POSSIBLE ORIGINS OF THS HBTHOXYL CARBON OF L I GUI N
FORMED BY HOHOBtTM VTTLGARE

By

John H ilb e r t F lo k s tr a

A THESIS

Subm itted t o th e School o f Graduate S tudies o f Michigan
S ta te C o lleg e o f A g r ic u ltu re and A pp lied Science
in p a r t i a l f u l f i l l m e n t o f th e requirem ents
f o r th e degree o f

DOCTOR OF PHILOSOPHY

Department o f Chemistry

1952

ProQuest Number: 10008302

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ACKNOWLBDC

The a u th o r wishes t o express h is a p p re c ia tio n to
D r. R ichard TJ. Byerrum f o r h is a d v ic e and a s s is ta n c e so
w i l l i n g l y g iv e n d u rin g th e work on t h i s problem, and to
s e v e ra l o th e r members o f th e Chemistvy Department f o r
t h e i r h e lp f u l suggestions} a ls o t o D r. E. H . Lucas o f
th e H o r t ic u lt u r e Departm ent, who so k in d ly fu rn is h e d
seeds and a s s is te d i n growing t h e p la n ts used i n t h is
in v e s t ig a t io n .
F i n a l l y , th e author wishes to thank th e TTnited
S ta te s Atomic Energy Commission f o r p ro v id in g a f e llo w ­
sh ip un der which t h i s work was done*
**********
* * * * * * * *

******
****
**
*

TABTE OF COTTTEfTTTS

D3TR0DTTCTI0I? . . .......................................
EXPKETOHTAL METHODS

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

R a d io a c tiv e Precursors
...........
P re p a ra tio n o f B a rle y P la n ts
...........
I s o la t io n o f L ig n in
...................
D em ethylation o f L ig n in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.................
D et© rialn ation o f R a d io a c tiv ity
RESULTS . . . . . . . ......................... . . . . .............
Experiments TTsing D#L~??ethionine .
..........................
O x id a tiv e D egradation Products o f L ig n in . . . . . . . . . . . . . . . . . .
Experiments TTsing F o r m a te
.............
DISCTTSSIOH
S W Y

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

BIBLIOGRAPHY
APPENDIX

...........

PAGE
1
3
3
3
6
7
9
10
10
IS
14
18
22
23
26

LIS T OF TAB IBS

TABLE

t
II

in
IT

PACE
COMPOSITION OF THE M H I M
affiTHIOIKHB E X P iR I1 « T S

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

4
12

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

te

COMPARISON OF ACTXTXT2SS OF LJOTIff FROM PLANTS ¥ m
m s m m t m m n th o se r m
form at t . . . . . . . . . . . . . . . . . . . . .

i?

fommn MPWMtmmn

.

SOLUTION . . . ............ . . . . . . . . .

INTRO IXTCT ION

INTRODUCTION

I n th© past s e v e ra l years many In v e s tig a to rs have shown the im ­
po rtance o f tra n s m e th y la tio n re a c tio n s i n th e anim al organism ( 1 ) # but
r e la tiv e ly l i t t l e

is known about th e r o le tra n s m e th y la tio n plays in

p la n t m etabolism *

Experiments re c e n tly un dertaken by Kirkwood and

M arion ( 2 ) , i n which th e y attem pted t o dem onstrate th e t r a n s f e r of m ethyl
groups from ohoXln© to th e b a r le y p la n t a lk a lo id , h o rd en in e, using ra d io ­
a c t iv e t r a c e r te c h n iq u e s , were unsu ccessful*

However, I n re c e n t months

i t has been shown by Brown ( 3 ) t h a t th© m ethyl carbon o f m ethionine may
be tr a n s fe r r e d to th© m ethyl group o f n ic o tin e i n tobacco (N ic o tla n a
r u s t ic a ) , and i t was p o s tu la te d t h a t a tra n s m e th y la tio n r e a c tio n m s in ­
vo lv e d *

It

seemed p o s s ib le th a t tra n s m e th y la tio n re a c tio n s m ight be

in v o lv e d I n th© m etabolism o f o th e r p la n t products*
Th© work d escrib ed h e re m s u n d ertaken i n an a tte m p t to study th©
p o s s ib le r o le o f tra n s m e th y la tio n i n th e fo rm a tio n o f l i g n i n I n th e
p la n t*

L ig n in , th e substance chosen f o r study i n t h is In v e s t ig a t io n , is

a c o n s titu e n t o f th e c e l l m i l o f p la n ts .

Although th© amount o f l i g n i n

presen t i n a p la n t in cre a s es as t h e p la n t grows o ld e r , th e r e is an
a p p re c ia b le amount p re s en t i n c o m p aratively young b a rle y p la n ts .
L ig n in is not a d e f i n i t e chemical compound, i t s com position v a ry in g
acco rd ing t o th e method used I n i t s

is o la tio n *

Hence a g re a t d e a l Is

unknown about i t s chem ical s tr u c tu r e and i t s fo rm a tio n i n th© p la n t*
Fuchs ( 4 ) suggested t h a t p e c tin , a p a r t i a l l y m e th y l- e s t e r if ie d p o ly g a l& e tu ro n ic a c id fo m e d i n p la n t c e l l w a lls , a c ts as th © p re c u rso r of

l i g n i n , sine© i t had been shown t h a t t h e p e rc en t ago o f p e c tin s in o ld e r ,
l i g n i f l e d p la n t tis s u e s was s m a lle r than I n young p la n t tis s u e s #

However,

as th e p la n t ages, whereas th e percentage o f p e c tin decreases, th e a c tu a l
amount o f p e c tin present in creases ( 5 ) *

F u rth e r , i t Is known th a t i n

l i g n i n t h e m ethoxyi groups form an e th e r lin k a g e , not an e s te r lin k a g e
such as Is found i n p e c tin , so a r a th e r profound chemical change would
have to t a k e p la c e i f

l i g n i n were to f o m from p e c tin .

A more reasonable

e x p la n a tio n of i t s appearance was o ffe r e d by H lb b e rt ( 6 ) #

On t h e b a s is

o f th e s tru c tu r e o f th e decom position products o f li g n i n , he p o s tu la te d
t h a t a fr u c to s e d e r iv a t iv e can condense w ith guai&col i n th e fo rm a tio n
o f lig n in #

Except f o r a

suggestion;, made by Klason ( 7 ) , t h a t form ald e­

hyde can a c t as a p re cu rso r o f th e mefehoxyl groups I n l i g n i n , l i t t l e

is

known about t h e i r o r ig in *
Since no s a t is f a c t o r y e x p la n a tio n f o r t h e appearance o f th© m ethoxyl
groups i n li g n i n has been o f f e r e d , i t seamed d e s ir a b le to le a r n w hether
tra n s m e th y la tio n was in v o lv e d i n t h e i r form ation#

Although I n a l l

p re v io u s ly re p o rte d tra n s m e th y la tio n stu d ies th e compounds in v e s tig a te d
contained m ethyl groups a ttac h ed to s u lfu r o r n itro g e n atoms, i t seemed
p o s s ib le t h a t t r a n s fe r o f a m ethyl group t o an oxygen atom could ta k e
p la c e , thus form ing a m ethoxyl group*
I t m s decided i n th e present study*

( a ) t o fe e d b a r le y p la n ts

a p p r o p ria te ly la b e lle d m ethionine and form at© , which a re known t o a c t as
m ethyl donors o r precursors i n th e anim al organism , ( b ) to is o la t e th e
li g n i n formed by th© p la n ts , and ( c ) ,

i f r a d io a c t iv it y is fo u n d , t o

perform d egradation s i n o rd e r to determ in e t h e lo c a tio n o f th e r a d io a c t i v i t y i n th e lig n in #

KXPS5IM3J1TAL METHODS

S X m i^ J T A L METHODS

R a d io a c tiv e Precursors
The r a d io a c tiv e tr a c e r s us ad i n t h i s work were D, L~meth 1o n i na con­
t a in in g

i n th e m ethyl group and th e sodltm s a l t o f C ^ “f o m i c a c id .

M ethio nine was synthesised by re a c tin g D,I^f>»ben*ylhoatoeyateine w ith
cl4«. jagrtjhyl io d id e which was obtained from T r& o e rla b , i n c . , under
a llo c a t io n from th e TTalted S ta te s Atomic Energy Commission.

The form ate

m s o b ta in e d from Dak Ridge K a t lo m l la b o ra to r y , a ls o under a llo c a t io n
from Mae tTnited S ta te s Atomic Energy Commission*
P re p a ra tio n o f B a rle y P la n te
A v a r i e t y o f Ho rd earn vu lg ar© known a© Bay b a rle y m s used i n t h i s
in v e s tig a t io n .

According

to P h illip s and (foes ( 8 ) th e percentage of

m ethoxyl i n b a r le y l i g n i n

in creased a t th e f a s t e s t r a t e when th e p la n ts

were t h i r t y t o f o r t y days

o ld .

i& oD cugall and Do Long ( 9 ) showed t h a t ,

w ith re sp ec t t o l i g n i n c o n te n t, n in e ty -fo u r -d a y -o ld p la n ts grown i n th e
greenhouse were a t about th e same stage o f m t u r i t y as fo r ty -o n e -d a y -o ld
p la n ts grown I n th© f i e l d .

Consequently Mi© seeds were p la n te d i n f l a t s

and a llo w e d to grow o u ts id e th © greenhous© f o r about t h i r t y days .

P la n ts

o f t h is age were from e ig h t t o tw e lv e inches t a l l , v a r ia tio n s probably
being due to seasonal e f f e c t s .

A commercial p la n t food m ix tu re was fe d

as re q u ire d in a w a te r s o lu tio n .
B efo re being fe d t h e r a d io a c tiv e compounds* th e p la n ts were g ive n
th© fo llo w in g tr e a tm e n t.

A© much o f t h e adhering s o il as p o s s ib le m s

c a r e f u lly removed from th e roots by shaking*

A f t e r soaking i n w a te r

f o r about t h i r t y m inutes, any d i r t l e f t on the ro o ts was washed away
under ru n n in g w a te r*

To reduce t h e number o f b a c te r ia p re s e n t, t h e

ro o ts were soaked i n a 0 .1 p ercen t s o lu tio n o f Wyandotte d e te rg e n t
g erm icid e Ho* 1528

1

f o r about t h i r t y m inutes*

Fo llo w ing a r in s e I n

d i s t i l l e d w a te r , th e roots o f each p la n t were placed i n 25 m l* of an
in o rg a n ic n u t r ie n t s o lu tio n i n a 50 m l. ilrleraneyer f la s k *

T h is n u tr ie n t

s o lu tio n was prepared by making a 1*3 d ilu t io n o f th e s to c k s o lu tio n ,
th© com position o f which is given i n T a b le 1*

As a f u r t h e r p re c a u tio n

TAB1.8 I
COMFQSITIOH Qp THE FOTKISBT SQUfTIOH

Calcium n i t r a t e *
€ a (M 0 g ),},l f .
Potassium c h lo rid e * K C I,“ 250
!.%.gno$iurn s u lfa te *
MgSC^, 250 mg.
Ammonium s u lfa te *
250 mg*
F e r r ic c h lo rid e * F e C l*, g rag*
Potassium dihydrogen phosphate* KHgPO^, 250 mg.
D i s t i l l e d w a te r to 1 1*

a g a in s t b a c t e r ia l c o n ta m in a tio n , th r e e drops of a one percent s o lu tio n
o f th e d e te rg e n t g erm icid e were added t o each f l a s k .

Each f la s k a ls o

contained a s u it a b le amount o f th e ra d io a c tiv e compound bein g s tu d ie d ,
as w i l l be described l a t e r *
I n th© p la n ts * m etabolism o f th e ra d io a c tiv e compounds th e r e m s a

1A

p o s s ib ili t y of C

■j

Og bein g lib e r a t e d .

The p la n ts wore grown i n a fum©

T h is m a te r ia l m s ob tained fro m , the Wyandotte Chemical C o rp .,
W yandotte, M ic h ., by th e M ichigan S ta te C o lle g e H o r t ic u lt u r e
Departm ent.

«*4 •«

h o o d ^ In o rd e r t o preheat th e contam in atio n o f th® a i r from t h i s source*
Two 36**inch, 30-•watt flu o re s c e n t tubes and a 1 0 0 -w a tt b u lb , about 15
inches above th© p la n ts , were used as a source o f l i g h t *

A lig h t

in t e n s i t y o f about 800 fo o t-c a n d le s a t th© leaves m s thus o b tained*
D uring th© fe e d in g ©srpsrimenbs, the li g h t m s tu rn e d on f o r about 18
hours out o f 84*

The volume o f th© n u trie n b s o lu tio n was k e p t f a i r l y

constant by th® a d d itio n o f d i s t i l l e d w a te r*

Is o l a t i o n o f L ig n in
D i f f i c u l t y m s encountered i n th e is o la t io n o f l i g n i n , usin g a
m o d ific a tio n o f th© s u lf u r ic a c id method of Ost and W ilkoning ( 1 0 ) *

In

t h is method th e d rie d p la n t tis s u e m s put in to 70 percen t s u lf u r ic a c id
f o r 16 hours a t 8 0 ° C* o r le s s *

The s u lf u r ic a e id m s then d ilu t e d to

about th r e e p e rc e n t, th e m ix tu re m s hydrolyzed f o r two hours a t 100° C *,
and f i l t e r e d , l i g n i n being th© p r e c ip it a t e ob tained*

Tifhen t h is method

m s used w ith th e s e young p la n ts , i t appeared t h a t com plete w e ttin g by
th e s u lf u r ic a c id m s not achieved*

I t m s f e l t t h a t an ext m e t I on o f

th e f a t t y m a te r ia l from th e t is s u e would perm it com plete co n tact by th e
s u lf u r ic a c id *

However, a f t e r an e th e r ©attraction m s performed on th©

t is s u e , th e percentage o f A lig n in '* present m s s t i l l found t o b© about
thro© tim es as h ig h as t h a t re p o rte d i n th e l i t e r a t u r e In work done w ith
comparable plant© ( 8 ) *
MacDougall and 0© Long ( 9 ) found th a t th© a b s o lu te methoxyl content
o f l i g n i n from d rie d p la n t tis s u e s m s g r e a te r 1han t h a t o f l i g n i n from
fre s h tis s u e s o f t h e same p la n ts *

They thought th a t t h is m s due t o th e

-5 -

in c lu s io n o f m efchoxyl-containing carbohydratss i n l i g n i n from th© d rie d
tis s u e s #

Th© fre s h p la n ts used i n th© presen t work were p r e -tr e a te d

b e fo re p la c in g I n contact w ith th© s u lfu x ‘io acid#

T h is tre a tm e n t con­

s is te d o f e x tra c tio n s i n a W aring B lendor u s in g v a rio u s s o lv e n ts , as
suggested by -%cDoug&ll and De bong (1 1 )#

E ther-satu rat@ d w a to r was

b e lie v e d by them t o be th e best n o n -acid s o lv e n t f o r removal o f n itro g e n c o n ta in in g m a te r ia l from young u n d rie d tissu es#

A f i v e p ercen t s o lu tio n

o f a c e tic a c id m s found e f f e c t iv e i n removing carbohydrates from young
tis s u e #

A c o n s ta n t-b o ilin g ( 1 * 2 ) m ix tu re o f ethanol and bensene m s used

as a s o lv e n t f o r f a t t y m a te ria ls p resen t I n t h e tis s u e s #

A c c o rd in g ly ,

i n the p resen t w ork, a pr© treatm ent o f t h e p la n t tis s u e s was adopted#
Th© fo llo w in g e x tra c tio n s w ere performed on th e tis s u e s i n a Waring
B lendort

two 1 5 -mi nut© e x tra c tio n s w ith e th e r-s a tu ra te d w a te r, one 2 0 -

m inute e x t r a c t io n w ith f i v e percent a c e tic a c id , two 15-m inute e x tra c tio n s
w ith 1*2 ethan o l-b em en e#

A s u ita b le q u a n tity o f 70 percent s u lf u r ic a c id

m s added to t h e d rie d f i b e r obtained a f t e r c a rry in g out th e above
procedure and th e r e a c tio n m ix tu re a llo w ed to stand f o r about 16 hours a t
f i v e degrees C#

Th is was don© i n th e co ld because i t had been shown t h a t

c a rb o n is a tio n o f hydrolysed carbohydrates takes p la c e a t h ig h e r tempera­
t u r e s , causing d i f f i c u l t y i n th e subsequent f i l t r a t i o n and producing an
im pure l i g n i n p re p a ra tio n ( 1 2 ) *

A t t h© end o f 16 h o u rs, th e s u lf u r ic a c id

was d ilu t e d t o th re e p e rc e n t, b o ile d g e n tly fo r two hours, keeping th©
volume c o n s ta n t by adding w a te r, and a llo w e d to o o o ij th® l i g n i n s e t t l in g
o u t*

The l i g n i n was th e n f i l t e r e d , using a f r i t t e d g lass f i l t e r , washed

th o ro u g h ly , and d r ie d i n a vacuum d e s ic c a to r a t room te m p e ra tu re .

The

l i g n i n c o n ten t of p la n ts t h i s age m e found t o be about 4 .5 percent on a
d ry w e ig h t b a s is .

Demefchylation o f L ig n in
To prove t h a t any r a d io a c t iv it y found i n th© l i g n i n m s lo c a te d i n
th e m ethoxyl carbon, i t was necessary t o s p l i t o f f th e m ethyl group and
o b ta in I t i n t h e f o r a o f a s o lid d e r iv a t iv e s u it a b le f o r coun ting*
A m o d ific a tio n o f th e method o f P h i lli p s ( 1 3 ) , In which th e m ethoxyl group,
tr e a t e d w ith h y d rio d ic a c id , y ie ld s m ethyl io d id e , was used*

I n t h is

method th e m ethyl io d id e is caused to re a c t w ith s i l v e r n i t r a t e and th e
p r e c ip it a t e o f s i l v e r io d id e is weighed*

However, sine® i n th© p resen t

work i t m s necessary t o re c o v e r th e m ethyl group, th © m ethyl io d id e
formed was swept in t o a s o lu tio n of t r i e th y l amine*

Previous work done i n

t h is la b o ra to ry showed th a t trie th y la m in © re a c ts q u a n t it a t iv e ly w ith
m ethyl io d id e to form m ethyl t r 1 ethylammonium io d id e *

T rie th y la m ia e is

a ls o le s s v o l a t i l e th a n trim ethylamine,vhkh:Was used by Simmonds and co~
w oikers ( 1 4 ) *
A m o d ified form o f th e apparatus d escribed by P re g l ( 1 5 , 3 ) m s used
f o r d e m e th y la tlo n *

About 60 mg* o f th e li g n i n t o be d ©methylated was

weighed in to c ig a r e t t e paper and placed in th e r e a c tio n v e s s e l w ith two
m l* o f ph en ol, which acted as a s o lv e n t f o r th e li g n i n , and fo u r m l* o f
47*3 p e rc e n t h y d rio d ic a c id *

A ttached t o t h i s was a gas-washing b u b b ler

c o n ta in in g 1*5 m l* o f th e f i v e percent CdSO^-WsgSgO^ s o lu tio n recommended
by F re g l to remove t h e h y d rio d ic a c id and io d in e *

A tu b e from th e

b u b b le r le d to th© re c e iv in g v e s s e l, which contained a f i v e percen t so lu ­
t i o n of tr lo th y la m ia © i n e th a n o l, cooled i n a m ethyl eolloGolvo-COg bath
to about -7 5 ° 0*
D uring dem obhylatlon t h e r e a c tio n f la s k m s issuersed i n a copper
o x id e b a th *

A stream o f n itro g e n was s lo w ly run through th© sid e -a rm o f

th e r e a c tio n f l a s k and through th e d e m e th y la tion assembly*

Stxperimeaitation

showed t h a t best re s u lts wore obtained when th© tem p eratu re o f the b ath
m s k e p t a t about 15 0° C* f o r 45 m inu tes, then ra is e d t o 2 0 0° C* and h e ld
th e r e f or 30 m inutes*

The b a th m s allo w ed to cool f o r 15 m in u tes , du rin g

which tim e a f a s t e r stream o f n itro g e n was passed through t h e a p p a ra tu s .
A t t h i s tim e t h e t i n o f th e d e liv e r y tube was rin s e d w ith ethanol in to th e
re c e iv in g v e s s e l*

T h is v e s s e l was th e n stoppered and a llo w e d t o stand

o v e rn ig h t a t room te m p e ra tu re *

The ethanol s o lu tio n m s ta k e n alm ost to

diyness by h e a tin g , th e la s t t r a c e o f ethanol and unre& eted amine removed
I n a vacuum d e s ic c a to r, and a w h ite s o lid , t h e m ethyl trleth ylai® ao n iu ia
io d id e , rem ained*

This was weighed and te s te d f o r r a d io a c t iv it y *

A m ethoxyl a n a ly s is o f li g n i n from t h i r t y - d a y - o ld b a rle y p la n ts was
made and th e m ethoxyl content o f t h e li g n i n m s found t o be about s ix
p e rc e n t, v a ry in g s l i g h t l y from one run o f p la n ts t o an o th er*
d e s ir a b le t o know i f

I t seemed

q u a n t it a t iv e demefchylation m s e ffe c te d , so d o -

m e th y la tio n o f v a n i l l i n , a compound o f known methoxyl coizfcont, was
perform ed*

T h is produced a 95 p e rs e rt reco very o f th e methoxyl group

as th e q u a te rn a ry compound under th e c o n d itio n s

used.

Assuming t h a t a

s im ila r s it u a t io n m aintained I n l i g n i n , t h is f ig u r e m s used as a
c o r r e c tio n f a c to r I n subsequent d e te rm in a tio n s *

D e te rm in a tio n o f R a d io a c tiv ity
A l l count;® w r a made using a Model 163 S c a lin g U n it m anufactured fey
Hu c l oar In strum ent and Chemical C o rp o ra tio n *

By r e f e r r in g t o a s e l f -

a b s o rp tio n curve prepared f o r t h e substance feeing counted, a l l counts made
w ere c o rre c te d to sero sample th ic k n e s s *
aluminum d isc s 3*83 square cm* i n a rea *

The e n s ile s w ere counted on
The d is c c o n ta in in g t h e sample

was placed #n th e to p s h e lf o f th e c o u n te r assembly o f th e S c a le r U n it*
Based on t h e measured a c t i v i t y o f a standard C*^ cample o f known a c t i v i t y ,
th® o v e r - a l l e f f ic ie n c y was found to fee 8*79 p e rc e n t*

resu lts

RESULTS

Experiments Using D ,L-M ethlonin©
I t has been known f o r some tim e t h a t m ethionine can a c t as a m ethyl
donor i n th© an im al ( 1 ) *

K e l le r and co-workers fe d m ethionine which m s

doubly la b e lle d i n th© m ethyl group w ith carbon-14 and deuterium to ra ts
and Is o la te d c h o lin e and c r e a tin e from th e r a t tis s u e s ( 1 6 ) *

W ith in

ex p e rim e n ta l e r r o r , th e r a t i o o f deuterium to carbon-14 i n m ethyl groups
o f c h o lin e and c r e a tin e m e found t o b© t h e same as t h a t I n th e m ethyl
group o f th e d ie t a r y m e th io n in e, proving t h a t m ethionine a c ts as a t r u e
transm sfchylating a g e n t*

Brown has shown t h a t th e carbon o f t h e m ethyl

group o f m ethionine can be tr a n s fe r r e d t o th e m ethyl group o f n ic o tin e
in th ® tobacco p la n t*

Thus i t appeared reasonable th a t m e th y l-la b e lle d

m ethionine oould g iv e r is e to th e m ethoxyl groups o f l i g n i n , and i t was
decided t o fe e d t h is compound to th© p lan ts#
An a tte m p t was made t o fe e d m ethionine to th © p la n t by immersing th©
ro o ts I n a n u t r ie n t s o lu tio n c o n ta in in g m e th io n in e , thus a llo w in g absorp­
t i o n t o ta k e p lac e through th e ro o ts *

A n alysis o f th e s o lu tio n was

necessary to determ ine th© ra t© o f uptake o f m eth ion ine by th© p la n t and
a ls o w hether d e s tr u c tio n o f th© m ethionine by b a c te r ia was ta k in g p la c e .
The c o lo r im e tr ic method o f a n a ly s is used measured th© red c o lo r produced
when m ethionine re a c ts w ith n itro p r u s s id e , as described by McCarthy and
S u lliv a n ( 1 7 ) .
l a oh of s ix b a r le y p la n ts m s fe d two nog. o f m eth ion ine i n 25 m l. o f
n u tr le n t s o lu tio n which co n tain ed th re e drops o f th© one percent d e te rg e n t

-1 0 -

g erm icid e s o lu tio n *

A fte r f o r t y - e ig h t hours th® p la n ts wore removed from

t h e s o lu t io n and a n a ly s is f o r m eth ionine m s made*

I t m s found t h a t In

two days th e a v era g e u p ta k e par p la n t o f m eth ionin e from s o lu t io n m s 70
p e r ce n t o f t h e two mg. o r i g i n a l l y p resen t*
observed i n th e medium w it h in t h i s tim e*

Wo b a c t e r ia l growth m s
As a fu r th e r check o f b a c t e r ia l

a c t i v i t y , sam ples o f t h e n u tr ie n t medium w ere in o c u la te d w ith ro o t
fragm ents*

A fte r f o r t y - e i g h t hours no d e c r e a se m s nobed i n t h e amount

o f m eth ion in e p resen t* compared w ith t h e uninooul&hed c o n tr o ls*
P la n ts which bad been prepared i n th e manner p r e v io u sly d e sc r ib e d
n

w ere fe d r a d io a c t iv e m ethionine*

S&oh f la s k co n ta in ed 2*01 % 1 0 ~ * m oles

o f m eth ion in e p o ss e s sin g a t o t a l a c t i v i t y o f S*6 x 10® coun ts p e r m inute
{e#p*m*) and th r e e drops o f t h e on# p ercent d e te r g e n t g erm icid e s o lu t io n
i n 25 ml* o f n u tr ie n t s o lu tio n *

The p la n ts w ere grown i n th e s p e c ia l l y

d esig n ed bbed under a r t i f i c i a l l i g h t a t roost tem perature f o r sev en days*
m

m entioned e a r lie r *

ob servab le*

At t h e end o f t h i s tim e no b a c t e r ia l growth was

As a fu r th e r t e s t of m eth ion in e u p tak e by th e p la n ts*

r e p lic a t e sam ples o f th e n u tr ie n t s o lu t io n w ere evaporated t o d iyn osg
a f t e r removing ih # p la in s a t th© end o f sev en days*

Only a sm a ll amount

o f r a d io a c t iv it y m s d e t e c t a b le i n th # r e s id u e , in d ic a tin g alm ost com p lete
a b so r p tio n o f m ethionine by t h e p la n ts*
The r o o ts o f th # plumbs w ere removed am! th e rem aining p art o f t h #
p la n ts g iv e n th # p r e v io u s ly d escrib e d s o lv e n t e x t r a c tio n su g g e sted by
MaeDougall and 0# bong*

b ig n in m s is o la t e d from t h e rem aining f ib e r by

th # W p ercen t s u lf u r ic a c id method*

A fte r h y d r o ly s is , f i l t r a t i o n and

w ashing, th # l i g n i n o b ta in ed p o sse sse d c o n sid e r a b le r a d io a c t iv it y as

-1 1

determ ined w ith a la b o ra to r y m o n ito r*
a c t i v i t y o f t h e l i g n i n in t o

I s o rd e r

t o b rin g th e ra d io ­

a rang© which could be ©ousted w ith th©

S c a le r

C h it# i t was necessary t o d i l u t e th e r a d io a c tiv e 1 ig n is w ith from s ix t o
te n tim es i t s own w e ig h t o f in a c t iv e l i g n i n o b ta in e d from s im ila r p la n ts
n o t fe d r a d io a c tiv e m a te ria l#

A weighed amount o f about 60 mg* o f l i g n i n

m s placed on a counting die© and counted as o u tlin e d p re v io u s ly *
The same sample o f l i g n i n used f o r counting m s used f o r d o m eth ylatlo n *
H a d !© a c tiv ity m s found i n th e q u ate rn a ry compound o b ta in e d from dem eth yla t io n o f th e lig n in #

S in ce l i g n i n is not a chem ical compound# th e

s p e c ific a c t i v i t y ©ou!& not

be expressed# as is customary# on a m olar o r

m lllim o l& r b a s is *

became necessaiy to express a c t i v i t y on

Henc© I t

a

reco very o f counts b a s is ; t h a t Is # b y a comparison o f th© t o t a l a c t i v i t y
o f th e <$sa t or n a ry compound ob tain ed from a c e r t a in amount o f l i g n i n w ith
th© t o t a l a c t i v i t y o f t h e l i g n i n used*
t i l th e Appendix*

A sample c a lc u la t io n i s shown

Recovery o f counts obb&lmd from l i g n i n Is o la te d I n two

experim ents is shorn i n Table I I *
TABUS I I

ymvLxmim $xmamw?&
' S p e ria iM E t
Ho*

oS airv’S f A c t i v it y
(©cunts p e r m inute)
M g n in
Q u aternary Io d id e

Per©©at pieoovery
o f Counts

1 (2 4 p la n ts )

em
46?

989
662

9 9 *0
92*9

2 (2 4 p la n ts )

710
866

979
864

9 0 .6
8 9 *6

—12**

Th@s@ d a ta show t h a t p r a c t ic a l ly a l l o f th© a c t i v i t y found i n ih o
li g n i n is recovered in th© q u ate rn a ry compound o b tain ed upon d ero ath ylatio n
under th © c o n d itio n s used.

T h is would in d ic a te t h a t th e a c t i v i t y i n th e

l i g n i n is lo c a te d i n th© methoxyl group*

V a r ia t io n i n th© percentage

recovery between experim ents may be noted*

I t was g e n e r a lly found i n

th e s e experim ents t h a t recovery o f counts from l i g n i n is o la te d from
p la n ts grown a t lo w er te m p e ra tu re (2 0 ° C«) was la r g e r th a n t h a t from
p la n ts grown a t h ig h e r te m p e ra tu re (2 5 -3 0 ° C .} * but no e x p la n a tio n f o r
t h i s o b s e rv a tio n is a p p aren t*

O x id a tiv e D eg rad ation Products o f L ig n in
L ig n in is o la te d from b a rle y p la n ts by th e u s u a l procedure was
analysed f o r n itro g e n * u s in g th e miero-IC^©1dahl method*

Th© n itro g e n

©outenb was abou t 2 *4 p e rc e n t, a v a lu e lo w er than th a t o b tain ed from
most l i g n i n p re p a ra tio n s *

However* th© p o s s ib ili t y escistcd t h a t t h is

n itro g e n was present i n t h e form o f p r o te in and t h a t th e r a d io a c t iv it y
observed i n th e l i g n i n was due t o th e presence o f m ethionine I n th e
p r o te in *

To determ ine d e f i n i t e l y mi e th e r o r not th© a c t i v i t y found was

a s s o c ia te d w ith nitrogenous compounds* an a tte m p t m s made to is o la t e
v a n i l l i n , s y rin g a ld e h y d % and s im ila r n ih ro g e n -f roe d e r iv a tiv e s formed
by o x id a tio n o f l i g n i n w ith a lk a lin e nib rob ©ms©nc* and t o t e s t them f o r
r a d io a c t iv it y *
To c a rr y p u t th is r e a c tio n , about 145 mg* o f l i g n i n Is o la te d from
p la n ts fe d r a d io a c tiv e m ethionine w ere placed I n a s ta in le s s e t e e l bomb
w ith one m l. o f nitrobensen© and 18 m l. o f

FaOH ( 2 0 ) .

a t 130*5 C ** w ith a g it a t io n * f o r about th re e h o u rs.

-1 3

T h is was heated

A f t e r c o o lin g , th e

a lk a lin e r e a c tio n m ix tu re mis steam d i s t i l l e d t o remove nitro genous
products#

The f i l t r a t e -was a c i d i f i e d t o pH 3 and th e r e s u lt in g m ix tu re

c o n tin u o u sly e x tra c te d w ith benzene f o r 48 hours*

The benzene s o lu tio n

was e x tra c te d te n tim es w ith 15 ml* p o rtio n s o f aqueous f i v e percent
NaHSOg s o lu tio n *

Th© b i s u l f i t e e x tr a c tio n was a c id i f i e d w ith th re e m l.

o f c o n c en trated s u lf u r ic a c id and t h e s o lu tio n fre e d from s u lf u r
d io x id e under reduced pressu re a t room te m p e ra tu re ( 2 1 )*

Th is s o lu tio n

wbm th e n e x tra c te d s ix tim es w ith 15 m l* p o rtio n s o f e th y l e th e r and th e
e th e r was th e n ©sraporated a t 60° C# ( 2 2 ) *

The re s id u e was e x tra c te d

w ith hot w a te r* f i l t e r e d * and mad© up to 30 ml*

D u p lic a te 10 m l*

a liq u o ts o f t h i s s o lu tio n were used f o r m ic ro -K je ld a h l n itro g e n de­
te rm in a tio n s *

W ith in th e experim en tal e r ro r o f th e d e te rm in a tio n no

n itro g e n was found*

Th© aldehyde m ix tu re was p r e c ip ita t e d from th© r e s t

o f th © s o lu tio n using 2 ,4 -d ittitro p h e n y lh y d ra z in e *

Th© p r e c ip i t a t e was

allo w ed t o stand o v e r n ig h t, f i l t e r e d , d rie d and counted*

Th© p h e n y l-

hydrazon© m ix tu re possessed a s p e c if ic a c t i v i t y o f 88 c*p*m* p e r mg.
C a lc u la te d as v a n i l l i n , t h i s would g iv e a s p e c if ic a c t i v i t y o f 192 c*p.m *
p e r mg*

Th© s p e c if ic a c t i v i t y o f th© l i g n i n used f o r o x id a tio n by

n itro b e n ze n e was s i so about 190 c .p ,m , per mg,

J&rpcri manta Using Formate
Recent experim ents show t h a t fo rm a te , o r a one-carbon compound
designated as "fo rm ate* appears t o p la y a r o le i n tra n s m e th y la tio n r e ­
a c tio n s *

There a r e two p o s s ib le ways i n which t h i s compound could e n te r

such re a c tio n s *

Formate mny a r is e from th e o x id a tio n o f m ethyl groups

and in some m n n e r a c t d i r e c t ly as a laethylsitirig a g e n t, o r i t may be

—1 4 —

reduced to fo rm a m ethyl group* which is then tr a n s fe r r e d *

S ie k e v its *

e t &1. ( IP, ), have shown th a t * i n th e a n im a l, th© m ethyl group o f
m eth ion ine may g iv e r is e t o fo rm ate*

On th © o th e r hand* acco rd ing t o

Welch and S&kaml ( 1 9 ) , fo rm a te can be reduced t o form th e m ethyl group
o f m ethionine i n ■fee anim al organism .
Two groups o f p la n ts * as c lo s e ly s i mi l a r as p o s s ib le * were grown
i n n u tr ie n t s o lu tio n under th e same c o n d itio n s , on© group being fe d
m eth ion ine and th© o th e r form ate#

By comparing th © a c t i v i t i e s o f l i g n i n

is o la te d from th© two groups o f p la n ts i t m s hoped t o o b ta in evidence as
to whether m ethionine o r form at© a c ts as th© more d ir e c t precu rso r of th e
mefehoxyl groups o f li g n i n *
Ho a n a ly t ic a l method could be found w h ld i would g iv e accuracy
comparable t o t h a t achieved I n the method used f o r t h e d e te rm in a tio n o f
m e th io n in e .

TXpon removal o f th e p la n ts from th e n u tr ie n t s o lu tio n a f t e r

th© seven day period# th© contents o f s e v e ra l o f th e fla s k s were
evaporated t o dryness*
resid u es*
©even day©*

N e g lig ib le r a d io a c t iv it y was found i n th e

Ho b a c t e r ia l growth m s observed I n th© f la s k s , even a f t e r
I t m s concluded th a t p r a c t ic a l ly com plete a b s o rp tio n o f

f o r m t © by th e p la n ts had ta k en p la c e w ith in t h i s tim e .
The p la n ts were fe d fo rm ate under th e same c o n d itio n s t h a t w ere
m aintained in th© m eth ion ine experim ents*

The fla s k s each con tain ed th©

n u tr ie n t s o lu tio n * d e te rg e n t g e m ie id © and 1*99 x 10"® moles o f fo rm a te ,
having a t o t a l a c t i v i t y o f 7*2 x 10® o.p*m*

The m olar c o n c e n tra tio n was

t h e same as t h a t of m eth ion ine— th e a c t i v i t y double*

As b e fo r e , th e

p la n ts were a llo w e d to grow i n th e s o lu tio n f o r seven days.

*1 5 *

As p re v io u s ly d e s c rib e d , th e p la n ts were processed and l i g n i n
is o la te d by th e 70 percen t s u lf u r ic a c id method*

The r a d io a c tiv e li g n i n

o b ta in e d was counted and demobhylated, and t h e qu atern ary compound formed
was counted*

The r e s u lts o b tained usin g two groups o f p la n ts a re shown

in T a b le I I I .

TABLE X I I
FORMATS EXPERIMENTS

Experim ent
Ho.

Observed A c t iv it y
(counts p e r m inute)
L ig n in
Q uaternary Io d id e

P ercent Recovery
o f Counts

1 (25 p la n ts )

243
241

283
256

7 1 .7
69*8

2 (26 p la n ts )

529
181

412
212

66.8
64*8

It

can be seen t h a t most o f th e a c t i v i t y found i n th e l i g n i n was re ­

covered by demethyl a t io n , showing t h a t i t m s present i n th e m ethoxyl
group*

The v a r i a t i o n between t h e two runs re p o rte d h e re may a g a in be due

to d if fe r e n c e in tem p erature a t which the p la n ts w ere grown, a lth o u g h
t h is was s m a lle r h e re th a n i n th e m eth ion ine experim ents*
As s ta te d e a r l i e r , a oou^arlson o f t h e a c t i v i t i e s o f l i g n i n o b tained
from p la n ts fe d m ethionine and th a t from p la n ts fe d form at© was mad©*
T o ta l a c t i v i t i e s , m olar q u a n titie s a d m in istered p e r p la n t and t h e s p e c ific
a c t i v i t i e s o f ih© l i g n i n is o la te d from each group o f p la n ts a re g iv e n i n
T a b le I V .

Th© s p e c if ic a c t i v i t y o f l i g n i n o b tain ed a f t e r a d m in is te rin g

—16—

TABLE IV

COMPARISON OP ACTIVITIES OF LIGFTN FROM PLANTS FED ?®?HIONIKE
AND THOSE FED FORMATE

Group
No.

Compound
A dm inistered

Moles Given
p e r P la n t

T o ta l A c t iv it y
Given
p e r P la n t

1

M ethionine

S .01 x 10**5

3 *6 x lO ^c.p.m * 385 o*p.m*/m g*

2

Format©

1 .9 9 x 1 0 -B

7*2 x I0^c#p*m* 2 9 .2 o*p*m*/mg*

S p e c ific A c t i v it y
o f L ig n in

m eth ion ine was about t h ir t e e n tim es as high as t h a t obtained, i n the
form at© e&perlment*

The m olar c o n cen tratio n s of m ethionine and format©

a d m in is te re d were t h e ©am©, but th e a c t i v i t y o f th e form ate m e tw ic e as
g reat#

14C onsidering t h i s , i t appears t h a t i f C
m ethyl m ethionine is

fe d t o b a rle y p la n ts , th© s p e c ific a c t i v i t y o f Hi© l i g n i n is o la te d is a t
le a s t one order of magnitude h ig h e r th a n t h a t o b tained a f t e r fe e d in g
C ^ - f o r a a t e , a l l c o n d itio n s being kept as n e a rly th e same as p o s s ib le i n
th© experim ents*

-1 7 *

DISCOSSKH

DISCUSSION

It

is dem onstrated i n th o s e experim ents th a t th e m ethyl carbon o f

m eth ion ine can a c t as th e p re c u rs o r of th e methoxyl carbon of li g n l n
produced by b a r le y p la n ts i n v iv o *

The r e s u lt s o b ta in e d g iv e a s tro n g

in d ic a tio n t h a t a tra n s m e th y la tio n r e a c tio n is in v o lv e d *

I n o rd e r to

prove c o n c lu s iv e ly t h a t such a re a c tio n has occurred, i* © * , t h a t th e
m ethyl group is tr a n s fe r r e d i n t a c t , i t w i l l be necessary t o conduct ex­
perim ents using m e th io n in e doubly la b e lle d w ith carbon-14 and deuterium
i n th e m ethyl group*
la b o ra to r y *

Such experiments w i l l soon be undertaken i n t h i s

I f a t r u e tra n s m e th y la tio n is proven, th e f i r s t in s ta n c e o f

tra n s m e th y la tio n t o an oxygen a t m w i l l be e s ta b lis h e d *
Formate can a ls o a c t as precu rso r o f th e methoxyl group o f l i g n l n
b u t t o a le s s e r e x te n t than t h e m ethyl group o f m ethionine*

The lo w e r

s p e c ific a c t i v i t y o f th e li g n l n o b tain ed from p lan ts fe d form ate as com­
pared w ith those fe d m ethionine in d ic a te s t h a t th e m ethyl group o f
m ethionine is tr a n s fe r r e d as such and does not go through a process of
o x id a tio n t o fo rm a te and subsequent re d u c tio n *

I f t h e l a t t e r w ere t r u e ,

th e l i g n l n fro m p la n ts fe d form at© would have a t le a s t as high a s p e c if ic
a c t i v i t y as t h a t from p la n ts fe d m ethionine*

From th ese re s u lts i t would

a ls o appear th a t fo rm ate is n o t tr a n s fe r r e d as such and then reduced*
A lthough Welch and Sakaml found t h a t f ormate may be reduced to t h e m ethyl
group o f m e th io n in e , ih e exact manner i n ishich Hi is ta k e s p la c e is not
known, but th e r e is some evidence t h a t th e r e a c tio n ta k e s p la c e v ia
s e r in e , ©thanol&mine, c h o lin e and befcain© ( 2 3 ) *

—IB —

It

is c o n ceivab le t h a t

c h o lin e and b e ta in © a c t as mefchyl&ting agents i n th e fo rm a tio n o f l i g n i n ,
m eth ion ine not n e c e s s a rily being fo m e d befor© traxisrnothylatlon takes
p la c e *

W hile most o f t h e fo rm ate p2*esent in th© p la n t probably Is o x id is e d

to carbon d io x id e , i t seems p o s s ib le t h a t some could be reduced t o methyl
groups and in v o lv e d i n mefchylation re a c tio n s *
I t Is n o t known why, i n d e m e th y la tio n o f l i g n l n from p la n ts fe d
fo rm a te , a lo w e r recovery o f counts is o b tained th a n from l i g n i n formed
by p la n ts fe d m ethionine*

I n s e veral th e o r ie s o f l i g n i n fo rm a tio n carbo­

h y d ra te s a r e named as l i g n l n precurso rs*

Sakaml (2 3 ) has shown th a t

fo rm a te and g ly c in e , through s e rin e , form p yruvate*

T h is p yru vate could

g iv e r is e t o carbohydrates, from which l i g n i n m ight co nceivably be formed*
The li g n i n formed would v e r y l i k e l y possess r a d io a c t iv it y from th e form ate
I n p o s itio n s e th e r than m ethoxyl groups, subsequent dem ebhyl&tioa of th e
l i g n i n g iv in g a lo w e r recovery o f r a d io a c t iv it y *

This is a p o s s ib le

e x p la n a tio n of t h e low r e s u lts ob tained*
Elwyn and co-workers found th a t i n th e synthesis o f m ethyl groups
from s e rin e t h e jB -carb o n does not go through th e o x id a tio n le v e l o f
fo rm ate *

The fS -carb o n o f s e rin e has an o x id a tio n s t a te midway between

t h a t o f format© and a m ethyl group*

Hence i t might be expected, i f th e

l a b i l e m ethyl group were t o be o x id is e d to t h i s stage and th e n reduced
a g a in t o a m ethyl group, t h a t th e s p e c if ic a c t i v i t i e s o f li g n i n from
p la n ts fe d form at© and those fe d m ethionine would b© of about th e same
o rd e r*

Since t h e d if fe r e n c e i n s p e c if ic a c t i v i t i e s is so la r g e , i t would

seem lo g ic a l t h a t th e m ethyl group is tr a n s fe r r e d w ith o u t t h is o x id a tio n
and re d u c tio n *

T h is aspect could b© f u r t h e r in v e s tig a te d by fe e d in g th e

-1 9 -

p la n ts la b e lle d s e rin e and comparing t h e a c t i v i t y o f th e li g n i n from
th e s e p la n ts wi th t h a t from p la n ts i n th e fo rm ate and m ethionine
experim ents*
I n suoh a comparison as t h a t made between li g n i n from p la n ts fe d
m eth ion ine and fo rm ate * s e v e ra l com plications may a ris e #

I n o rd e r t h a t

a f a i r comparison can be mad© i t is necessaiy# among o th e r fa c to rs *
t h a t t h e ra te s o f u p ta k e of chemicals fe d be about th e same and t h a t th e
s i 2 © o f th e pools o f th e s e chem icals i n the p la n t remains r a th e r constant#
I t is p o s s ib le t h a t a v a r i a t i o n I n thes© fa c to rs could a p p re c ia b ly a f f e c t
th e r e s u lts and c o n c lu s io n s .

However* th e co n d itio n s were kept as

c o n s ta n t as p o s s ib le * and s in es a d iffe r e n c e in s p e c ific a c t i v i t i e s of
mor© th a n one o rd e r of magnitude m s observed# th e s e conclusions appear
to be v a l i d *
The r e s u lts o b ta in e d i n th e presen t study d i f f e r g r e a t ly from th o se
o f Kirkwood and Marion#

They found t h a t r a d io a c tiv e form at© fe d to

b a r le y p la n ts m s a p p re c ia b ly in co rp o rate d in t o the m ethyl groups o f
hordenlne* but t h a t r a d io a c tiv e m ethyl groups o f c h o lin e fe d to th©
p la n ts war© n o t*

I n t h i s la b o ra to ry Wing (2 4 ) found t h a t , i n 24 hours*

an e x te n s iv e d e s tr u c tio n o f c h o lin e i n n u tr ie n t s o lu tio n in o c u la te d
w ith ro o t fragm ents had ta k e n p la c e * in d ic a tin g b a c t e r ia l a c t iv it y #
Since t h e above authors make no m ention of precautions ta k en a g a in s t
b a c t e r i a l growth# i t

seems pro b ab le t h a t th e e h o lin e was destroyed by

b a o t e r ia l a e tio n b e fo re i t Gould be absorbed by th e p lan ts#
T h at th © r a d io a c t iv it y was present in t h e mebhoxyl group and n o t I n
m eth ion ine o r any o th e r nitrogenous component m s proved by p r e c ip it a t in g

"*

20 —

aldehydes formed upon o x id a tio n of l i g n l n as 2 * 4 -d in itro p h en ylh vd raao n es
from a n ib ro g e n -fr© o s o lu tio n .

For a convenient b a s is o f comparison*

th e a c t i v i t y found i n th® phenylhy&mson© m ix tu re was c a lc u la te d as
s p e c if ic a c t i v i t y o f v a n i l l i n and t h is was found t o b© on th e same o rd e r
as t h a t o f t h e l i g n l n which m s oxidized#

I f much o f th e r a d io a c t iv it y

i n th e l i g n i n were a s s o c ia te d w ith th e n itro g e n p re s e n t, th e s p e c if ic
a c t i v i t y o f t h e v a n i l l i n would be a p p re c ia b ly low er th a n t h a t o f the
l i g n i n used f o r o x id a tio n #
Th© re s u lts o f the presen t study g iv e evidence t h a t i n II* vu lg ar®
tra n s m e th y la tio n is in v o lv e d i n fo rm atio n o f lig n in *

Although th e r e Is

evidence t o d is p ro v e £h@rebovf s statem ent t h a t l i g n i f i c a t i o n Is m erely
an accum ulation o f m ethoxyl groups o r a proc©as o f m e th y la tio n (2 5 )# i t
is p rob ab ly tr u e t h a t most th e o rie s o f l i g n i n fo rm a tio n do not p la c e
due emphasis upon the im portance of th e r o le tra n s m e th y la tio n m y p la y
i n th e process*

Evidence f o r d ir e c t m e th y la tio n appears to be more

convincing than t h a t supp orting Klasonf s th e o ry th a t form aldehyde acts
as t h e p re c u rso r o f mefchoxyl groups*

B efo re th© process o f li g n i n

fo rm a tio n is understood th o ro u g h ly a g re a t deal more experim ental work
is necessary*

"Tore l i g h t could probably be dried on th© problem by

fe e d in g p la n ts carbohydrates la b e lle d in th e carbon c h a in a n d /o r i n
mefchoxyl groups*

D e te rm in a tio n o f th© lo c a tio n of r a d io a c t iv it y I n

l i g n i n synth esized by these p la n ts would g iv e a f u r t h e r in d ic a tio n o f
t h e manner i n which th© li g n i n is produced#

•*21**

s irm n Y

s ir m a Y

R a d io a c tiv e m eth ion ine m s ad m in is te re d to b a rle y p la n ts *
is o la te d from th ese p la n ts possessed r a d io a c tiv ity #

L ig n in

D egradation

showed t h a t p r a c t ic a l ly a l l o f th is a c t i v i t y m s lo c a te d In the
m othoxyl groups*

L ig n in w ith a lo w e r le v e l o f r a d io a c t iv it y m s a ls o Is o la te d from
b a r le y p la n ts which had been fe d r a d io a c tiv e form ate*

Most o f t h is

a c t i v i t y was found i n th e msthoxyl p o s itio n *

On th e basis o f these r e s u lts I t is p o s tu la te d t h a t th e m ethyl group
I s tr a n s fe r r e d as an e n t i t y , w ith o u t o x id a tio n and re d u c tio n ta k in g
p la c e *

I t Is f u r t h e r p o s tu la te d t h a t fo rm a te , as such, is not tra n s ­

f e r r e d , b u t t h a t re d u c tio n t o a m ethyl group is f i r s t undergone*

BIBLIOGRAPHY

BIBLIOGRAPHY

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Chem., 1 3 4, 787-8 (1 9 4 0 ).
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Can. J . Chem., 2 9 , 30-6 (1 9 5 1 ).

( 3 ) S . A* Brown, ^S tudies on M e th y la tio n R eactions in P la n ts : Th© O r ig in
of t h e M ethyl C arton o f N ic o tin e Formed. by H ic o tla n a R u s tlc a ”
Ph. 0 . T h e s is , M ichigan S ta te C o lle g e , 1951.
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(1 0 ) H. Ost and L . W llk e n in g , Ch@iQ.-3tg., 3 4 , 461 (1 9 1 0 ).
(1 1 ) D. IfecB ougall and W. A. Dehong, Can. J . Research, 268, 457-63 (1 9 4 8 ).
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E d ., 4 , 2 1 6 -7 (1 9 3 2 ).
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Chem., 1 4 9 , 519-25 (1 9 4 3 ).
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E d it io n , P. B la k is to n 's Bon and C o ., I n c . , P h ila d e lp h ia , 1937,
pp. 1 7 1 -8 4 .
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APPENDIX

Th© fo llo w in g forcmala was used f o r th e c a lc u la tio n of percentage
reco very o f counts*

Cc (q u a t# ) /

% recovery *

____ n.r

a

C0 ( l i g n i n ^

.....X..... 1Q.Q.. , , , x 200,
x

9g

where Cc ( q u a t , ) 9 observed count o f qu aternary compound (c#p#m#)

a » f r a c t i o n o f xaaximm s p e c ific a c t i v i t y a t sample th ic k n e s s
used— from s e lf-a b s o r p tio n curve f o r q u atern ary compound,

C0 ( l i g n i n ) * observed count o f li g n i n (c#p#m«),

b 3S f r a c t i o n o f maximum s p e c ific a c t i v i t y a t sample th ickn ess
used— from s e lf-a b s o r p tio n curve f o r l i g n i n ,

jy?S - c o r r e c tio n f a c t o r , based on 95^ recovery o f m ethoxyl
groups from v a n i l l i n .

Sample c a lc u la tio n s
Co ( q u a t * ) *
a

»

CQ ( lig n in ) ®
b

n

% reco very *

741 c.p#m*
#584
467 c#p*m«
#525

i f Z ~ ~ ------ 2------i2 2 ---- x 100 =
^6V * 3 2 S
x
95

92.