t r a n s f e r r e d t o n i c o t i n e , i n a
m anner a n a lo g o u s t o t r a n s m e th y la tio n r e a c t i o n s a lr e a d y demon­
s t r a t e d i n th e a n im a l.
4 . The s i g n i f i c a n c e o f th e s e f in d in g s as r e l a t e d to c e r t a i n a s p e c ts
o f a l k a l o i d b io g e n e s is i s d is c u s s e d .

-2 6 -

REFERENCES
(1 )

du V ig n ea u d , V ., J . P . C h a n d le r, M. Cohn and G. B. Brown,
J . B io l# Chem., 154, 787-8 (1 9 4 0 ).
du V ig n e a u d , V. , M. Cohn, J . P . C h a n d le r, J# R. Schenck and
S . Simmonds, J# B io l. Chem., 140, 625-41 (1 9 4 1 ).
S c h e n ck , J . R ., S# Simmonds, M. Cohn, C. M. S te v e n s and V. du
V ig n e au d , J . B i o l . Chem., 149, 355-9 (1 9 4 3 ).

(2 )

H o ro w itz , N. H ., D. Bonner and M. B. H oulahan, J . B io l . Chem.,
1 5 9 , 145-51 (1 9 4 5 ).
H o ro w itz , N. H ., J . B i o l . Chem., 162, 413-9 (1 9 4 6 ).

(3

V ic k e r y , H. B ., B i o lo g ic a l S ym posia, £ , 3-19 (1 9 4 1 ).

(4

Dawson, R . F . , Am, J* B o tan y , 3 2 , 416-23 (1 9 4 5 ).

(5

R o b in so n , R ., J . Roy. Soc* A r t s , 96 , 795-

(6

B a rre n s c h e e n , H. K. and T. v . V a ly i-N a g y , 2 . p h y s io l. Chem.,
2 7 7 , 97-113 ( 1 9 4 2 ).

(7

K irkw ood, S . and L. M ario n , Can. J . Chem., 2 9 , 30-6 ( 1 9 5 1 ).

(8

M in tz, J . A ., J . B i o l . Chem., 182, 489-99 ( I 9 6 0 ) .

(9

Dawson, R. F . , A dvances i n E n zy m o l., 8 , 209-33 (1 9 4 8 ).

10

Ja m e s, W. 0 . , i n Manske, R. H. F . and H. L. Holm es, ”The
A lk a lo id s ” , Academic P r e s s , Hew Y ork, 1950, v . 1 ,
p p . 1 5 -9 0 .

11

K l e i n , G. and H. L i n s e r , P l a n t a , 20, 470-5 (1 9 3 3 ).

12

Dawson, R. F . , P la n t P h y s i o l . , 14, 479-91 (1 9 3 9 ).

13

du V ig n eau d , V. and W. I . P a t t e r s o n , J . B io l. Chem., 109,
97-103 (1 9 3 5 ).

14

M e lv i l l e , D. B ., J . R. R ac h ele and E. B. K e l l e r , J . B io l.
Chem., 169, 419—26 ( 1 9 4 7 ).

-2 7 -

(1 9 4 8 ).

K e l l e r , E . B ., j . R# R ac h ele and V. du V ig n eau d , J* B io l.
Chem ., 177, 733-8 ( 1 9 4 9 ).
Dawson, R. P . , Am. J . B o ta n y , 2 9 , 6 6 -7 1 , 813-5 (1 9 4 2 ).
M cC arthy, T . E . and M. X. S u l l i v a n , J . B i o l. Chem., 141,
871-6 (1 9 4 1 ).
S m ith , C. R ., I n d . Eng. Chem., 34, 251-2 (1 9 4 2 ).
H en ry , T . A ., ”The P la n t A lk a lo id s ” , The B la k is to n C o .,
P h i l a d e l p h i a , 1949, p . 3 7 .
L a i b l i n , R ., Aii n . , 196, 129-82 (1 8 7 9 ).
H e r z ig , J . and H. M eyer, M o n atsh ., 15 , 613-26 (1 8 9 4 ).
P r e g l , P . , ” Q u a n t i t a t i v e O rg an ic M ic r o a n a ly s is ” , 4 th Eng. E d n .,
The B la k is to n C o ., P h i l a d e l p h i a , 1945, p p . 1 5 6 -6 0 .
Simmonds, S . , M. Cohn, J . P . C h an d ler and V. du V ig n eau d , J .
B i o l . Chem ., 149, 519-25 ( 1 9 4 3 ).
C a lv in , M ., C. H e id e lb e r g e r , J . C. R e id , B. M. T o lb e r t and P . F .
Y ankw ich, wI s o t o p i c Carbon” , Jo h n W iley & S o n s, Hew Y ork,
1949, p . 1 0 5 .
du V ig n e a u d , V ., W. G. V e rly and J . E , W ilso n , J . Am. Chem. S o c .,
7 2 , 2819-20 (1 9 5 0 ).
S ak am i, W ., J . B i o l . Chem., 176, 995-6 (1 9 4 8 ).
W eissb a c h , A ., D. Elwyn and D. B. S p r in s o n , J . Am. Chem. S o c .,
72, 3316 (1 9 5 0 ).
M ack en zie, C. G ., J . B i o l . Chem., 186, 351-68 ( 1 9 5 0 ).
G r a n t, W. M., A n a l. Chem., 19, 206-7 ( 1 9 4 7 ).
W ing, R. E . , U n p u b lish ed r e s u l t s .

-2 8 -

APPENDIX I
The fo rm u la u s e d in c o r r e c t i n g t h e o b se rv e d count t o z e ro sam ple
t h i c k n e s s w as:
Cc . M

W• b
w h ere A^ s

maximum s p e c i f i c a c t i v i t y ( c o u n ts /m in u te /m illim o le )

CQ -

o b s e rv e d c o u n t ( c o u n ts /m in u te )

M -

m o le c u la r w e ig h t o f compound

W s

w e ig h t o f sam ple c o u n te d

b

f r a c t i o n o f maximum a c t i v i t y a t t h e sam ple

=

t h i c k n e s s u se d ( T ) — o b ta in e d from s e l f - a b s o r p t i o n curve-

Sam ple c a l c u l a t i o n :
N i c o t i n e p i c r a t e — CQ = 142 c .p .m ., W =

2 6*4 m g ., M » 620,

T = 9 .0 m g ./cm .^
A^ s

142 x 6 2 0
2 6 . 4 x 0 .4 6 5

-2 9 -

s

7 .1 7 x 10^

c.p .m ./m M .