The preparation of synthetic mustard oil glycosides and the specificity of the myrosinase system
by Robert D Gaines
A THESIS Submitted to the Graduate Faculty I N PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in Chemistry
Montana State University
© Copyright by Robert D Gaines (1960)
Abstract:
Synthetic mustard oil glycosides were prepared and characterized IN ORDER TO DETERMINE THE
SPECIFICITY OF THE GLYCOSIDIC ENZYME FOUND IN THE MYROSI NASE SYSTEM.
THESE GLYCOSIDES CONTAINED IN THE GLYCONE MOIETY GLUCOSE, GALACTOSE,
MANNOSE AND XYLOSE. THE SYNTHETIC GLUCOSE COMPOUND WAS FOUND TO BE
IDENTICAL WITH THE NATURALLY OCCURRING MUSTARD OIL GLUCOS I DE,
GLUCOTROPAEOL I N.
During purification of the myrosinase system, there was observed TWO ACTIVE FRACTIONS, A
THIOGLUCOSIDASE AND A FACTOR POSSESSING SULFA-TASE ACTIVITY. A BRIEF
CHARACTERIZATION OF THE LATTER FACTOR WAS MADE AND INDICATED A
DEFINITE SPECIFICITY FOR SULFATE ESTERS OF THE TYPE EXISTING IN THE MUSTARD
OIL COMPOUNDS.
The SPECIFICITY OF THE MUSTARD THIOGLUCOSIDASE WAS FOUND NOT TO BE AS
ABSOLUTE AS PREVIOUS REPORTS HAD INDICATED. THE ENZYME SHOWED A
DEFINITE PREFERENCE FOR THE GLUCOSE MOIETY AS THE GLYCONE GROUP, BUT IT
POSSESSED AN OVERALL ACTIVITY VERY SIMILAR TO THE β-GLUCOSIDASE OF
ALMOND EMULSIN. IT WAS FOUND THAT THE MUSTARD ENZYME WOULD, UNDER
CERTAIN CONDITIONS, HYDROLYZE β-GLUCOSIDES AND β-THIOGLUCOSIDES, IN
ADDITION TO THE MUSTARD OIL DERIVATIVES. THESE RESULTS WERE INTERPRETED
AS MEANING THAT THE ACTUAL DIFFERENCE BETWEEN A THIOGLUCOSIDASE AND A
GLUCOSIDASE IS PROBABLY RATHER LIMITED. THE PREPARATION OF SYNTHETIC MUSTARD OIL GLYCOSIDES
AND
THE SPECIFICITY OF THE MYROSI NASE SYSTEM
BY
ROBERT D. GAINES
A THESIS
Subm
itt e d
to
Gr a d u a t e
the
Faculty
IN
PARTIAL
FULFILLMENT
FOR T H E
Doctor
of
OF
THE
DEGREE
Ph il o s o p h y
in
R E Q U I RE M E N T S
OF
Ch em istr y
at
Mo n t a n a
A
pproved
College
;
jor
D
epartment
XAMINING
Dean,
St a t e
Gr a du
M MlTTEE
Di v i s i o n
Bozeman
J
une
Mo n t a n a
,
1960
A
I
WISH
J.
Go e r in g
IN
GRADUATE
In
Sc ie n c e
IN
T
for
h is
,
o u n d a tio n
hanks
are
s in c e r e
g u id a n c e
I
would
l ik e
,
whose
research
also
DR.
AND TO
in a lly
agement,
INDEED
TO CONVEY MY DE E P E S T
and
GRATITUDE
c o n s id e r a t io n
DR.
TO
d u r in g
KENNETH
my
years
SCHOOL.
INSTRUMENTAL
F
TIM E
to
express
my
a p p r e c ia tio n
grant
made
p o ssible
to
the
the
work
Na t i o
nal
d e s c r ib e d
T H E S l S.
ADVICE,
THE
THIS
a d d it io n
F
THIS
AT
cknowledgements
,
I
to
GREAME BAKER
Dr .
FOR
J.
Do n ald
Reed
for
h is
HI S GENEROUS A S S I S T A N C E
welcome
DU RI NG
WORK.
would
PATIENCE
DIFFICULT
extended
AND
TO
l ik e
to
thank
HELPFULNESS,
my
w if e
WITHOUT
COMPLET E MY GRADUATE
,
El
iz a b e t h
W H I CH
PROGRAM.
IT
,
for
WOULD
her
encour­
HAVE BEEN
—
Table
L
is t
of
Ta
L
is t
of
F
A
bstract
I.
T
b le s
4_ '
Contents
of
.........................................................................
ig u r es
I
and
l lu s tr a tio n s
6
............... .. ............................................................. ..
7
..............................................................................................................................
Pr e p a r a t i o n
of
A .
I
............... .. .....................................
10
B.
Ex
................................................................
16
he
ntr o d uc tio n
p e r im e n t a l
Sy n t h e t ic
Mu s t a r d
O
Gl
9
il
y c o s id e s
1.
T
he p r e p a r a t io n
of p h e n y l a c e t o h i o h y d r o x a m i c
2.
T
he p r e p a r a t io n
of
3.
T
he p r e p a r a t i o n
of t h e
4.
Sa
p o n if ic a t io n
of
5.
Su
l f o n a t i on
the
of
<x -
acetohaloglycoses
the
. ...........................
a c id
. . . . . .
......................................
a c e ty lth io g ly c o s id e s
t h i ohydro xamic
18
19
............... .....
a c e t y lt h io g ly c o p y r a n o s id e s
22
. . . . . .
Sa
7.
I
p o n if ic a t io n
so latio n
of
n a s tu r t iu m
C.
II.
TfiE
I
nfrared
Sp
d ata
e c if ic it y
of
the
the
acetyl
mustard
glucotropaeolate
seeds
ion
o il
g ly c o s id e s
24
...
My r o s i n a s e
the
S y s t e m ........................................ ..
A.
I
B.
Exrer
i m e n t a l ... ......................................................................................... ..
1.
A
ssay
2.
Su
3.
Pr e p a r a t i o n
4.
I
5.
Sp
ntr o d uc tio n
. , . . ...................................... .................... .. .................................................... ...
procedures
bstrates
n h ib it io n
e c if ic it y
of
of
p u r if ic a t io n
My r o s i n a s e
the
by
Su lf a t a s e
of
m y r o s in a s e
..........................
P h o s p h a t e ............ .. ................. ..
Fac to
r
31
32
44
44
47
47
......................................................................
. .............................. .. ........... ................................................................ ...
and
28
from
............ .. .................... ............................ ............................. ..
......................................... .............. .. ......................................................................
of
24
a c id
A C E T Y L G L Y C O S I D E S . . . ....................................................... ................................
6.
10
............................................... ..
48
48
56
57
-
Table
6.
Sp
e c if ic it y
Contents
of
of
the
T
.
T
he
p u r if ic a t io n
B.
T
he
EFFECT
a
MUSTARD
C • Dl SCUSS
O IL
5-
OF
(Co n t . )
h io g lu c o s id a s e
of
almond
of
e m u ls in
Mu
stard
^ -
EMULSIN ^ - G L U C O S ID A S E
............... ..
g lu c o s id a s e
58
. .
65
ON T HE
G L Y C O S I D E S . . ............................................ .................... ..
I O N ......................................................................................................................... ..
65
71
111 . S u m m a r y . ........................................................... .. ............................................................................................. ..
83
IV.
86
L
ite r a tu r e
C
it e d
........................................................ .................................................... ...
—
L
I .
T
he
Re a c tio n s
||.
Sa
p o n if ic a t io n
I I I .
Pu
r if ic a t io n
IV.
Hy d
Fr
V.
V I.
T
T
he
V II.
Hy d r o
Ef f e c t
T
he
Pu
X.
T
he
Gl y
of
...........................................
26
...................................................................................................
54
T
h io g ly c o s id e s
Separate
by
Co m b in e d
and
My r o s i n a s e
n h ib it io n
c o s id e s
im e
T
-
sulfonates
M y r o s u l f a t a s e ............ .. ..
by
h io g lu c o s id a s e
T
h io g lu c o s id a s e
on
of
of
Mu s t a r d
of
A
Em
O
lmond
u ls in
il
Em
Gl y c o
u ls in
/5-G l u
T
h io g lu c o s id a s e
/S-Gl u
of
ANDyS-Gl u
by
My
r o s in a s e
c o s id a s e
by
................
..............................
Mu s t a r d
O
c o s idase
A
Va
r ia t io n
on
A
62
64
66
il
68
c t iv it y
... ............................................................
Glyco ne
60
Sy n t h e t ic
s id e s
c o s idase
59
Va r i o u s
on
. . .................................................................................................................................
nase
T HE E f f e c t
Ox
............................................ ' • ........................................................................
r if ic a t io n
My r o s i
12
in ig r a t e
55
Mu s t a r d
ly s is
Co m p a r a t iv e
of
X II.
I
r o s in a s e
Mu s t a r d
of
h io g ly c o s i d e s
IX .
........................................
the
S
ilver
.................................' .............................................................................................................
c t iv it y
Hy d r o
S
and
of
in ig r in
of
T
he
X l.
S
of
A
T
My
of
ly s is
bstrates
V III.
in ig r in
Pr o d u c t s
Tables
of
.......................
a c t io n s
he
Su
r o ly s is
S
of
is t
6—
70
lmond
^ - G L U C O S l D A S E . . . . ................................................i ............................................. .. ................. .. ...
73
-
L
F
ig u r e s
is t
of
ig u r es
and
I
llu s tr a tio n s
:
1.
Sa
p o n if ic a t io n
of
A
2.
Chromatography
of
Pa
3.
El e c t r o p h o r e s i s
I
F
7-
llu s tr a tio n s
:
I
.....................................................
25
............................................
51
...............................................
53
.................................................. .. ........................................... ..
32
c e t y lt h io g ly c o s id e s
Pa t t e r n
nfrared
Pu
r t ia l l y
of
Spec
Pu
tra
r if ie d
r if ie d
....'.
My r
My r
o s in a s e
o s in a s e
1.
P H E N Y L A C E T O T H l O H Y D R O X A M I C AC I D
2.
P H E N Y L A C E T O T H I OHYDROXAMI C
3.
P H E N Y L A C E T O T H I OHYDR OXAMI C AC I D - S - j B - D - 1 - T E T R A A C E T Y L G A L A C T O P Y R A N O S I DE
4.
P H E N Y L A C E T O T H I O H Y D R O X A M I C A C I D - S - J 3 - D - 1 - T R I A C E T Y L X Y L O P Y R A N O S I DE
5.
P H E N Y L A C E T O T H I OHYDROXAM I C A C I D - S - ^ - D - I - T E T R A A C E T Y L M A N N O P Y R A N O S I DE
6.
P H E N Y L A C E T O T H l OHYDROXAMI C AC I D - S - J S - D - I - G A L A C T OPYRA N OS I DE
7.
P H E N Y L A C E T O T H I OHYDROXAMt C
8.
P H E N Y L A CETOTH I OHYDROXAM I C A C l D - S - ^ - D - I - M A N N O P Y R A N O S I D E
9.
P H E N Y L A C E T O T H I OHYDROXAMI C AC I D- S- JSj- D - I - GL U C O P Y R A N O S I DE
AC I D - S - ^ - D - I - T E T R A A C E T YL G L U C O P Y R A N O S I DE
AC ID -S -^-D -I-X Y LO P Y R A N O S ID E
10.
T
etr am eth ylam m o n iu m
tetraacetylglucotropaeolate
(
s y n t h e t ic
)
11.
T
etr am eth ylam m o n iu m
tetraacetylglucotropaeolate
(
natural
I 2.
S - / 3 - D - 1 - T E T R A A C E T Y L G A L A C T O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAM I C AC I D -
)
0 - T E T R A M E T H Y L A M M O N I UM S UL F O NA T E
13.
S - / 3 - D - 1 - T R I A C E T Y L X Y L O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAMI C
T E T R A M E T H Y L A M M O N I UM
I 4.
A C ID-O -
SULFONATE
S - j S - D - l - T E T R A A C E T Y L M A NN OP Y R A NO S Y L - P H E N Y L A C E T O T H I OHYDRtl XAM I C AC I D O - T E T R AME T H Y L A MM ON I UM S U L F O N A T E
15.
S - / 3 - D - 1 - M A N N O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAMI C AC I D - O - T E T R A M E T HYL AMMO NI UM
SULFONATE
—
I 6 .
S-^/3 -D -l
- G A L A C T O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAM I C A C I D - O - T E T R A -
MET HYL AMMONI UM
I 7 .
8~
SULFONATE
S -/3- D - 1 - X Y L O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAM I C A C I D - O - T E T R A M E T H Y L AMMON I UM S U L F O N A T E
18.
I E T R A M E T H Y L A M M O N I UM GLWCOTROPAEOL ATE
(SYNTHETIC)
19.
T E T R A M E T H Y L A M M O N I UM G L U C O T R O P A E O L A T E
(NATURAL)
-
A
Sy n
th e tic
IN
ORDER TO
IN
T HE
mustard
DETERMINE
MYROSINASE
MOIETY
GLUCOSE,
GLUCOS I DE,
Du r in g
TWO A C T I V E
p u r if ic a t io n
FRACTIONS,
INDICATED
EXISTING
T
he
IN
A
IT
BRIEF
AS
TO T H E
OF
and
CONTAINED
THE
c h a r a c te r ize d
GLYCOSIDIC
XYLOSE.
W ITH
m y r o s in a s e
S P EC IFIC ITY
THE
THE
ENZYME
IN
T HE
SYNTHETIC
NATURALLY
FOUND
GLYCONE
GLUCOSE
OCCURRI NG
MUSTARD
system
AND A
FOR
,
there
FACTOR
OF T HE
LATTER
SULFATE
was
observed
POSSESSING
E ST ERS
SULFA-
FACTOR WAS MADE'
OF THE T Y P E
COMPOUNDS.
MUSTARD T H I O G L W C O S I D A S E WAS FOUND
REPORT S
FOR T HE
HAD
INDICATED.
GLUCOSE M O I E T Y
A C TIV ITY
VERY
AS
SIM ILAR
THE
THE
ENZYME
GLYCONE
NOT TO
SHOWED
GROUP,
TO T HE ^ 3 - G L U C O S I D A SE
T HE MUSTARD
ENZYME WOULD,
UNDER
HY D RO L Y Z E ^ - G L U C O S I DES AND ^ - T H I O G L W C O S I DE S ,
MUSTARD
PRET ED AS M E A N I N G
SIDASE
the
I T WAS FOUND T H A T
CONDITIONS,
ADDITION
of
PREVIOUS
PREF EREN CE
EMULSI N .
CERTAIN
MANNOSE AND
IDENTICAL
O IL
P O SSESSED AN O V E R A L L
ALMOND
GLYCOSIDES
CHARACTERIZATION
MUSTARD
SPEC IFIC ITY
DEFINITE
THESE
prepared
OF T HE
A THIOGLWCOSIDASE
A DEFINITE
THE
BE A S A B S O L U T E
A
were
G L U C O T R O P A EOL I N .
TASE A C T I V I T Y .
AND
g ly c o s id e s
GALACTOSE,
BE
bstract
SP E C IFIC ITY
SYSTEM.
COMPOUND WAS FOUND TO
O IL
o il
THE
9-
T HAT
O IL
T HE
AND A G L U C O S I D A S E
IS
DERIVATIVES.
ACT UAL
THESE
DIFFERENCE
P RO BABL Y
RAT HER
R E S U L T S WERE
BETWEEN A T H I O G L U C O LIM ITED .
IN
INTER­
BUT
OF
—I 0 —
Pa r t
T
Pr e p a r a t i o n
he
Sy n th e tic
of
A o
T
he
MIDDLE
THE
d is c o v e r y
OF
T HE
SEEDS
OF
COMPOUNDS
GANIC
HAVE
RECOGNIZED A S S O C I A T I O N
O IL
LIBERATED
THAT
IF
BRING
DID
THE
W ITHIN
1831
T HE
FROM
F REE
IN
T HE
PLANTS
IN
DENATURATION
FROM
CALLED
WAS
PRESENT
PL A NT
SEEDS
OF
CRUSHED
PREVIOUSLY
FROM
OF T HESE
( l) .
BECAUSE
FAM ILY,
r e c o g n ize d
THEY
TREATED W IT H
OR­
THESE
OF T H E I R
CRUCI FERAE.
that
B L A CK M U S T A R D ,
T HE RE WAS NO MUSTARD
the
SOME T H I R T Y
OILS
SEEDS.
to
ISOLATED
MATERIALS
OF T HE MUSTARD
(3)
dates
OCCURRENCE
MUSTARD
Faure
also
THE
AND AT
PLANTS
and
SEEDS WERE
OF
PROVIDED
AS
A D ER IVATIVE
THE
KNOWLEDGE T H A T
FIRST
OF
allyl
BUT WAS
ALSO
OBSERVED
REAGENT S WHI CH
OIL
LIBERATED
OF
COMPOUNDS,
HOWEVER,
UNTIL
AS T H E
SEEDS.
POTASSIUM
BUS S Y
A WATER-SOLUBLE
SALT,
P-HYDROXYBENZYL
T HE MUSTARD
PRODUCTS
THESE
B L A C K MUSTARD
S IN ALBIN ,
CONJUGATION
ISOLATING
WAS T R E A T E D W I T H
ALLYL
WITH
(2)
T IM E,
ISOLATED
COMMONLY
is o th io c y a n a te s
BY
OF W A T E R .
KNOWN ABOUT
SUCCEEDED
OCCUR
PROTEIN
ISOLATION
he
IN
NOT
MACERATED
ADDITION
O IL,
Robiq u et
Gl y c o s i d e s
il
ISOTHIOCYANATE
REPORTED,
WHEN WATER WAS ADDED TO T HE
ABOUT
"T
o ccurring
BEEN
ARE
O
ntro ductio n
S I NCE T H A T
FREQUENTLY
ISOTHIOCYANATES
MUSTARD
WAS
MUSTARD.
BEEN
and
Mu s t a r d
CENTURY WHEN A L L Y L
B L A CK
HAS
Boutron
THE
LAST
I
naturally
ISOTHIOCYANATES
ORGANIC
LONG
of
I
GLUCOSE
(4).
OILS
OCCURRED
VERY
LITTLE
1 8 4 0 WHEN B U S S Y
A CRYSTALLINE
DEMONSTRATED T H A T WHEN T H I S
PROTEIN
I S O T H I O C Y A N A T E WAS L I B E R A T E D .
THE
E XT RACT
FROM T H E
G L U C O S I DE WAS
MUSTARD
(5)
GLUCOSIDE
GLUCOSIDE
GROUND
SEEDS ,
NAMED AS THE
SALT
-
OF A COMPLEX
THE
OR G A N I C
HYDROLYSIS
OF T H I S
SULFATE
AS A
F UR T H E R
ATTEMPT
IS
KNOWN AS
WAS
l
hey
cyanate
ALLYL
,
that
CARRIED
T
he
LARGELY
OUT
fir s t
THE
MOLECULAR
S IN IG R IN
HYDRATE
the
AND
NUMBER
a
TABLE
I
proposal
PROVIDED
RAPID
RESIDUE.
S T RUC T U RE S FOR
IN
VERY
BUT
FOR
of
NO
OF T H I S
AMOUNTS
to
(7)
be
COMPOUND.
allyl
OF
BUT
AND KORNER
s in ig r in
p r in c ip a l ly
ILLUSTRATES
MADE
SOME T I M E ,
AND BY W l L L
formula
SMALL
D ET ECT ED
G L U C O S I D E WHI CH TODAY
OF R E A C T I O N S
(9)
T HAT
T HAT
is o t h io
SUL FUR
THE
REACTIONS
was
made
VIEW
( I )
AND
S IN IG R IN
OF
­
AND
SIN IG R IN
OF
STRUCTURAL
MOIETY
RESULTS,
SILVER
Ga d a m e r
GADAMER
(8)
CONF I RMED
AS C ^ g H ^ g O g N S g K ' H g O .
SIN IG R IN
UNIT
ACTION
WAS
by
WAS A MONOHYDRATE AND
RE P R E S E N T E D
BY T H E
OF T H E S E
AND K o R N E R .
CLEAVAGE
THIS
SULFATE
s in ig r in
BY W l L L
A F F O RD E D
T HE
S IN IG R IN
(6)
produced
for
ENZYMATIC
EVIDENCE
SHOWED T H A T
BUSSY ALSO
FOR
INVESTIGATORS.
AS M E A N I N G
he
L A N GE
RESPONSIBLE
MYRONAT E,
OF T H I S
e m p ir ic a l
FORMULA WAS CORR ECT L Y
ISOTHIOCYANATE
T
ST RUC T U RE
A L T HOUGH
OF B l R K E N W A L D
INTERPRETED THE
GLUCOS I D E .
EARLY
EVIDENCE
OBSERVATION
MYROSIN.
POTASSIUM
d eg r adatio n
KHSO^,
structural
ON THE
OF
LUDWIG
observed
WERE F OR M E D.
THE
HE
BY
en zym atic
BY T HE
T HE
INVESTIG ATED
GLUCOSE AND
CYANIDE
PRODUCT
ENZYME
T H E WORK OF B U S S Y WAS D I S P U T E D
CONFI RMED
AND
AND THE
COMPOUND WAS C A L L E D
ELUCIDATE
Korner
and
found
TO
SI N I G R I N .
C ioH igO io N S gK
T
MYRON I C A C I D ,
DECOMPOSITION
EVENTUALLY
Wil
ACID,
11-
OF
S I N I GRATE
YIELD
WAS P RESE NT
SILVER
NOT A T T A C H E D
GADAMER
TO
ALLYL
I N THE
NITRATE
TO THE
ON
CARBO­
PROPOSED T HE F OL L O W I N G
( I I ):
-
12-
Table
T
he
Re a c tio
ns
of
My r o s i n
S
in ig r in
+
MgG
I
and
^
S
ilver
S I NlGR I n •
H+ , A
------------- >
AGNOg
^
in ig r a t e
C H g - CHCHg NCS +
(trace
H2 O ,
S
OF S
CH2= C H C H 2 CN +
KHSQ^ +
and
CgHigGg
CHg CHCHgCN)
Hg S +
KHSO4 +
CgH^Og
C 4 H ^ O 4 NS2 A G g 4- C g H - j g O g
BaCl ,
CHgCM=CHCOOH + BaSO4 + CgH12Og
Ba (OH),
CH2=CHCH2NCS + BaSO4 + CgH12Og
(
small
-Cl " ,
a
^a m o u n t )
-> CHg=CHCHgNCS + 2AgCl + HgSO4
S I LVER
Si NI GRATE
'A
c id ic
R e a g e n t s —>
CH2=CHCH2CN + H2SO4 + S +
A g2 S
-
13S-Ag
CH2=CHCH2N=Cx
1OSO3Ag
CH2=CHCH2N=Cx
1OSO3K
II
S I NALBlN
BE
AND A L L
OF T H E
SAME
OTHER
STRUCTURE,
C O RR ESPON DI NG TO T H E
De
ALL
s p it e
OF T HE
WITHOUT
THETIC
SIDES
MUSTARD
the
fact
REACTIONS
REVISION
FOR
FROM T H E
G L U C O S I D E S WERE ASSUMED
BUT W I T H
PARTICULAR
that
the
SOME V A R I A T I O N
MUSTARD
Ga d a m e r
KNOWN FOR T HE
NEARLY
APPROACH TO T H I S
( i l l )
O IL
SIXTY
PROBLEM
SILVER
ISOTHIOCYANATE
O IL .
structure
MUSTARD
YEARS.
O IL
could
not
COMPOUNDS,
SCHNEIDER
PREPA RED A
SALTS
OF T H E
BY ANALOGY TO
SERIES
ET A L
account
IT
for
REMAINED
(lO )
IN A SYN­
OF T H I O U R E T H A N E
GLUCO-
OF T H I O U R E T H A N E S AND A CET O BRO MOG L U CO SE.
111
T
compounds,
hese
blance
LINE
TO T HE
MUSTARD
THIOURETHANE
MODE OF
AND
modeled
IT
OIL
INTEREST
THESE
T HAT
OTHER ( B - I - T H I O P Y R A N O S I D E S
COSI DES.
T HE A C T I O N
P - T H I OPYRA NOS I D E S
FOR T HE
OF
g er
and
IN
NOT
structure
CHEMICAL
CLEAVED
,
showed
BEHAV IO R.
BY M Y R O S I N .
no
THE
resem­
CRYSTAL­
FROM T H E I R
G L U C O S I DES WERE T A K E N TO BE j B - T H I O P Y R A N O S I D E S
THE
OPTICAL
C L OS E L Y
(12)
ROTATIONS
RESEMBLE
NITRATE
MUSTARD
OF THE
Lundeen
Ga d a m e r
GLUCOSIDES
SILVER
AND T HE
^-CONFIGURATION
Et t l i n
the
G L U C O S I D E S WERE
PREPARATION,
I S OF
after
O IL
NAT UR AL
TO
THOSE
OF T H E S E
OF T HE MUSTARD
L I B E R A T E Ot-D-GLUCOSE
G L U C O S I D E S WAS T A K E N
GLUCOSIDES
subjected
COMPOUNDS AND
s in ig r in
AS
OIL
GLU-
FROM THE
EVIDENCE
( ll) .
to
r e d u c tiv e
cleavage
-
14-
AND HYDROGENATION OF THE OLEF I NI C BOND AND OBTAINED N-BUTYL AM I NE AS A
PRODUCT OF THE REACTION.
THESE WORKERS ALSO SHOWED THAT WHEN S I N I G R I N
WAS HYDROLYZED WITH 3 N HgSO^,
ACETI C AC I D AND HYDROXYLAMINE
IN
ORDER TO
COSE MO I E T Y }
OLYSIS
ADDITIONAL
POLYGALITOL
SHOWN TO
GLUCOS I DES WERE
FINDING S,
IN AD D I T I ON TO GLUCOSE AND INORGANIC SULFATE.
EVIDENCE
S IN IG R I N TETRAACETATE
AND Y I E L D E D
PREVIOUSLY
OI L
OBTAIN
THE PRODUCTS OF THE REACTION WERE VINYL
AS A
ESTABLISHED
Et t l i n
GLUCOSl DES
EXIST
g er
and
AS
WAS
FOR T HE
S U B J E C T E D TO R A N E Y
TETRAACETATE.
SIX
STRUCTURE
MEMBERED
SlNCE
RING
proposed
a
new
NICKEL
P O L Y G A L I T OL
(13),
THE
I - T H I O G L U C O P Y R A NOS I D E S .
Lundeen
OF T HE
structure
HYD ROGEN HAS BEEN
MUSTARD
BASED
for
GLU­
OIL
ON T H E I R
the
mustard
( I V ).
/ S-C6H11Q5
R-C.
^N-OSO3K
T
h is
structure
s a t is f ie d
CONCURRED W I T H
T HE
not
PREVIOUS
SUGGESTED A REARRANGEMENT
UCTS
BY T HE A C T I O N
I
TO
order
ESTABLISH
GLUc o s i d e s ,
Et t l i n
COMPOUNDS.
T
STITUTED
THE
he
and
PROPOSED
THIOHYDROXAMIC
PREPARATION
REVEALING
g er
OF A
A LOGICAL
the
newly
EXPERIMENTAL
OF T H E
OBTAINED
n
only
LOSSEN
e s ta b lis h e d
e v id e n c e
WORK ON SI N I G R I N .
TYPE
( l 4)
,
but
also
T H E S E AUTHORS
TO ACCOUNT
FOR T HE
PROD­
OF M Y R O S I N .
t h e ir
proposed
Lundeen
attempted
STRUCTURE
A CID.
structure
( IV )
CAMBI
STABLE S -B EN Z Y L
APPROACH TO T HE
the
for
the
s y n th e s is
MAY BE R E C O G N I Z E D
(15)
SOME YEARS
DERIVATIVE
PROBLEM.
OF
USING
of
mustard
one
of
these
AS A Dl SUB­
EARLIER
HAD REPORTED
SUCH A COMPOUND,
THE
o il
SO-CALLED
T HUS
CLASSICAL
-
methods
OF G L Y C O S I D E
SYNTHESIZING
A COMPOUND,
R E S P E C T S TO T H E
BENZYL
MUSTA1RD
HYD RO L YZ ED
It
T HE
IS
BE USED
IN
THE
NAT UR AL
O IL,
ETTLIN GER
AND
G LUCOT ROPAEOL I N ,
MATERIAL
WAS F I R S T
(16).
DETECTED
BY GADAMER
THE
WORK TO F URT HER
IN
OF A
OF T H I S
SERIES
ESTABLISHING
HYDROLYSIS
OF T H E S E
OF MUSTARD
T HE
IDENTICAL
(17)
ISOTHIOCYANATE,
O IL
T HE
AND
OF THE
IN
IN ALL
GLUCOSIDE
WHICH
ENZYME
OF
SHOWN TO BE
GLUCOSE AND
APPL Y THESE
GLYCOSIDES,
S P E C IFIC ITY
COMPOUNDS.
SUCCEEDED
G L UC OT ROPAEOL I N ,
BENZYL
PURPOSE
lUNDEEN
W H I C H WAS
BY M Y R O S I N TO Y I E L D
SYNTHESIS
TO
SYNTHESIS,
15-
KHSO4 .
METHODS FOR
IN
TURN ARE
SYSTEM
INVOLVED
-
T
he
methods
REQUIRED A
THESE
SUITABLE
WOULD
ACID
YIELD
A
QUITE
BY T R E A T I N G
ACID.
T HE
Ex p e r i m
in
the
ental
s y n th e s is
SIM ILAR
USED
TH IS
BY
of
the
AC I D A S T HE
INACCESSIBLE,
PRODUCT
CHOSEN WAS T H A T
THIOHYDROXAMIC
B.
THIOHYDROXAMIC
COMPOUNDS ARE
T HAT
T HE
in v o l v e d
16-
AND A
STARTING
TO A N A T U R A L L Y
ETTLIN GER
AND
D ITH IO -A C ID
g ly c o s id e s
LUNDEEN,
SlNCE
WAS R E Q U I R E D
OCCURRI NG
PREPARED
WITH
o il
MATERIAL.
DERIVATIVE
COMPOUND WAS F I R S T
C O RR ESPON DI NG
mustard
GLUCOSIDE,
PHENYLACETOBY C A M B I
HYDROXYLAMINE
(llT .
15)
HYD ROCHL OR -
I DE.
T
he
general
method
in vo lved
is
to
react
a
salt
the
AN ACET OBROMOGLYCOSE AND
(OR
A C E T Y L - G L Y C O P Y R A N O S Y L - P H E N Y L A C E T O T H I O H Y D R O X A M I C AC I D TO F U R N I S H
A
PRODUCT
SIM ILAR
BROMO- SUGARS
SIDES
(18)
(1 9 ).
TO T H E
HAVE
LONG
BY T H I S
T h is
TYPE
r e a c tio n
is
NATURALLY
BEEN
OF
a
REACTION,
ACID,
he
D I TH I O - AC I
WAS PREPA RED
EXCESS
OF
CARBON
ACT ED
RAPIDLY
CRYSTALLINE
T
T HE
he
TH I
d
BY
r eq u ir e d
DISULFIDE
for
(20).
AQUEOUS
GLYCOSIDES
I ON.
AND T H I O G L Y C O -
T HE K O N I G S - K N O R R
d is p la c e m e n t
and
ACETO-
y ie l d s
REACTION
the
^ -
s e r ie s
INVERSION.
BY T HE A D D I T I O N
WITH
KNOWN AS
b im o le c u la r
S -^-D -I-T E T R A
G L U C OT R O P A E O L A T E
USED TO S Y N T H E S I Z E
FROM T HE < ?- ACETOBROMOGLYCOSE
T
OCCU RRI NG
RESULTING
t h i o h yd r o xam ic
AC I D W I T H
T R I-)
SULFONATE THE
of
t h is
OF
THE
SYNTHESIS,
BENZYL
MAGNESI UM
ETHEREAL
HYDROXYLAMINE
d it h io
-
ph en ylac etic
CHLORIDE
TO AN
D I T H I O - P H E N Y L A C E T A T E RE­
HYDROCHLORIDE
TO F U R N I S H
P H E N Y L A C E T O T H I OHYDROXAMIC A C I D .
o h yd ro xam iC
CORR ESPON DI NG
a c id
ACETYLATED
reacted
WITH
T HE
TH I O G L Y C O S I DE .
acetohaloglycose
THE
REACTION
to
YIELDED
fu r n ish
ONLY
ONE
-
iso m er
,
S U P P O S E D L Y THE
AND A L S O
T
BY E T T L I N G E R
he
(21)
TO F U R N I S H
I ON S WERE
CATION
T HE
AS
y ie l d s
INDIVIDUAL
AS
(LlT=
T HE
FOR T HE
he
.
ACETYLATED
AS T HE
MUSTARD
OBSERVED
REACTIONS
VARIED
NO P A R T I C U L A R
T
fig u res
hese
COMPOUNDS W I L L
r e a c tio n
SUL F U R
OIL
BE
as
IN
OBTAINING
CONSIDERABLY
as
DISCUSSED
in vo lved
SALTS,
in
the
WITH
these
other
T HE
p h en ylac eto th io h y
I ON.
THE
BE CA U S E T HE
AND
data
SHOULD
NOT
p e r t a in in g
PREPARATIVE
p r e p a r a tio n s
A cetohaloglycose
ugOAc
WoSO^(CH3 ) 4
MeOH
NH
3
.S- Sug
NOSO"ft(CH3 ) 4
.S-SugOAc
NOSO3
-
LARGER
PRODUCT.
BE T A K E N
ON O B T A I N I N G
to
M AX­
the
P ROCEDURES.
may
be
.
KOH
F O L L O WS :
/S -S
15)
GLYCOSIDIC
A CRYSTALLINE
E M P H A S I S WAS PL A CED
well
(LlT=
BY C A M B I
TR I OX I D E - P Y R I D I NE ADDUCT
GLYCOSIDE
T ETRAMETHYLAMMONI UM
SINCE
overall
AS
16).
BY USE OF A
PROVED TO BE MORE ADV A N T A G E O US
CONCLUSIVE
T
AND LU NDEEN
SULFONATED
ISOLATED
YIELDS
IMUM
CONFIGURATION,
S-jS-D-l - t e t r a a c e t y l g l y c o p y r a n o s y l -
r e s u l t ing
D R O X A M I C AC I D WAS
ANTI
17-
d e p ic te d
—
As
METHODS
IN
IT
WAS
NE C E S S A RY TO
INVOLVED
ADDITION
IN
TO T HE
MANY
8.2 5
GM.
RE F E RE N CE
STIRRED
SUCH A RA T E
VIGOROUSLY
STIRRING
FRESHLY
CONTINUED
,
37 .5
IN
OF 3 0 . 5
T HAT
ADD ITION ,
WHICH
VIGOROUSLY W IT H
m l
STIRRED
WHILE
GM•
WATER WERE A D D E D .
I NG
TWO PHASE
RED
ETHER
THE
SOLUTION
PHASE WAS
C ongo R ed
STAND
ADD ITION
MATERIAL
OF
dry
ether
UNDER A
BENZYL
ADDITION
was
BE G I V E N
added
NITROGEN
RAPIDLY.
T HE
THE
ONE
IN
15 0 ML.
HOUR,
THE
THE
AND THE
WAS C O M P L E T E .
SOLUTION.
COLD,
UNDER
100
m l
.
SOLUTION
10$ HgSO^
W H I C H WAS REMOVED BY
IN
ICE
D ITH IO -
IN
AND R A P I D L Y
150
1 0 MINUTES
ML.
OF
I CE
AND THE
LONGER.
RESULT­
T HE DARK
PHASE AND T H E WATER
of
ether
.
T
he
OF THE
POTASSIUM
EXTRACTION.
ACID­
c om bined
EX T R A C T E D W I T H
PRODUCED A L AR GE
ETHER
OF
ROOM T E M P E R A T U R E .
VACUUM AND
A C ID IF IC A TIO N
O0 C . ,
STIRRED
FOR A FEW M I N U T E S
w ith
ABOUT
THE M I X T U R E WAS
WAS COOLED
AQUEOUS
T HE
DURING
AT
ABOUT
ATMOS­
M I X T U R E WAS
CHLORIDE
OF
■
to
ETHER.
ETHEREAL
OVERNIGHT
FROM T H E
extracted
TO CONGO RED W I T H
S E M I-SO LID
of
ETHER
REQUIRED
WAS S T I R R E D
E X T R A C T S WERE CO NCENT RAT ED
E XT RACT
.
H Y D R O X Y L A M I NE H Y D R O C H L O R I D E
ETHER
K 2 CO3
PROCEDURES W I L L
OF T HE
WORK.
REFLUXED
SOLUTION
to
20$
m l
DRY
STIRRER.
IFIED
OF A C O L D ,
1 0O
DISULFIDE
ABOUT
SEPARATED
and
COMBINATIONS
REAGENT WAS SLOWLY ADDED TO A C O L D ,
REQUIRED
OF
OF
ETHER
CARBON
A MAGNETIC
15
in
HOUR A F T E R
DITHIOPHENYLACETATE
he
THE
ORIGINAL
T HE A D D I T I O N
P H E N Y L A C E T A T E WAS ALL OWED TO
T
.,
T HE
ONE-HALF
OF
OR TO USE
REACTIONS,
OF THE
I OO M L .
D U RI NG
ML.
MODIFY
A C ID :
PREPA RED G R I G N A R D
SOLUTION
THIS
c h lo r id e
OF M A G N E S I UM
PHERE AT
SLIGHTLY
OF T H E S E
PHENYLACETOTHIOHYDROXAMIC
Benzyl
18—
1 0O M L .
CARBONATE
QUANTITY
THE
OF
ETHER
-
WAS
s o lu tio n
CENTRATED
T HE
DRIED
W A S H I NG
THE
DRYING
AGENT W I T H
10 0 ML.
125
A fter
c h ill in g
reported
A
cetobromoglucose:
p r e p a r a tio n
T he
OF
VACUUM.
m in u t e s
(L
,
25
HAD BEEN
SATURATED W IT H
SOLUTION
S T RA W- CO L O RE D
SHAKEN
IN
AND A G A I N
i t
product
18
GM.,
was
DRY
W ITH
I CE W A T E R .
T
s ir u p
ether
up
POINT
A
:
FOR
THE
compound
PREPARATION
dry
SOLVENT.
AND T H E M E L T I N G
This
in
CALCIUM
THE
VACUUM,
cetobromoxylose
OF T HE
AND
FILTRATE
HEXANE WERE A DD E D,
ACID
f ilt e r e d
BEGAN TO F O R M .
and
was
OF T HE
THE
OF T H I S
the
WAS
PRODUCT WAS
prepared
from
MATERIAL
AFTER
LEA VIN G
and
A
dried
under
MELTED
AT
46
a c e tic
a n h y d r id e
ROOM T E M P E R A T U R E .
DRYING
VISCOUS,
GM.,
89° C .j
BE
of
300
M L.O F
SODI UM
THE
CO L O RL E S S
OF
REPORTED
tetraacetate
PRESE NTE D
SE P A R A T E D
SOLUTION,
c r y s ta llize d
80#
CH L O R ­
BICARBONATE
L A Y E R WAS THEN
product
/S-x y l o s e W ILL
.
SA T U R A T E D
CHLOROFORM
CHLORIDE.
YIELD
m l
MIXED WITH
ICE WATER,
S OL VEN T WAS REMOVED UNDER
OF T HE
OF
B R O M I DE AT
S I R U P WAS C O O L E D ,
T HE
EVAPORATION
VOLUME
ML.
150
in
HYDROGEN
SUCCESSION W IT H
ANHYDROUS
taken
FILTE R IN G
AND T HE M A T E R I A L
placed
DRIED
was
was
WAS ADDED AND
15).
.
AND
he
OVER
the
GM.,
glucose
OFORM AND
AFTER
THE
200
AFTER
M L.,
AND CON­
Q-Ac e t o h a lo g ly c o s e s :
nhydrous
RESULTING
,
33 % ,
WAS ABOUT
of
100
SULFATE.
BENZENE,
FILTERED
PHENYLACETOTHIOHYDROXAMIC
72-75° C.
,
he
WHICH
30
for
YIELD
he
UNDER
CRYSTALS
T
A
ML.
SULFATE,
BENZENE,
M AGNESI UM
CO L O R L E S S
T
ML.
DRIED W ITH
LARGE
74° C . ;
50
AGAIN
WAS REDUCED TO
VACU UM.
OVER ANHYDROUS M A G N E S I UM
UNDER . VACUUM TO ABOUT
SOLUTION
19-
on
SIRUP.
r a p id
THEORETIC AL,
87-89° C. (2 2 ).
.
F IR S T.
T
he
procedure
-
D -(+)-X Y LO S E ,
TO T HE M I X T U R E
REFLUXED
SOLID
ON A WATER
ON A
ABSOLUTE
T
he
C.
T
ET HANOL
on
THE
REFLUXED W IT H
9
y ie ld e d
30
.
THE
MINUTES
WAS T A K E N
^-D -
of
UP
IN
-
MIXTURE"WAS
A
THE
AND THE
HEATING
SMAL L
CARBON FOR
xylose
A C E T A T E AND
AFTER
ANHYDRIDE
WERE REMOVED BY
DECOLORIZING
gm
SODI UM
ANHYDRIDE.
EXCES S A C E T I C
RESIDUE
AND
OF F USED
MINUTES,
THE
17
ML.
-
x y lose,
SOLUTION.
FORM M I X T U R E
SOLUTION
DISSOLVED
OF
DRIED
MELTING
AT
compound
D - (+ )-G
A C E T A T E AND
d isso lved
RESULTING
POURED
THE
AMOUNT
OF
A FEW M I N U T E S .
tetraacetate
WITH
INTO
AND T HE
100-101°
C.
m l
.
of
,
M .P.
PRODUCT
ICED
THE
CRYSTALLIZED
THE
ROOM T EMPER­
YIELD
T HE
SODI UM
SEPARATION,
VACUUM.
SOLUTION.
AT
THE
RESULTIN G
UPON
WAS
a c e t ic
B R O M I DE WERE
CHLOROFORM.
I CE WATER,
AFTER
UNDER
g la c ia l
DRY HYDROGEN
10 0 ML.
I CE W A T E R .
EVAPO RAT ED
6
in
S O L U T I O N WAS KEPT
SUCCESSION W IT H
ETHER TO T H E
cetobromogalactose
Thi s
AND
was
AC I D S A T U R A T E D W I T H
IN
ETHER,
PETROLEUM
.,
AND T HEN
AND A G A I N
IN
gm
THE
WAS WASHED
FORM L A Y E R WAS
5
OF A C E T I C
FOR TWO HOURS,
MIXTURE
45
BATH.
A T URE
A
FOR ABOUT
REACTION
TO T H E
PRODUCT
OF A C E T I C
T HE
ADDED
D ITIO N
ML.
SM.
(23).
AC I D AND
WAS
50
D U R I NG
c o o lin g
etraacetyl
BONAT E
WAS ADDED TO 5
HAD D I S S O L V E D .
STEAM
filt r a t e
120°
BATH
AC I D FORMED
MIXTURE
GM.,
WERE ADDED
MATERIAL
ACETIC
10
20 -
CHLORO­
BICAR­
CHLORO­
SIRUP
CA R E F U L
AD­
4.3
OF
GM.
(24).
:
was
also
alactose
,
100
OF A C E T I C
ML.
WAS R E F L U X E D
20
prepared
FOR
GM.,
was
from
placed
the
w ith
acetate
1 0 GM.
of
of
the
FUSED
A N H Y D R I D E WERE ADDED TO T HE
ONE HOUR
ON A WATER
BATH,
AFTER
sugar
SODI UM
MIXTURE.
WHICH
.
THE
THE
EXCESS
-
A C E T I C ANHYDRIDE
AND T H E
REMOVED
ON A
STEAM
10 0 ML.
OF A B S O L U T E
BATH.
THE
AND
17
M.P1 130° C.
MELTED
AT
T
GM.
he
DARK
OF
ANHYDRIDE
IOUSLY
SA T U R A T E D W I T H
AND
POURED
WAS WASHED W I T H
WITH
after
10 ML.
several
T HE
WITH
WERE
REMAIN
INTO
100
AFTER
MIXTURE
OBTAINED,
M .P .
AT
ML.
REMOVAL
WAS T A K E N
FILTERING
THE
UP
IN
OF THE
ABSOLUTE
MIXTURE
WERE
AND
COOLI NG
OBTAINED,
from
COLD
82°
C.
THE
O0 C .
ROOM T EM P E R A T U R E
CHLOROFORM.
SODI UM
AND
DRYING
THE
GM.
THE
SIRUPY
AFTER
OF
MIXTURE
THE
water
OF
10 ML.
BEING
PREV­
RESULTING
FOR TWO HOURS,
THE
BICARBONATE
VACUUM.
8.5
ACID,
B R O M I D E AT
SO LID IFIED .
(l:5 ),
WAS ADDED TO A M I X T U R E
ACETIC
OF
SEPARATING
AND G R A D U A L L Y
ETHER-LIG RO IN
COMPLETE T HE
SO LIDIFIED,
r e c r y s ta lliz a tio n s
GM.,
GLACIAL
SOLVENT WAS REMOVED UNDER
LIGROIN
10
DRY HYDROGEN
I CE W A T E R ,
I CE W A T E R .
WAS P A R T I A L L Y
I MPURE ^ - G A L A C T O S E - P E N T A A C E T A T E
S O L U T I O N WAS ALL OWED TO
WAS
AFTER
T HE R E A C T I O N WERE
(25).
ACETIC
IT
DU R I NG
COLORED R E S I D U E
J3-D-GALACT0SE-PENTAACETATE,
WHICH
FORMED
RESIDUE
CHARCOAL.
product
C.
141.5°
WHEN THE
THE
HEAT ED W I T H
FILTR A TE ,
ACID
ETHANOL WERE ADDED TO
AC I D AND A N H Y D R I D E .
ALCOHOL
ACETIC
21 -
CHLOROFORM
AFTER
SOLUTiq N
SOLUTION
AND A G A I N
CHLOROFORM
SOLUTION,
RESIDUE
WAS T R I T U R A T E D
RECRYSTALLIZATION
FROM AN
C R Y S T A L L I N E Q - A C E T OB R O M O G A L A C T O S E
(26).
A cetochloromannose:
Due
THE
to
T HE
DIFFICU LTY
CORR ESPON DI NG
CHLORINE
IN
OBTAINING
DERIVATIVE
C R Y S T A L L I N E Q- A CE T OB R OM O M A NN OS E ,
WAS,USED
IN
PREPARING
THE
MANNOSE
ML.
DRY
GLYCOSIDES.
D - ( + J-Ma n n o s e ,
10
GM.,
WAS SHAKEN W I T H
A MIXTURE
OF
67
-
AND
p y r id in e
SUGAR
HAD
50
OF A C E T I C
DISSOLVED.
REFRIGERATOR
THICK
ML.
O IL
22 -
ANHYDRIDE,
T HE R E S U L T I N G
FOR TWO DAYS AND T HEN
PRECIPITATED
STALLIZATIO N
FROM
95$
AND
O0 C . ,
UNTIL
ALL
S O L U T I O N WAS ALLOWED T O
POURED
SO LIDIFIED
ET HANOL
AT
INTO 2 5 0
AFTER
YIELDED
7.2
ML.
OF
STAND
I N THE
I CE W A T E R .
GRINDING WITH
SM.
OF THE
WATER.
A
RECRY­
OF ^ - P E N T A . A C E T Y L M A NNOSE,
M.P. 117° c. (27).
J3-MANNOSE-PENTAACETATE,
5
GM.,
FORM AND T R E A T E D W I T H
2.4 5
CHLOROFORM.
PRECIPITATE
INTO
SOLUTION
A WATER
THE
A
YEL LOW
UPON W A R M I N G .
BATH,
THE
SOLUTION.
YELLOW
THE
ETHER AND
CAREFULLY
CO L O RL E S S
CRYSTALS
THE
OF
MIXTURE
FOR 3 0
CRYSTALLINE
Pr e p a r a t i o n
T
ALL
he
of
the
OF T H E
REFLUXING
A THICK
MINUTES,
WERE
MELTING
AT
81 .5 °
MATERIAL,
VOLUME
IT
W ILL
BE
IN
25
ML.
C.
WENT
4
WAS T A K E N
SOON FORMED,
AND
FOR
OF PET ROLEUM
DRIED.
DRY
BACK
HOURS ON
UP
IN
ETHER.
AND A F T E R
FROM
HARD,
C O O L I NG
THE
YIELD
WAS 3 . 7
was
id e n t ic a l
GM.
(28).
th io g ly c o s id e s
the
DRY CHLORO­
AND WAS F I L T E R E D
used
prepare
OF
THE MIXTU RE
:
to
ML.
RAPIDLY
a c e ty lth io g ly c o s id e s
DESCRIBED
I N MORE G ENERAL
in
T ERMS TO
REACTANTS.
10 ML.
OF A CET ONE AND
OF 3 * 1
N MET HANOL I C P O T A S S I U M
ON T H E
SIRUPY
FILTERED
25
BUT
APPEARED
OF a - A C E T OC H L O R OMANNOSE
PHENYLACETOTHlOHYDROXAM I C A C I D ,
BASED
AGAIN
A LARGE
IN
TETRACHLORIDE
SOONED FORMED,
PRECIPITATE
TREATED W IT H
THEREFORE,
INCLUDE A L L
OF T I T A N I U M
AFTER
FILTR ATE,
PRODUCT
procedure
CASESj
GM.
WAS D I S S O L V E D
PARTIALLY
HY D RO X A M I C A C I D ,
3
GM. ^ ( I 0 $
NEUTRALIZED
HYDROXIDE.
DISSOLVED
EXCESS ) ,
WAS
BY THE A D D I T I O N
ACETOHALOGLYCOSE,
IN
10 ML.
DISSOLVED
OF
0.9
4.98
IN
ML.
EQUIVALENT
ACET O NE WAS ADDED TO THE
r e a c t io n
MIXTURE
m ix t u r e
.
A fter
WAS POURED
INTO
EACH
INDIVIDUAL
23 -
s t ir r in g
at
15 0
SOON BECAME A S E M I - S O L I D
RECRYSTALLIZATION
-
ML.
OF
room
I CE W A T E R .
MASS AND WAS
PROCESSES
temperature
VARIED
T HE
SEPARATED
7
for
OILY
REACTION
BY F I L T R A T I O N .
SOMEWHAT AND W I L L
GM.
r e s id u e
was
a ir
dried
o ver n ig h t
FROM A CHL OROFORM- C ARBON T E T R A C H L O R I D E
l iz e d
OF
CRYSTALLINE
MATERIAL,
MELTING
AT
16 4 °
and
MIXTURE.
C.
(llT .
S-ff-D-1-T R I A C E T Y L X Y L O P Y R A N O S Y L - P H E N Y L A C ET OTH I OHYDROXAM
T
h is
m a te r ia l
ETHER
MIXTURE,
YIELD
WAS
BEING
ABOUT
he
was
r e c r y s t a lliz e d
F OLLOWED
SOMEWHAT
0.8
fir s t
from
BY R E C R Y S T A L L I Z A T I O N
LOWER T H A N THAT. O B T A I N E D
GM.
galactose
METHODS USED
AFTER
AND M E L T I N G
compound
FOR T HE
AT
was
GLUCOSE
RECRYSTALLIZATION
1 61 °
a
then
THE
WAS
and
DER IVATIVE.
C.j
l in e
he
mannose
product
MIXTURE,
FIN ALLY
0.5
-
GM.,
.
I
t
AND T HEN
DRIED
d e r iv a t iv e
was
I C A C ID :
chloroform- petroleum
ETHANOL.
GLUCOSE
THE
very
r e c r y s t a lliz e d
YIELD
WAS ABOUT
d if f ic u l t
r e c r y s ta lliz e d
AT
VACUUM FOR
76°
C.
ABOUT
72
T HE
COMPOUND,
A C ID :
by
the
T HE OBSERVED M E L T I N G
R E C R Y S T A L L I ZED R E P E A T E D L Y
UNDER
MELTED
f ir s t
was
1.9
C.
prepared
16 0 °
­
16).
FROM A B S O L U T E
FOR THE
recrystal
Y I E L D WAS
0 .7 5
S-/3-D- I -T E T R A A C E T Y L M A N N O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAMI C
T
FOR
C A C ID t
5-J3-D-1-T E T R A A C E T Y L G A L A C T O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDR OXAMI C
T
THE
COMPOUND.
s e m i- s o lid
he
the
PRODUCT
BE D I S C U S S E D
S-/3-P-1 -T E T R A A C E T Y L G L Y C O P Y R A N O S Y L - P H E N Y L A C E T P T H I OHYDROXAMI
T
hours,
from
to
an
o b t a in
ether
-
FROM A B S O L U T E
HO UR S.
THE
same
POINT
GM.
AC ID :
as
a
crystal
petroleum
ALCOHOL
CRYSTALLINE
­
ether
AND
PRODUCT,
Sa
p o n if ic a t io n
T he
of
general
the
a c e ty lt h io g ly c o p y r a n o s id e s
procedure
PRODUCTS WAS TO D I S S O L V E
0 .1 -0 .2
GM.,
Ge n e r a l l y
.
I
SPECIFIED
ACETIC
about
TIM E
part
SILVER
,
2
one
THE
SMAL L
SYSTEM
NITRATE
N NaOH
-
AT
remove
AMOUNT
hour
AND
M L.,
is
s u f f ic ie n t
MAY BE
acetyl
W H I CH
to
T HE SPOTS MAY
parts
.
F
ig ur e
I
-
the
s a p o n if ic a
ALIQUOTS
ON PAPER
IN
BE D E V E L O P E D
ONE
AT
A BUTANOLUSING
the
deacetyl
S-j9-D-1 - T E T R A A C E T Y L G L Y C O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAM
AT I ON OF
­
AN
5 N NH4OH -
PART,
illu s t r a t e s
the
PREVIOUSLY
BY RE M O V I NG
THEM
0.1 N AgNO^
from
GLYCOSIDE,
HAD BEEN
complete
FOLLOWED
CHROMATOGRAPHI NG
(4 :3 :1 ).
groups
OF THE A C E T Y L A T E D
C. (2 9 ).
SOLUTION;
two
10
the
O0
REACTION
INTERVALS
ACID-WATER
ALKALINE
one
DRY AM M O NI A
DESIRED,
f
to
I N ANHYDROUS M E T H A N O L ,
SATURATED W IT H
t io n
A
used
:
IC
ACID.
After
AND T HE
hour
RESIDUE
GENERALLY
FROM T HE
Su
one
the
RECRYSTALLIZED
90-95$
of
the
OF T HE
OF
DROPWI SE
62
C H L ORO S U L F O N I C
MATERIAL
CATOR .
Eq
ML.
ACID.
OF DRY
AND WAS F I L T E R E D ,
TABLE
I!
to
dryness
ALCOHOL.
under
THE
S U M M A R I Z E S T HE
vacuum
YIELD
DATA
IS
OBTAINED
TH I OGLYCOS I D E S .
TRIOXIDE
THE
evaporated
FROM A B S O L U T E
t h i o h y d r o x a m ic
• T HE P Y R I D I N E - S U L F U R
ADDING
was
OF T H E O R E T I C A L .
DEACETYLATION
l f o n a t i on
m ix t u r e
a c id
a c e t y lg ly c o s id e s
ADD ITION
PYRIDINE
ADDUCT
IN
:
PRODUCT WAS PREPARED
350
ML.
CHLOROFORM
S E P A R A T E D AS A W H I T E
WASHED W I T H
TO
BY
38.5
GM.
CRYSTALLINE
CHLOROFORM AND D R I E D
IN
A DESIC­
( L i t . 2 1 ).
u iv a le n t
amounts
of
the
acetylated
g ly c o s id e
and
the
Py
r id in e
-SO3
25 -
SOLVENT MIGRATION
-
TIME
F
ig ure
I.
Sa
T HE F I G U R E S
I
-
"C "
p o n if ic a t io n
AT
ACETYLATED
PRODUCTS;
4
IN MINUTES
-
REPRESENTS
THE
RIGHT
GLUCOSIDEj
COMPLETELY
A GLUCOSE
of
a c e t y lt h io g ly c o s id e s
OF THE
2-3
-
DI AG R A M
PART I A L L Y
A C E T Y L AT ED
DEAC E T Y L AT ED PRODUCT .
CONTROL.
.
REPRESENT:
T HE
-2 6 -
Table
Sa
p o n if ic a t io n
Pr o d u c t s
I I
of
T H E T H I OGLYCOS I DES
Y I ELD
Pr o d u c t
M.P.
(uNCORR. )
S—
JB—D—I - G L U C O P Y R A N O S Y L -PATHA1
0.19
GM.
121° C.
S-p-D-1
0.18
GM.
170-175° C.
S-J3-D-1- X Y L O P Y R A N O S Y L - P A T H A
0.20
GM.
120-125° C.
S—
J3—D—I - M A N N O P Y R A N O S Y L - P A T H A
0.09
GM.
SlRUPY
-
galactopyranosyl
1PATHA- P H E N Y L A CETOTH
-PATHA
I OHYDROXAM I C A C I D
RESIDUE
-
WERE
adduct
STIR
SUSPENDED
OVERNIGHT
IDE
(10$
THE
PH
KEPT
AQUEOUS
TO
On
ICE.
AMOUNT
SOLUTION)
CONSTANT
COOLING,
BY
Sa m p l e s
the
ANALYSIS.
THE
TETRAMETHYLAMMONIUM
Pr o p o s e d
Y
AGITATION
AND
COOLED
ie l d
:
AL C OH OL
GM.,
p o in t
Op
tic a l
r o t a t io n
:
S-j3-E>-1 - T
ICE
:
181°
ound
47$
about
:
BATH.
AQUEOUS
IN
OBTAINED
TO B R I NG
MIXTURE
THE
PHASE
WAS
RESULTING
CHILLED
IN
AND WAS
FROM. A SMALL
A DESICCATOR.-
g ly c o s id e s
FOR
HYDROX­
were
CARBON AND
FOR T H E S E
recrystal
­
HYDROGEN
COMPOUNDS:
(llT .
16)
:
C.
(
for
:
based
on
uncorrected
2 2.5 _
D
Ca l c u l a t e d
F
o il
SUBMITTED
DATA WAS
AN
WAS R E C R Y S T A L L I Z E D
DRIED
mustard
AND
IN
BASE T HE
OUT AS A GUMMY R E S I D U E
RESIDUE
AND
OF THE
T E TR AA C ET YLGLUCOTROPAE OLA TE:
lt in g
n a ly s is
THE
ALLOWED TO
REACTION MIXTURE
AND THE
SETTLED
FILTER ED,
structure
0.5
ETHER,
acetylated
Me
A
WITH
PRODUCT
FOL LOWI NG
AND THE M I X T U R E
ENOUGH T E T R A M E T H Y L - A M M O N I U M
ADD ITION
T HE
FROM A B S O L U T E
l iz e d
PYRIDINE
THE
ETHANOL,
of
DRY
WAS ADDED TO T HE
DECANTATION.
OF HOT
ML.
DURING
WAS WASHED WELL
SEPARATED
5
ROOM T E M P E R A T U R E .
NEUTRALITY.
UNDER
SOLUTION
AT
IN
27-
p h e n y l a c e t o t h io h y d r o x a m ic
a c id
)
_ n .6 + 0 . 6
in
alcohol
(c=0.6;2
dm.
C ggH ggO igN gSg:
C,
4 8 . Oj
H,
5.8 4
C,
4 7 .6j
H,
5.59
ETR A AC ET Y L G A L ACT OP YRA NOS Y L - P H E N YL AC ET OT H I OHYDROXAM I C A C I D - O -
TETRAMETHYLAMMONIUM
SULFONATE:
tu b e
)
-28Pr o p o s e d
Y
ie l d
0.2
:
Me l t i n
structure
g
gm
p o in t
about
19^
147° C.
(
.,
:
Op
tic a l
r o t a t io n
An
a l y s is
:
:
:
based
on
p h e n y l a c e t o t h i o h y d r o x a m ic
uncorrected
g22.5_
+
o.2
a c id
)
in
( c= 0 . 6 ;
alcohol
I
dm
.
tu b e
)
D
Ca l c u l a t e d
for
C H O N S :
26
F
ound
38
13
2
C, 4 8 .Oj H, 5.84
2
C,
:
S-J3-D-1 - T E T R A A C E T Y L M A N N O P Y R A N O S Y L - P H E N Y L A CETOTH
T ETRAMETHYLAMMONI UM
Pr o p o s e d
48.2
j
H,
5 .9 4
I OHYDROXAM I C A C I
D-Or .
SULFONATE:
structure
:
?Ac
OAc
/ = X
N O SO :A(C H A
Y
ie l d
:
Me l t i n
Op
g
tic a l
0 .2 5
gm
p o in t
:
.,
r o t a t io n
about
24$
65-66°
C.
:
g,2
(
based
on
p h e n y l a c e t o t h io h y d r o x a m ic
uncorrected
+
0.9
in
)
j
very
alcohol
h yg r o sc o pic
(c= 1.0j
I
dm
.
m a te r ia l
tu be
D
A
n a ly s is
:
Ca l c u l a t e d
F
S -^ -D -I - T R
ound
for
:
CggHggO^gNgSg:
C,
48 .0;
H,
5.84
C,
47 .9;
H,
5.7 0
I A C E T Y L X Y L O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAMI C AC I D - O -
T ETRAMETHYLAMMONI UM
SULFONATE:
a c id
)
—2 9 —
Pr o p o s e d
structure
:
QAc
f e r
N O S O ^(C H 3)4
Y
ie l d
Me l t i n
Op
A
:
g
tic a l
n a ly s is
0.45
gm
p o in t
:
r o ta t io n
:
p o n if ic a t io n
T
he
same
ound
of
THESE
IS
COMPOUNDS
:
-1 7 .7 + 1 . 1
Op
A
g
tic a l
n a ly s is
p o in t
r o t a t io n
:
ound
ac etyl
was
-
mustard
used
in
o il
the
187° C.
:
tu b e
g
p o in t
:
tic a l
THE
of
DATA
(LIT .
these
compounds
OBTAINED
FOR
-15.1 + 2 . 1
for
16)
w ith
in
C13H30OgN2S2 :
d e c o m p o s itio n
water
( c= 0 . 4 ;
I
dm
.
tu b e
C, 44.8; H, 6.22
C, 44.9; H, 5.91
■
I OHYDROXAM I C A C I D - O - T E T R A M E T H Y L -
1 9 0°.C.
:
:
Ca l c u l a t e d
F
ound
:
=
(
uncorrected
-1 1 .7 + 1 . 0
for
),
w ith
in
water
C13H33O3N2S2 :
c,
d e c o m p o s it io n
(
c=0 . 6 ;
CO
n a l y s is
r o t a t io n
)
:
d e a c e t y la tio n
( u n c o r r e c t e d ),
q
pp
A
.
SULFONATE:
Me l t i n
Op
dm
BELOW:
S-J3-D-1 - G A L A C T O P Y R A N O S Y L - P H E N Y L A C E T O T H
AMMONI UM
I
C, 47.7; H, 5.88
g ly c o s id e s
UNSULFONATED ANALOGUES.
:
(c=0.4;
alcohol
C, 47*6; H, 5.71
Ca l c u l a t e d
F
in
C23H34O11N2S2 :
for
a c id
)
:
GIVEN
:
p h e n y l a c e t o t h io hydro xam ic
uncorrected
T E T R A M E T H Y L A MM ON I UM G L U C O T R OPAEOL A T E :
Me l t i n
on
(
procedure
THEIR
based
189° C.
the
A S WAS USED FOR
48$
about
Ca l c u l a t e d
F
Sa
.,
I
dm
H, 6.22
C, 44.1; H, 6.65
.
tu be
)
)
-
30 -
S-J3-D-1 - M A N N O P Y R A N O S Y L - P H E N Y L A C E T O T H
I OHYDR OXAMI C
ACID-O-TETRAMETHYL-
AMMON I UM S U L F O N A T E !
Me l t i n
Op
p o in t
tic a l
very
:
r o ta t io n
:
h yg r o sc o pic
„
^D
Ca l c u l a t e d
F
ound
’ =
for
,
no
m e ltin g
-3 .2 + 1.0
in
po in t
(c= 0 .I j
water
C18H30OgN2Sg:
could
C, 44.8; H,
be
I
o btain ed
DM .
Si
n a ly s is
S-J3- D - I
:
CO
A
g
tu be
)
N, 5.8
c , 45.2; H, 6.61; N, 5.7
:
- X Y L O P Y R A N O S Y L - P H E N Y L A CETOTH I OHYDROXAM I C A C I D - O - T E T R A M E T H Y L -
AMMONI UM
SULFONATE:
Me l t i n
Op t ic
g
p o in t
al
:
Hy g r o s c o p i c ,
r o t a t io n
:
2 2 .5_
melts
-2 1 .9 +
110-115°
from
0.6
in
water
C .;
d ec
( c= 0 . . 8 ;
.
I
at
160°
.
tu be
dm
c.
)
D
A
n a ly s is
:
Ca l c u l a t e d
F
A
EVEN
ll
Mic
a n a l y s is
t
the
ON SHORT
Geller
I
of
MAY
.
BE
PO SITIVE
ound
compounds
r o a n a l y t i cal
OPTICAL
THAT
ROTATIONS.
GAVE
NEGATIVE
ROTATIONS
HIGH
PO SITIVE
ROTATIONS,
SIDE,
+819°,
OPTICAL
ROTATION
was
THE
d r ied
SEVERAL
IN
FOUNDED TO ASSUME
q u it e
ALCOHOLIC
H,
6.19
3,
44 .7;
H,
6.42
and
SINCE
n ia
,
N ew Y o r k ,
vacuum
IS
NOT AN
SlN CE
THESE
DATA WAS
up
water
TO THE
elemental
to
GLYCOSIDES
UNUSUAL
THE
for
p r e v io u s
THE A N A L Y S I S .
GAVE
SMALL
PHENOMENON WHEN THE
DEACETYLATED
THE q - T H I O G L Y C O S I D E S
HAVE
PRODUCTS
EXTREMEL Y
E T H Y L T E T R A A C E T Y L - a ( - D - 1 - T H I OGL UCOPYRANO-
E T H Y L - a - D - 1 - T H IOGLUCORYRANOSIDE,
T HAT
plcked
COMPOUNDS WERE S U B M I T T E D
under
SOLVENT.
E .G .,
4 5 .I j
OF T HE A C E T Y L A T E D
THIS
AND
C,
hyg r o sc o pic
L a b o r a t o r y , Ba r d o
COMPOUNDS ARE
and
were
TO A I R .
m a te r ia l
NOTICED
CiyHggOgNgSg:
:
EXPOSURE
Ea c h
for
+605°,
IT
SEEMS WELL
COMPOUNDS ARE ^ B - T H I O P Y R A N O S I D E S .
OBTAINED
T HE
FROM T H E SCHWARZKOPF M l C R O A N A L Y T I C A L
3 1—
—
L a b o r a t o r y .,
ISOLATION
Wo o d s j
N ew Y o r k .
seed
,
100
gm
MINUTES
A SoXHLET
USING
IN
BY F I L T R A T I O N
GM.
T HE
THROUGH
OF A M B E R L I T E
F ORM.
El u
t io n
was
ETHANOL
Y I ELDED
NAT UR AL
G L U C O S I DE WAS
T
he
PYRIDINE,
POINT
T HE
natural
AND
TO THE
A SoXHLET
500
RESIDUE
CELI TE.
Co n c entratio n
THE
RESIN,
0.29
GM.
of
T ropeaolum
EXTRACTOR.
M L.,
AND THEN
SOLUTION.
DISSOLVED
AQUEOUS
.05
effluent
,
was
R E S I D U E WAS B O I L E D
EXHAUSTIVELY
50
METHANOL
ML.
defatted
EXTRACTED
S O L U T I O N WAS
WATER
AND
PURIFIED
S O L U T I O N WAS PAS S E D THROUGH
PREVIOUSLY
w ith
majus
THE
THE
IN
SEEDS:
CONVERTED TO T H E
N
tetramethylammon
and
CHLORIDE
i
um
r e c r y s ta lliz a tio n
hydro xide
from
BY A M I X E D
MELTING
POINT
AND
95$
THE
I NFRARED
MATERIAL.
was
PRODUCT
SYNTHETIC
,
NASTURTIUM
OF T E T R A M E T H Y L A M M O N I UM G L U C O T R O P A E O L A T E .
SYNTHETIC
GAVE A
the
ID EN TIFIED
m a te r ia l
FROM
n a s tu r t iu m
ac co m plishe d
.
THE
IN
I ON
SAME METHANOL
IR-4B
s o lu t io n
SPECTRUM W I T H
of
METHANOL,
EVAPORAT ED TO D R Y N E S S ,
5
.,
CARBON T E T R A C H L O R I D E
FOR T H I R T Y
IN
,
OF T HE G L UC O T R O R A E O L A T E
Gr o u n d
WITH
de
acetylated
IDENTICAL
TETRAACETYL
IN
w ith
a c e t ic
INFRARED
a n h y d r id e
IN
DRY
SPECTRUM AND M E L T I N G
GLUCOTROPAEOLATE•
-
C.
I
THE
n
order
I NF RARED
to
SPECTRA
GROUPED TOGETHER
THE
DEFINITE
DIFFERENCES
EASILY
I.
r e c e iv e
FOR
OF T HE
nfrared
full
AMONG THE
ABOUT
BY
Da t a
value
from
PREPARED AND
DISCUSSION.
S IM ILA R ITY
BROUGHT
the
I
32 -
a
c o m p a r a tiv e
ISOLATED
T HE F O L L O WI N G
VARIATION
OF
THE
COMPOUNDS
I NFRARED
T H I O G L Y C O S I DES,
AL T HOUGH T HE
GLYCONE
MOIETY
P H E N Y L A C E T O T H I OHYDROXAM I C AC I D
WAVELENGTH IN MICRONS
TW
HAVE
SPECTRA
RECOGNIZED.
TtiS
s ta n d p o in t
1600 1566 iloo
i3oo Tzoo
WAVENUMBER IN KAYSERS
TToS-
iooo
,
BEEN
DEPICT
SPECIFIC
CAN BE
33
W AVELEN G TH
IN
M IC R O I
'7 :7Er - 3
W AVEN UM BER
2.
Ph e n y l a c
3.
P h e n y l a c e t o t h 1o h y d r o x a m i c
e t o t h io h y d r o x a m ic
3500
K AYSERS
tetraacetyl
-
g l u c o p y r a n o s i de
d-S -^ -D -1 -
tetraacetyl
-
g a l a c t o p y r a n o s i de
a c i
W AVELENG TH
4000
IN
AC 1 d -S -^ -D -1 -
IN
M ICRONS
food
300
W AVENUMBER
IN
KAYSERS
900
800"
34
W AVELEN G TH
IN
M IC R O N S
I
Ttoo ieoo iioo
4.
Ph e n y l a c e t o t h I
o h y d r o x a m ic
a c id
5.
Ph e n y l a c e t o t h i
o h y d r o x a m ic
a c id-
-S -^-D -I-
S -£-D -1-
W AVELENG TH
2000
1900
1800
moo
t r i a c e t y l x y l o p y r a n o s i de
tetraac etylm an no pyr ano sid e
IN
M ICR O N S
800
1700
W AVENUMBER
IN
KAYSERS
TOO
35
6.
P H E N Y L A C E T O T H I O H Y D R O X A M l C AC I D - S - ^ - D - I - G A L A C T O P Y R A N OS I DE
7.
P H E N Y L A C E T O T H I OHYDROXAM I C AC I D - S -J3- D - I - X Y L O P Y R A N O S I DE
W AVELENG TH
IN
M ICRONS
12
=
4500
3
4000
2500
2000
W AVENUMBER
IN
KAYSERS
13
14 15 16
36
W AVELEN G TH
8 .
PHENYLACETOTHlOHYDROXAMlC
9.
P H E N Y L A C E T O T H I OHYDROXAM I C AC I
1900
M IC R O N S
ACID-S-^-D-I-M ANNO PYRANOSIDE
D-S-J3-D-1 - G L U C 0 P Y R A N 0 S
W AVELENG TH
2000
IN
IW b
1700
1600
1500
W AVENUMBER
IN
M ICRONS
1400
IN
1306
KAYSERS
1206
UOO
I DE
37
W AVELENG TH
IN M ICR O N S
7___________ 8
T600
1500
WAVENUMBER
1400" 1300
1200
IN K AYSER S
12
TiOO
1000
900
800
10.
T
etr am eth ylam m o n iu m
tetraacetylglucotropaeolate
(
s y n t h e t ic
11.
T
etr am eth ylam m o n iu m
tetraacetylglucotropaeolate
(
natural
W AVELENG TH
2000
1900
feoo
IN
M ICRONS
1700
W AVENUMBER
IN
KAYSERS
13
)
)
14 15 16
700
600
38
W AVELENG TH
IN
M IC R O N S
1500
W AVEN UM BER
1400
1300
1200
IN K AYSERS
1100
12. S -P -D -I - T E T R A A C E T Y L G A L A C T O P Y R A N O S Y L - P H E N Y U A C ETOTH
O-T ET RAMET HYLA MMONI UM S UL F O NA T E
13. S-p-D -1 - T R
1000
900
I OHYDROXAMI C
I A C E T Y L X Y L O P Y R A N O S Y L - P H E N Y L A C E T O T H I OHYDROXAMI C
T ET R A M E T H Y L A M M O N I UM
S UL F O NA T E
W AVELENG TH
IN
M ICR ON S
■I
W AVENUMBER
IN
m
KAYSERS
800
700
AC I D -
A C I D-O-
39
I
4.
S - p - D - 1 - T E T R A A C E T YUMANNOPYRA N O S Y L - P H E N Y L A C ETOTH
T ETRAMETHYLAMMONI UM
I OHYDROXAMI C
AC I D - O -
S UL F O NA T E I
I 5 . S -p -D -1 -MANNOPYRANOSYL-PHENYLACETOTH
I OHYDROXAMI C
AC I D - O - T E T R A M E T H Y L -
AMMON I UM S U L F O N A T E
W AVELENG TH
IN
M ICRONS
6_________________ 7____________ 8
"! too iioo
fsoo
WAVENUMBER
moo
IN
1300 izw
KAYSERS
ilod
1000
5o5
555
755“
40
W AVELEN G TH
16.
IN
M IC R O N S
S-p-D-1-GALACTOPYRANOSYL-PHENYLACETOTHI OHYDROXAMICACID-O-TETRAMET HYL AMMONI UM S U L F O N A T E I
I 7 . S-j3-D-1 - X Y L O P Y R A N O S Y L - P H E N Y L A C E T O T H
I OHYDROXAM I C
ACI D-O -TETR A-
M E T H Y L A M M ON I UM S U L F O N A T E
W AVELENG TH
IN
M ICRONS
12
4000
3500
3000
2000
T ro o
Ttoo
Troo
14 15 16
TW
iro o "
W AVENUMBER
13
IN
KAYSERS
600
41
W AVELEN G TH
,0° : : :
Jn
;:
~ ^ r .
r
IN
M IC R O N S
7
I
zSjfrjE J j i j
I
:
8
I ;
I0
I
12 13 14 15 16 100
I I I I I
0 #
90
.
p E
I)
80
IEE W :TTj :
A
EIrElEr
p i s t\
I i
E s \
5
s i „ Lj \
t6mE A
I
%
S I
=E--
A
I
L
70
i l
60
SI
i !
I
S i-
I/
L
> -i ■: ’
30
. ... .,
n
:;
: .:-:h TTtl-
-TS
50
E
EEiEiESTTt=
TTTI
—
T
.
SCio
4 !O
O
40X)
3500
3CX)
25»
2C»
1900 If»
17»
16»
1500 14M 13»
W AVENUM BER
18.
T
19.
T E T R A M ETHYLAMMON I UM G L U C O T R O P A E O L A T E
etr am eth ylam m o n iu m
glucotropaeolate
(
ir o o
1700
1200 11»
KAYSER S
s y n t h e t ic
)
( NA T U R A L )
W AVELENG TH
1900
IN
.
IN
M ICRONS
TSOO 1400 1300 1200 fiOO"
W AVENUMBER
IN
KAYSERS
.
10»
20
Ii-I1
<O
10
U!
8
X)
0
6CK)
-4 2 I
t
a t iv e l y
is
,
very
BECAU SE
compound.
INFRARED
S IDIC
hey
Sugars
in
were
also
THE
OF
DIFFERENCE
REACTION
HAD T A K E N
HAD REACHED
T
here
NA T U R A L
are
useful
AND
SYNTHETIC
120
SPECTRUM
OF T H E
CM""I
F E R E N C E S ARE
P RO B A B L Y
D IFFIC U LT,
T HE
M I NO R
A
nother
HYDROXYL
BAND , I N T HE
SPECTRA
UATION,
3000
s tu d ie d
LITTLE
WORK
HAS
SPECTRA
GIVEN
AND
FOR T HE
IT
IN
NOT
WAS
a
even
THIS
tent
TYPE
c o m pr eh en sive ly
HERE WERE USED
PURPOSE
POSSIB LE
CERTAIN
,
BEEN REPORT ED
g iv e n
OF
­
OF
by
ON G L Y C O PRIMARILY
ID EN TIFICATION .
sequence,
r e a c tio n
TO D E T E R M I N E
SITUATIONS
d iff e r e n c e s
CM""^
ARE
MATERIAL
SPECT RA
DUE TO T HE
SPECTRAL
T HE
AS T HE A C E T Y L A T E D
BE
e
.
g
.,
WHETHER A
WHETHER T HE
THAN
IN
T HAT
MORE
FOR T HE
OF THE
TO
REACTION
SUFFICIENTLY
be
PRONOUNCED
NATURAL
IM PURITIES
out
is
the
THE APPEARANCE
E S T E R S WAS C O N S I D E R E D
SAMPLE.
THIS
S A MPL ES A F T E R
THESE
SAMPLES.
G L YCO SYL
WAS
the
ABSORPTION
SO T H E Y WOULD
brought
CM"1 .
OUT.
of
IN
PRODUCT,
D IF­
IT
IS
NOT
COULD
BE
e x is te n c e
OF T H I S
TO
BE THE
I N D E E D FOUND TO
BE THE
EXTREME
THE
D U P L I C A T E T H E KBR
EVEN M I N U T E
should
spectra
GLUCOSIDES.
CONDITIONS,
3300-3400
the
POINTED
CONSIDERABLY
ANALYSIS.
that
BAND AT
OF WATER W I T H I N
MUST
NON-UNIFORMITY
P EAKS
feature
OF T HE
between
OF THE A C E T Y L A T E D
ABSORPTION
OF A STRONG
RESULT
OUT W I T H
been
EVEN UNDER R E P E A T E D
USED FOR T H E
.
AND
CARRIED
a s s ig n m e n t
not
f o llo w in g
obvio u s
SYNTHETIC
IN
id e n t ic a l
BEEN
GLUCOStDES WHICH
AND
PARTICULARLY
CHANGE THE
in
SPECT RA
PLACE,
several
I
PELLETS
VERY
frequency
COMPLETION.
P EAKS AT
VERY
have
INFRARED
IN
group
WORK HAS
COMPARISON,
very
FROM T HE
make
general
SPECTROSCOPY AND
PURPOSE
to
SO L I T T L E
COMPOUNDS.
FOR T HE
T
d if f ic u l t
DRYING
DO NOT
S IT ­
SHOW AN
-4 3 APPREClABLE
IT
1740
MAY
CM"^
BAND
BE
ARE
IN
THIS
OBSERVED T H A T
COMPLETELY
T HE A B S O R P T I O N
WELL
ON T H E
GLUCOSE AND
CM- 1 ,
W H I CH
T HE
ESTER
ABSORPTION
REMOVED . I N T HE
PEAK A T
FOR T HE J 3 - P Y R A N O S I DE
840
AREA.
880
X Y L OS E
(30).
DEACETYLATED
CM“ ^ - 9 0 0 C M
^}
DER IVATIVES,
As
NONE
W H I CH
IS
OF T H E S E
IN ITIA L
INDICATION
SHOWS UP
COMPOUNDS
FOR T HE
1380
CM- 1
AND
PRODUCTS.
SU P P O S E D L Y
C H A R A C T E R I Z E S T HE Qf- C O N F I G U R A T I ON
T E R I O N WAS USED AS AN
PEAKS A T
PARTICULARLY
CHARACTERISTIC
EXHIBIT
(llT .
30),
PRODUCTION
A PEAK AT
THIS
CR I­
OF THE j 3 -
GLYCOS I D E .
T
MAKES
T HE
he
r a r ity
IT
PRACTICALLY
VARIATION
V A L UE
CAN BE
EXAMPL E
CITED
of
in fo r m a t io n
OF T H E
OBTAINED
ABOVE
c o n cer n in g
IM POSSIBLE
GLYCOSYL
TO
INTERPRET
RESIDUES.
FROM T H I S
the
PORTION
IN
in fr a r e d
spectra
of
T HE
CHANGES
OBSERVED
VIEW
OF T H I S
FACT,
OF T HE
SPECT RUM,
sugars
FOR
LITTLE
OTHER THAN THE
-4 4 -
Part I I
T
Sp
he
e c if ic it y
A.
My r o s i n a s e
MUSTARD
O IL
AEOLACEAE,
is
the
WAS T H E
T HE
F ACT
THAT,
NOT
MUSTARD
O IL
COMPLEX
OR G A N I C
BE
ABOUT
RELEASE
GLUCOSIDE
ENZYME W H I CH
MYROSIN
system
ACID,
PROTEIN
OF T HE
FROM B L A C K
r e s p o n s ib le
AS
IT
the
cleavage
of
I T WAS NOTED
the
TROP-
PREVIOUSLY
MUSTARD WERE T R E A T E D W I T H WATER T HEY
ALLYL
ISOTHIOCYANATE.
SEEDS WERE
VOLATILE
O IL .
MUSTARD AND
5).
ALSO
PREVIOUSLY
D E N A T U R A T I ON,
MATERIAL
IS
for
OF T HE CRUC I F E R A E ,
(S i).
MYRON I C AC I D ( l l T .
OR M Y R O S I N A S E ,
AS
O IL,
GROUND
H Y D RO L Y Z E D T H I S
ILLUSTRATED
BLACK
MUSTARD
WHEN T HE
BRING
CAUSE T H E
OF
System
ntro ductio n
MANY R E P R E S E N T A T I V E S
SEEDS
VO LATILE
R EAGENT S W H I C H
DID
IN
My r o s i n a s e
the
CA P P A R I DACEAE AND R E S E D A C E A E
T H A T WHEN MACERATED
RELEASED
I
enzyme
G L U C O S I DES
of
TREATED WITH
THE A D D I T I O N
BUS S Y F I R S T
NAMED
IT
NAME
BECAME M Y R O S I N . 1
THE
ON MUSTARD
O IL
OF WATER
ISOLATED
AS T H E
T H U S THE
NOW NAMED,
OBSERVED
SALT
T HE
OF A
FOR THE
ACTION
OF
G L U C O S I D E S MAY
F OL L O W S :
.S-C6H11O5
------ ^ RNCS + JB-D-Gluc o s E
H2O
NOSO3
A
lthough
the
a c t iv it y
R E C O G N I Z E D WELL
ENZYME
1926
SYSTEM
(34).
T
of
m y r o s in a s e
OVER A CENTURY AGO,
UNTIL
hese
THE
on
the
VERY
P R E L I M I N A R Y WORK
workers
suggested
that
HSO^
4
+
mustard
LITTLE
OF
the
WAS
o il
KNOWN ABOUT
VON E u L E R
enzyme
g lu c o s id e s
AND
was
THIS
ERIKSON
composed
was
IN
of
two
I
T
hroughout
WITH
the
lit e r a t u r e
S l N l G R INASE
(32)
the
name
m y r o s in
AND M Y R O S I N A S E
(33).
has
been
used
analogously
-4 5 E N T l T l ES,
(35)
A THIOGLUCOSID ASE
REPORT ED THE
MERCURIC
SEPARATION
ACETATE,
THEY
T HE
C L EAVAG E
OF T H E
AGE
OF
THESE
FIN DING S
ACT. S I M U L T A N E O U S L Y
T
by
he
Et t l i n
T HE
REVISION
g er
A
HYDROLYSIS
OF T H E
REARRANGEMENT
BY T H E
OF
BY A D S O R P T I O N
EACH
TH I O G L U C O S I D A S E
ENZ YME,
AND THE
SANDBERG AND H O L L Y
STRUCTURE
e s ta b lis h e d
I . E .,
CLEAV­
(36)
T HERE WERE TWO ENZYMES WHI CH
some
HYDROLYSIS
FOR T HE MUSTARD
doubt
AND T H A T
T HE
as
OF T H E S E
A TH I O G L U C O S I D A S E ,
SUBSTRATES
FOR
F URT HE R E D T H I S
PROPOSAL
BY
POSTULATING
MENT
AN A NAL OGY TO THE
G L U C O S I DES
n ec essity
MOL ECU L E
SIMILAR
N A G A S H I MA
A M E C H A N I SM
OBSERVATION
the
COMPOUNDS.
RESIDUAL
HYDRbXAM I C A C I D S .
to
OIL
THEY
WAS R E S P O N S I B L E
UNDERGOES A REARRANGEMENT
OBSERVED
U TILIZIN G
PREPARATIONS
WITH
SUBSTRATE.
ENZYME,
GLUCOSE M O I E T Y
BY F R A C T I O N A T I O N
A C TIV ITY
SULFATASE.
OF THE G a d a m e r
SINGLE
THEIR
CONCLUDED T H A T
SYSTEM FOR T H E
POSED T H A T
OF T H E
AND
ON T H E
L undeen
and
TWO ENZYME
BY T H E
NEUBERG AND SCHOENEBECK
ENZYMES
INDIVIDUAL
GLUCOSE M O I E T Y
SULFATE
TWO
P UR IFIED
OBSERVED T H E
INORGANIC
SULFATASE.
OF T H E S E
AND F URT HER
ON A L U M I N A .
CONFI RMED
AND A
REMOVAL
LoSSEN
AND UCHI YAMA
FOR T H I S
OF HURD
PRO­
FOR T O T A L
AFTER
TO T HE
of
(38 )
(37)
REARRANGE­
ON HYDROXAMI C
ACIDS.
./S-C 6H i1O5
NOSO3K
R-C
■ -M'
NHOSO3K
> R-C-N-OSO3K
RNCS + KOSO3"
Gm
e lin
and
V
ir ta n e n
(L
i t
.
31)
reported
that
the
m y r o s in a s e
system
46in
THE
MANY
GF T HE C R U C I F E R A E
PRODUCTION
WORKERS
I
t
OF T H I O C Y A N A T E S
SUGGEST T H E
COORDINATION
IS
M Y R O S I NASE
OF T H E
THE
HYDROL I ZES T H E
EXISTENCE
CLEAVAGE
PURPOSE
OR ENZYMES P RESE NT
OF A YET
PRODUCTS
OF T H I S
SYSTEM AND TO
W ITHIN
RATHER
PORTION
DETERMINE
THE
THE
SYST EM.
THAN
MUSTARD
OIL
GLUCOS I DES W I T H
ISOTHIOCYANATES.
UNDETECTED
FACTOR
THESE
INVOLVED
I N THE
OF M Y R O S I N A S E .
OF T H E
THESIS
SP E C IFIC ITY
TO
INVESTIGATE
OF T HE
ACTIVE
THE
ENZYME
-4 7 -
B.
A
ssay
procedures
I
t
was
HYDROLYSIS
cyanate
•
p r e v io u s ly
or
B U F F E R WERE
SPECIFIED ,
AND T H E
UT ES
REACTIONS
IFIC
CITRATE
T
USING
AC TIV ITY ,
1 0 MG .
T H I OCYANAT E WAS
SATURATED W IT H
Gr
o te
'
s
Pr
LIGHT
o te in
PROTEIN
Pr
EITHER
1.0
OF
MMo
CARBAZOLE
-
2 .0
OF
mg
TO A V O I D
OF
20$
BY
IN
AT
BOILING
BENZIDINE
UNLESS
c.
FOR
WATER
a c id
d e te r m in e d
0-
of
S UB S T RA T E
HOURS
M IN­
IN
OF
SPEC­
PERIODS
SUBSTRATE.
(39),
(4 0 ).
SULFATE
c o lo r im e tr ic a lly
.
FOR 5
method
CORR ESPON DI NG
m l
FOUR
INCUBATION
OF T H E
I SOTHI
OTH ERWI SE
ACID.
BY T R E A T M E N T W I T H
CONVERTED T O T H E
0 .5
DETERMINATION
U TILIZATIO N
AS
en zym atic
AND AN
in
TRICHLORACETIC
E .G .,
by
ENZYME AND
37°
USED AND THE
ETHER AND
then
BOTH
d in it r o s a l ic y l ic
PRECIPITATIO N
were
KHSO4
SOLUTION.
FOR
HEATING
o b t a in e d
substrate
INCUSATED
SUBSTRATE,
the
of
WAS USED
COMPLET E
by
.
ENZYME
S U B S T R A T E WERE
was
run
q u a l it a t iv e l y
ON A BECKMAN MODEL
MEASUREMENT WAS MADE
o te in
BY
ML.
AS T H E
OF
products
I BO­
METHANOL
THIOUREA.
by
use
of
(41).
a n a ly s is
280
PH 6 .2 ,
DRY A M M O N I A ,
reagent
,
ML.
EX T R A C T E D W I T H
d e r iv a t iv e s
AT
0.2
d e te r m in e d
SULFATE
the
V E S S E L S WERE
S IN IG R IN
was
INORGANIC
h io u r ea
a c t iv it y
S T OPPED
THE A D D I T I O N
Gluco se
T
of
REACTION
he
that
G L U C O S I DES WERE G L U C O S E ,
BUFFER,
SHORTENED A P P R E C I A B L Y
AND
OIL
a n a ly s is
REACTIONS
OR BY
m e n tio n e d
INCUBATED W IT H
SOLUTIONS.
ental
:
OF MUSTARD
F
Ex p e r i m
bound
was
abso r ptio n
SPECTROPHOTOMETER.
BY T HE METHOD
carbohydrate
BY T H E METHOD
DU
by
OF LOWRY
d e te r m in e d
OF G U R I N AND HOOD
(43).
color
of
u ltr avio let
QUANTITATIVE
(4 2 ).
!m
e t r ic a lly
w ith
-4 8 “
Su
:
bstrates
'
S a l I C I N,
AMYGDAL I N ,
PHLOR I DZ I N AND
PURCHASED FROM T H E C A L I F O R N I A
OTHER
Nu
SUBSTRATES
t r it io n a l
T
he
B
EXCEPT
T HE
io c h e m ic a ls
f o llo w in g
CORPORATION
t h io g ly c o s id e s
were
SIDE
O^-B EN Z Y L T H I OGL UC OF UR A N OS I DE
for
B
y s ta llin e
OF ANY
T
CARBOHYDRAT E
he
c ribed
other
IN
THE
T HE
BY THE
B odgson
and
Pr e p a r a t io n
mustard
PREVIOUS
AT
40°
C.
OF
CHEESE
CIPITATED
o il
Spencer
and
J
uncea
I
t
(50)
ac c o r d in g
to
the
was
FOR ABOUT
MATERIAL
A
(45),
was
a - E T H Y L T H I OGLUCOPYRANOS I DE
j 3 - P H E N Y L T H I OGL UCOPYRANOS I DE
from
Ca
the
chromatographed
Corporation
lif o r n ia
and
(48).
shown
to
be
free
were
OF T H I S
USED
IN
or
is o la t e d
as
des
­
THESIS.
CHECKING
TO S M I T H
AND
prepared
FOR THE A C T I V I T Y
(49).
OF A S U L F A -
G L U C O S E - G - S O ^ WAS PREPARED
P-NITROPHENYL
SULFATE
BY T H E
METHOD
OF
(51).
fin e ly
CLOTH.
(47),
g ly c o s id e s
p u r if ic a t io n
,
s y n th e s ize d
purchased
SECTION
OXIMES
OF O H L E
De f a t t e d ,
a s s ic a
was
PREPARED A C C O R D I N G
METHOD
FROM THE
IM PURITIES.
S U L FO N A T E D
T A S E WERE
Br
s i n i g r in
Research.
io c h e m ic a l
PURCHASED
ALL
3 - E T H Y L T HI O GL UCOF URANOS I DE AND j? - E T H Y L T H I OGL UCOFU R A N O -
p - E T H Y L T H I OGLUCOPYRANOS I DE
Cr
WERE
RESEARCH.
Co r p o r a tio n .
CITED:
(46),
FOR B I O C H E M I C A L
THlO G LYCO SIDES
REFERENCES
(44),
P - N I T R 0 P H E N Y L - J 3 - D - G L U C 0 S I DE WERE
ground
m ix e d
12
n
of
m y r o s in a s e
seed
w ith
,
300
900
m l
HOURS AND T HEN
EQUAL
REMOVED
VOLUME
BY
:
GM.,
.
from
w ater
.
Or
T he
ie n ta l
m ix t u r e
EXPRE SSED THROUGH
OF 8 0 #
yellow
SEVERAL
AL C OH OL WAS ADDED
CENTRIFUGATION.
THE
CL EAR
was
mustard
,
in c ubated
LAY ERS
AND THE
PRE­
SUPERNATANT,
-4 9 CONTAINING
THE
WAS A DE Q UA T E
MATERIAL
T
he
T
h is
crude
TO P R E C I P I T A T E
was
he
A
crease
THE
AFTER
p p r o p r ia te
FOR 2 4
IN
ITY
A
SIM ILAR
Ea c h
MANN ER,
PROTEIN
he
LY ALL
OF T H E
REMOVAL
OF T HE
YIELDED
220
ether
stored
2 .0
s o lid
in
ALCOHOL,
PROTEIN.
THE
S U PER NAT ANT
and
GM.,
a
fin a lly
W HI CH
PROTEIN
DISCARDED.
a ir
d e s ic c a t o r
d is s o lv e d
sulfate
BY F A C T O R S
TO P R E C I P I T A T E .
SULFATE
WATER
WAS
am m onium
SOLUTION
10 ML.
THE
d r ie d
for
.
several
OF
BY
SALT
AND T H E
DIALYZED
PROTEIN
m l
added
to
PERCENT
AGAINST
in
UNTIL
­
NO
PROTEIN
D ISTILLED
PREC IPITATIO N
SOLUTION
.
WAS C E N T R I F U G E D
PRECIPITATED
AND D I A L Y Z E D
OBTAINED
10
100
in
were
MIXTURE
AND T H E
FRACTION
FRACTION
A C T IV IT Y
MG.
SALT
PHOSPHA TE
BUFFER,
CHROMATOGRAPHY
OBTAINED
PURIFIED
NOET HYL
PH 7 .0 ,
COL U MN.
AT
70#
AND WAS R E T A I N E D
BY D I A L Y S I S ,
OF T HE
N ,N -D l E T H Y L A M I
he
of
AND T HE
OE 9 5 #
OF A C T I V I T Y .
OF AMMONI UM
IN
REMAINING
and
be
VOLUMES
WATER
WAS T R E A T E D
CHECKED
FOR A C T I V ­
CONTAINED
ESSENTIAL­
ON SI N I G R I N .
T
T
may
LOSS
OF THE
APPEARED
SUSPENDED
HOURS.
alcohol
amounts
EACH A D D I T I O N
OF T HE
PREPARATION,
SATURATION
COLLECTED,
w ith
p r e p a r a tio n
ENZYME
MATERIAL
MOST
2 .5
CENTRIFUGATIO N
APPRECIABLE
crude
WATER.
BY
washed
enzyme
MONTHS W I T H O U T
F URT HER
WAS T R E A T E D W I T H
WAS C O L L E C T E D
pr o tein
T
ENZ YME,
FOR
ENZYME
(DEAE),
AND ALLOWED TO
HEIGHT
he
am m onium
sulfate
5
GM.,
GRAVITY
OF T H E
fr a c tio n a t e d
WITH
PU R IFIC ATIO N .
AL C OH OL
AND
AFTER
DRYI NG
PREPARATION.
COLUMN WAS T HE N WASHED THOROUGHLY W I T H
T
FURTHER
PRECIPITATIO N
CELLULOSE
THE
SATURATION
WAS D I S P E R S E D
PACK
I N A l
CM.
IN
DIAMETER
COLUMN WAS A P P R O X I M A T E L Y
T HE
enzyme,
PHOSPHATE
50
mg
.,
15
CM.
BUFFER.
was
d is s o lv e d
in
-5 0 5
MU.
OF
WASHED
BUF F ER
SEVERAL
GRADIENT
ELUTION
TIMES
PHO SPHA T E
WITH
A MIXING
FLASK
FROM T HE
COLUMN AND
INCREASING
PORTIONS
R A N G I NG
BY MEANS
CONNECTED TO T HE
CHECKED
e lu t io n
T HE
IONIC
was
FROM
OF
OF
BUFFER
7 .0
PM
COL U MN.
CONCENTRATION
OF T HE
AND T HEN
5 .7 .
OF 3
ML.
ABSORPTION
u s in g
COLUMN WAS A G A I N
AT
phosphate
SOLUTION WITH
ELUTED W I T H
GRADIENT
RESERVOIRS
SAMPLES
attempted
THE
TO PH
TWO BUF F ER
FOR U L T R A V I O L E T
also
COLUMN.
FEEDING
WERE
280
I NTO
COLLECTED
MU.
buffers
SODI UM
w h ile
CH L O R­
(5 2 ).
IDE
S
in c e
SULFATE,
phosphate
T HE
48
SOLUTION.
T
HOURS,
AVOID
MEANS
USING
SEPARATION
2
T HE
T
he
BOUNDARY
APPARATUS.
^PVP
-
Po
OF
sulfate
T
he
PARTIALLY
CITRATE
DIALY SIS
IN
ENZYME,
AT
in o r g a n ic
AGAIN ST
AGAIN ST
MANNER
FURTHER
DISTILLED
30$ PVP^
A
LOST
OBTAINED
USING
ENZYME
ENZ YME,
purchased
from
50
A
ELUTION
CONSID­
PROCEDURES
I T WAS FOUND T H A T
OF T HE
ON A P E R K I N - E L M E R
,
THIS
A CONSTANT
COMPONENTS
PURIFIED
ly v in y lp y r r o lid o n e
BY
of
DIALYZED
BUFFER.
f r a c tio n a t e d
ELECTROPHORESIS
d e t e r m i n a t i on
P RO CE D URE .
OF T HE
D I AG R A M
-
.
PREPA RED
CITRATE
OF THE
ELUTION
am m onium
WHEN
D IALY SIS
GRADIENT
OR BY MEANS
t h e
CONCENTRATED
INACTIVATION
SATISFACTORY
REPRESENTS
THE
w ith
F R A C T I O N S WERE
SOLUTION
D U R I NG
THIS
WERE A T T E M P T E D
BY T H I S
AND T HE N
ENZYME
he
ERABLE A C T I V I T Y
To
in t e r f e r e s
ENZYME-CONTAINING
FOR
WATER
BY
SMALL
BUFFER,
WAS A C C O M P L I S H E D
Gr a d i e n t
DEAE
AND P A S S E D THROUGH THE
PH,
6 .2 ,
YIELDED
ENZYME
SYSTEM.
CITRATE
BUFFER,
was
also
MODEL
MG.,
ntara
checked
38-A
A
FIGURE
PH
FOR
6 .2 .
PURITY
ELECTROPHORESIS
WAS D I S S O L V E D
Ch e m
ELUTION
ic a ls
IN
5
ML.
-5 1 -
!.0 "
'0 0
FRACTION -NUMBER
F
2.
ig ur e
on
a
DEAE
buffer
,
pH
Chromatography
column
6 .2 .
.
El u
t io n
of
p a r t ia lly
was
made
p u r if ie d
w ith
0.1 M.
m yr o s in a s e
c it r a t e
-5 2 OF
(
VERONAL
changed
he
WHEN
10-5
CM. 2
T he
I
t
for
-.8 6 3
TO
is
F URT HER
AMMONI UM
BASED
VOLT-1
of
the
III
OF T HE
P U R IFICA TIO N,
72
hours
.
T
BUFFER,
SEC
VERONAL
he
YIELDED
BUFFER
r e s u ltin g
enzyme
TWO P E A K S ,
FIG URE
PH 8 , 5 ,
( SMALL
fr a c tio n s
OF T HE
ORDER TO
AT
0 ,I
PEAK)
3.
IONIC
AND 2 . 5 8 8
X
o b ta in e d
HYDROLYSIS
OF
d ur in g
p u r if ic a
SI N I G R I N ,
­
ARE G I V E N
I . E .,
RESOLVE
WAS A C H I E V E D
AND A N A L Y S I S
EXPERIMENT,
OF T HE
AND
T HAN T H E
DATA
FOR
a c h ie v e d
an
ap p r o x im a t e
SYSTEM.
It
MAY A L S O
BE
observed
SEPARATION
EXTREME
ITS
BY
OF THE
COMPONENTS
OF THE
ESSENTIALLY
ELIMINATED
ANY T O T A L
TABLE
INDICATES
THIOGLUCOSID ASE
T HUS
OF NEUBERG
A S E WAS A T WO - EN Z Y M E
SPECIFIC
REACTION
SUBSTRATE.
OBTAINED
SYSTEM.
L O SS
INCUBATION
OF T H E
DEFINITELY
PRO PO SAL S
was
FRACTION
THIS
This
PRODUCTS
there
SUBSTRATE.
StRATE.
COMPONENT
that
ENZYME
SULFATE-PRECIPITATED
COMPONENT
T HE
CM. 2
Table
from
EACH
I NG
VERONAL
ON T HE T O T A L
TO CHECK
T HE
IN
a c t iv it ie s
PURIFICATIO N
HYDROLYSIS
OTHER
of
F RESH
SEC.- I .
e v id e n t
T HAT
YSIS
total
AGAIN ST
ELECTROPHORESIS,
PEAKS
X IC T ^
DIALYZED
I I I .
F OL D
n
a
ENZ Y M E ,
4000
I
. )
hrs
s p e c if ic
I N Table
AND
OF T H E
VO LT-1
OF T H E
tio n
PH 8 . 5 ,
SUBMITTED
M O B ILITIE S
STRENGTH WERE
,
24
every
SO LUTION,
T
BUFFER
FAR
A C TIV ITY
OF T HE
MIXTURES
IV
T HE
PRESE NT
OF A C T I V I T Y
THE
BASED
FOR
EACH
ON T HE
T O T HE
M Y R O S I NASE
RESULTS
HYDROL­
OF T H I S
OR ENZYME
SYSTEM.
EVIDENCE
EARL Y WORKERS T H A T
OBSERVATION
SUBr-,
EACH
OF T HE
OF A FACT OR
CONVINCING
OTHER
NECESSARY
S U B S T R A T E WJ TH
PRESENCE
IN
WAS
RELATIVE
S U M M A R I Z E S T HE
GAVE RAT HER
DT AIL AND
IT
F A V OR ­
MYROSI N -
OF T H E S E WORKERS
-5 3 -
F
ig ur e
A
sc en d in g
3.
strength
;
Ele c t r o p h o r e s is
pattern
of
boundary
in
veronal
buffer
T
.,
49
(
im e=
I
hr
m in
.
see
,
p u r if ie d
pH
text
m y r o s in a s e
8 .5 , 0.1
for
io n ic
m o b il it ie s
).
.
Table
Pu
r if ic a t io n
of
V olume
Fr a c t io n
(
m l
)
111
My r o s i n a s e
Ac
t i
v ITY^
u n its
/
md
Pr
o t e in
mg/ ml
Sp . Ac
u n its
t iv it y
/
mg
PROTEIN
Wa t e r
E t OH
extract
p r e c ip it a t io n
(NH4 )2SO^
fr a c tio n a t io n
400
.203
29 .0
.007
100
23.8
25 .0
.954
30 .4
10
669
22 .0
DEAE
(
small
peak
)
8
N.T.H.
2.1
DEAE
(
large
peak
)
6
N.T.H.
6.1
- - - -
DEAE
C O MBI NED
13
12.8
3.8
27 .4
fr a c tio n s
El e c t r o p h o r e s is
(
small
peak
)
0.5
N.T.H.
I .25
—
El e c t r o p h o r e s i s
(
large
pea k
)
0 .5
N.T.H.
3,05
- - - -
El e c t r o p h o r e s is
c o m bin ed
0.8
61 . 7
2.15
28.7
fr a c tio n s
I
One
UNlT=THE
SUBSTRATE
N.T.H.
-
IN
AMOUNT
I
OF
HOUR AT
NO'. T O T A L
ENZYME
37°
HYDROLYSIS,"
PRODUCTS OF T HE MUSTARD
OIL
REQUIRED
T<)
HYDROLYZE
50$
(5 .0
M G.)
OF
C.
THIS
INDICATES
T HAT A L L
GLUCOSI DE WERE NOT
OF THE
OBTAINED.
CL EAVAGE
.
Table
Hy d r o
ly s is
En z y m e
Systema
My r o s i n a s e
M I CROGRAMS
F r a c t io n s
M l CROGRAMS
185
O
O
No. 2
195
O
<10
No. I
O
75
O
No. 2
O
81
O
No. I
235
108
159
No. 2
250
100
150
T
h io u r ea
peak
peaks
peak
d e s ig n a te s
ID EN TIFIED
WITH
NEGATIVE
NO. I
20
M ic r o g r a m s b
Com bined
No. I
Larg e
bT wo
and
peak
m b i ned
T HE
Separate
by
I N O R G A N i c SO4
Sm a l l
a
in ig r in
Glucose
Large
Co
S
of
IV
AND
MG.
OF
the
FRACTION
REP R E S E NT
SI N I G R I N WERE
Z ero
t im e
AND MEASURED A G A I N S T
p o s it iv e
m ig r a t in g
TH I G L U C O S I DASEJ
MIGRATING
No. 2
MINUTES.
THE
THE
DESCRIBED
DUPLICATE
REAGENT
were
PLANKS.
run
PEAK
I N T HE
ENZYME
INCUBATED W IT H
controls
fr a c tio n
SMALL
THE
on
IS
TEXT.
FRACTIONATIONS.
ENZYME
ALL
FOR
SAMPLES
-5 6 -
pH CHANGE
T HAT
ENZYMES,
AN
A
pH
at
l
THE
6.2
FOR T H E
pH
and
PH
AND ANY
T
T HE
SYSTEM.
THE
S O L U T I O N S WERE
CONFIRM
THIS
OF T HE
COMPONENT
VARIATION
ONE FACTOR
SUBMITTED
APPEARED
DER IVATIVE
samples
were
GLUCOSE AND
INORGANIC
OF
WERE
S IN IG R IN
T E C T E D AS A
INVOLVED
AND TO
IN
THIS
TO
A
PAPER
A
ETHER
AMOUNT
IDENTIFIED
FOR THE
at
pH
also
run
At pH 6.2
WHILE
PRODUCT.
RETAINED
p r e v io u s
AT
by
these
AT
THE
THE
BY
LOWER
A C T IV IT Y
STRONG
ALL
PRESENT,
CONVERTED TO
EVAPO RAT ED TO DRY­
ITS
R
T HE ALCOHOL
TO THE
METHOD
V A L U E AS A L RUN A T
PH 6 . 2 .
REACTION
RUN AT
PH
and
OF THE
PH
FOR
MIXTURE
values
ALL
fir s t
EXTRACTED WITH
CHROMATOGRAPHY A C C O R D I NG
SPOT,
SI N I G R I N
were
OF A L C O H O L .
D ET ECT ED
BE
AT
EXTRACT
M I X T U R E S WERE
SMALL
IF
CAREFULLY
COULD
OBSERVED,
COMPONENTS WAS BASED
IN
FAIN T,
REACTION
My r o s i n a s e
the
UP
T HE
in c u b a t io n
W H I C H WAS VERY
ORIGINAL
I
of
after
EXTREMELY
IN
I NCU BATED W I T H
FROM THE
30$
of
AND
REACTION
(5 3 ).
APPROXIMATELY
n h ib it io n
vessels
PRESENT
SULFATE.
HYDROLYSIS
SYSTEM WERE
SYST EMS WERE T HE N
THE
TAKEN
AND R U B I N S T E I N
p lic a t e
Mu c h
A C T IV IT IE S
ISOTHIOCYANATE,
PH
ISOTHIOCYANATE
RESIDUE
NO T H I O U R E A
r e a c t io n
HIGHER
NESS AND T HE
THIOUREA,
he
ENZYME
ODOR OF A L L Y L
RUN AT
LOW
3 .0 .
D ER IVATIVE.
Du
TO
OF MORE T HAN
PURIFIED
T HE T H I O U R E A
LYL
IN THE
WAS D E V I S E D
EXISTENCE
OF T H E
FOR T HE
OF K J A E R
VARIATION
HYDROLYSIS.
SYSTEM
ETHER
T HE
I QUOTS
CHECKED
A
EXPERIMENT
SUBSTANTIATE
ENZYMATIC
EFFECTS
analyzed
3 .0 .
for
HYDROLYSIS
PRODUCTS
ONLY GLUCOSE WAS DE­
OF THE
T H I O G L U C O S I D A S E WAS
PH 3 .0 .
Ph o s p h a te :
b e lie f
that
m y r o s in a s e
ON T HE F A C T
THAT
T HE
RELEASE
was
OF
I
composed
INORGANIC
of
two
SULFATE
-5 7 FROM T HE
OF
S U B S T R A T E WENT
GLUCOSE A T T A I N E D
IN
ATTEMPTING
PHAT E MUST
IABLY
IN
THE
PH
PHOSPHA TE
BUFFER,
Ph o s p h a t e
was
RESULTANT
MIXTURES
ABOUT
RETICAL
T
AN
EXPERIMENT
SHOWED
BEEN
OUT
the
FIN DING
he
VIOUSLY
THE
of
TO
OF T HE
THEORETICAL
MAXIMUM.
USE
PHOSPHATE
OF
SULFATE
THE A C T I V I T Y
AND
the
SEPARATELY
method
IN
F
of
THE
BE
OF T HE
CITRATE
is k e
(54).
PHO SPHA T E
OF
INORGANIC
T HE
ORDER TO A V O I D
USING
THIS
CITRATE
Su lf a t a s e
AS THE
WAS
S IN IG R IN
BUFFER,
pH
n a ly s is
IN
6 .2 .
of
the
SYSTEM
THE
INHIBITED,
PROCEEDED TO THE T H E O ­
EFFECT,
ALL
PROCED­
Factor;
POSSESSING
SULFATASE
SULFATASE
ETHEREAL
(55).
P-NITROPHENYL
ENZYME.
IT
BUFFERS.
T HE
GLUCOSIDES
PHOS­
SYSTEM WAS A P P R E C ­
A
INHIBITORY
REPORT ED T H A T
TOWARD
REL E A S E
DETERMINED,
BUFFERED
SULFATE
S P EC IFIC ITY
FOR THE
CAN
IN
T HE
O IL
BUFFERS,
INCUBATED W IT H
DETERMINE
G L U C O S E - 6 - S O 4 AND
SUBSTRATES
by
WHEREAS
SYSTEM WAS
OF A FACTOR
NO A C T I V I T Y
MUSTARD
ENZYME
6.2 ,
COMPLETION,
INORGANIC
BUF F ER
RELEASE
IN
CARRIED
e c if ic it y
BEF ORE
TO
TO T HE TH I O G L U C O S I D A S E WAS C O N S I D E R A B L Y
THE
MAXIMUM.
THE
SHOWED T H A T
ATTRIBUTED
URES WERE
Sp
removed
41$, W HILE
60-70$
TO A V O I D
CITRATE
INCREASED.
A C T IV IT Y
ONLY
BE REMOVED
OBSERVED T H A T
ESSENTIALLY
No
A C TIV ITY
OF T H I S
ENZ Y M E ,
SULFATES
FACTOR.
CALLED
OR S U L F A T E
I N PARTIAL
REPETITION
S U L F A T E WERE
IT
HAS P RE ­
MYROSULFATASE,
ESTERS
OTHER THAN
OF. T H I S
PREPARED AND
H Y D R O L Y S I S WAS
NECESSITATED
WORK,
CHECKED AS
OBSERVED W I T H
EITHER
MATERIAL .
I
IN
T HE
?
n
order
MUSTARD
to
more
OIL
closely
GLUCOSIDES,
d u p lic a t e
SEV E R A L
the
type
SULFONATED
of
sulfate
ester
O X I M E S WERE
found
PREPARED
-5 8 AND
CHECKED AS
OF T H I S
T
results
OR AT
NOTICED
P O S S IB ILITY
OCCURRI NG
n
SIDASE
AND
ROUGH
ALREADY
THESE
THE
RESULTS
to
presence
ENZYMATIC
USUALLY
USED AS
the
FOUND
ON T H E
of
the
T
IN
T HE
IF
ANY,
SUBSTRATES.
COMPONENT
P A R T I C U L A R L Y WHEN THE
THIS
WOULD
IN
VELOCITY
PO SSIBLE.
M E C H A N I SM W I L L
MYROSINASE
O IL
A
INDICATE
T HE
THE
NAT URALL Y
ONLY WHEN A
MORE
BE BROUGHT
THIS
OF
PURITY,
REPORT ED
TO
HAVE
ARE
GIVEN
above
table
DETAILED
OUT
IN
THE
DIS­
OF
TYPE
MANY
OF
it
GLYCOSIDIC
IT
SUBST RAT ES
may
OTHER T HAN
ON COMPOUNDS
MAY A L S O
COMPOUNDS
ENZYME
TH I OGL UCO-
NECESSARY
REPORTS
EXPERIMENT
ON T HE
OF T HE
WAS F I R S T
PREVIOUS
OF T H E
IN TABLE
:
S P EC IFIC ITY
OF M Y R O S I NASE
NO EF F E C T
,
stard
SYSTEM,
ON V A R I O U S
A C TIV ITY
As
Mu
of
GLUCOSIDES.
CRITERION
F REE
IS
EVALUATE THE
A C TIV ITY
MUSTARD
TH I S E X P E R I M E N T
M AXI MUM
second
I T MAY BE
ONLY WHEN T HE
SUBSTRATES.
AT
a
A C T IV IT Y .
USED T OG E T H E R ,
SUBSTRATE
h io g lu c o s id a s e
PROPERL Y
POSSIBLE
the
REACT
of
WORK.
EXISTING
RELATIVELY
IS
SYSTEM ARE
OF A P O S S I B L E
ORDER TO
F rom
as
POSSESSING
OR PERHAPS
ATTACK
LITTLE,
NAT UR AL
doubt
F A C T O R S MAY BE BOUND TOGETHER
SYNTHETIC
VERY
ILLUSTRATES
T HAT
OF T H I S
CHECK FOR
A FACTOR
ENZYME
ENZ Y M E ,
e c if ic it y
I
V
ARE
INTERPRETATION
Sp
TABLE
GLUCOSIDES
SIMULTANEOUS
CUSSION
l it t l e
MA X I MU M A C T I V I T Y
OF T H E
OIL
leave
LEAST
T HAT
FRACTIONS
MUSTARD
SUBSTRATES.
EXPERIM ENT.
hese
ENZYME
POSSIB LE
THE
HAVE
TO
NATURAL
INDICATED
OTHER THAN
THE
SERVE AS A
CHECKED WERE THOSE
(56).
THE
RESULTS
OF
VI .
be
seen
that
the
m y r o s in a s e
ENZYME'S OTHER T HAN THOSE W H I C H
system
is
SHOW , B - G L U C -
-5 9 Table
T
he
Hy d
r o ly s is
Sulfate
of
Ox
im e
-
V
sulfonates
Compound^ ^
M icrograms
4
cetophenone
o x im e
o xim e
ll
of
the
p o t a s s iu m
24
HRS.
170
40
115
30
100
35
80
-O -
S U L F ON I C AC I D
1
A
HRS.
SO4
-O -
SUL FON I C AC I D
Cyclohexanone
n o r g an ic
O XIM E-0-
SUL F ON I C A C I D
A
I
in
r-
S i NI GR I N
METHYL-N-AMYL
My r o s u l f a t a s e
by
sulfate
s a lts
compounds
were
run
as
the
.
2
2 .0
MG.
P UR IFIED
S A MPL ES WERE
SULFATASE
I NCU BATED W I T H
FRACTION
AT
0.2
37° C.
ML.
OF T H E
-6 0 Table
T
Su
Ef f e c t
he
bstr ate
'
Mu s t a r d
of
M ic r o g r am s
S ugar
4
Ce
L
T
Vl
h io g lu c o s id a s e
R ed u c in g
Va r i o u s
on
Substr ate
Su bstr ates
M icro g ram s
Sugar
ib e r a te d
HRS.
24
HR S.
4
O
O
Ch
Ma l t o s e
O
O
Lactose
O
O
Me
O
O
Sucrose
O
Trehalose
Pe c t ic
L
HRS.
R ed uc in g
ib e r a te d
24
HRS .
O
O
I NUL I N
O
O
Q-ME-D-GLUCOSlDE
O
O
Q-ME-D-MANNOSIDE
O
O
O
- M e - D - X Y L O S I DE
O
O
O
O
j S - i p - D - G L U C O S I DE
O
O
actd
O
O
Ge
n t i o b i ose
135
370
y l o p e c t in
O
O
Am
y g d a l in
140
560
A mylose
O
O
Ph
lo r id z in
120
347
Glycogen
O
O
Sa
l
105
965
Xylan
O
O
P —1\]0 2 ~ (p —D ™
560
1030
Am
l l o b io se
l l ib io s e
■
it in
ic iN
GLUCOS I DE
T
In
,
5
'
r e a c t io n
m ix t u r e s
OF T H E
PURIFIED
T H ! OGLUCOSIDASE
ITING
REDUCTION,
REDUCI NG
STANDARD.
SUGAR.
mg
.
the
T HE
AC TIV ITY
ALL
SUGAR
of
substrate
PREPARATION.
WAS MEASURED
IN
were
in c u b a t e d
FOR T HOSE
TERMS
w ith
0.2
COMPOUNDS
OF AN
INCREASE
MEASUREMENTS WERE REFERRED TO A GLUCOSE
ml
EXHIB
IN
-6 1 OS I DASE A C T I V I T Y .
T HE MUSTARD
AND MAY
BE
A C TIV ITY
WAS
RESPONSIBLE
ON S Y N T H E T I C
SPECIFIC
T HUS
FOR T HE
HOWEVER,
G L U C O S I DES T H I S
BY
FAR
(5 7 ).
THAT
MAY MORE
SPECIFICALLY
BINDING
he
large
INDICATES
LITTLE
EFFECT
DOES A F F E C T
COLYTIC
IN
OF T H E
WOULD
RAT E
OF
UNDER
T HESE
SAME
OF MUSTARD
USING
SE V E R A L
RESULTS,
NOT
BE
OF T H E
STRICT
A L T HOUGH
SPECIFIC
BY THE
ENZYME
AC TIV ITY
HYDROLYSIS,
AS
AND
ITS
OF THE
SUBrN O N -T H I O-
IMPLIED
SURPRISING,
T H I O-LINKAGE,
BUT
OF THE
PROPOSED
SUBSTRATE.
o il
LINKAGE
g lu c o s id e s
OF THE M O L E CU L E
ENZ Y M E .
AS WOULD BE
RELATIVE­
STRENGTH
mustard
PORTION
OTHER
BEEN
SOMEWHAT
RELATIVE
IS
NAT UR AL
HAS
FOR T H E
occ ur r in g
AGLYCONE
THERE
V II.
WAS O B T A I N E D
BE AS
CONTRAST
MOLECULE
IMPLY
TO T HE
FOUND
CONSIDERABLY
T HE MUSTARD
IN
T HE
S P E C IFIC ITY
IN
FIRST
OF T HE
O IL
PART
VARIABLE
THE
MORE
AGLYCONE
NAT URAL
THIS
HAS
VARIATION
E X PEC T ED FOR A G L Y ­
GLYCOSIDES,
OF T H I S
MOIETY,
PRODUCTS
S P EC IFIC ITY
IS
ENZYME TOWARD CHANGES
THE
AND
T HE
PORTION
GLUCOSE.
TOWARD T H E
WERE USED TO
IN
GLYCONE
ALWAYS
INVOLVED
SYNTHESIZED
THESIS,
OF
FOR
CONDITIONS,
I N THE
NOT
HYDROLYSIS
WHEN CHECKED
FOUND
naturally
ABSOLUTE
OBSERVED.
LINKAGE
BE A F F E C T E D
of
FOR T HE
AS A / S - T H I O G L U C O S I D A S E
T HE TM I O G L U C O S I D A S E
MAY
THESE
VARIATION
THE
CHARACTERIZED
IN TABLE
ENERGY BETWEEN T H E
ON T H E
RESPONSIBLE
ENZYME.
MOIETY.
CRIBED
T HAT
ENZYME MAY
number
T HAT
SHOWN
THIOGLU COSID lC
INDICATE
T
AS
AS A C T I V I T Y
T HE
BEEN
THlO G LYCO SIDES
SP E C IFIC ITY
P R E V I O U S WORK
AND T HE
HAS
IS
FOR T HE A C T I V I T Y
FOUND,
APPEARS
S T R A fES;
ENZYME W H I C H
he
G L U C O S I DES
NO A C T I V I T Y
IT
LY
O IL
T
ISOLATED
SUGAR
AS
DETERMINE
GLYCONE
THIS
PORTION
DES­
THE
OF THE
-6 2 Table Vl I
Ac
t iv it y
of
Mu s t a r d
T
h io g lu c o s id a s e
on
Sy n th e tic
T
h io g lyc o sid e s
i
Substrate
M icrograms
SUGAR
4
L
R ed uc in g
ib e r a te d
HR S .
24
I
C - E T H Y L T H I OGLUCOFURANOS I DE
O
O
C - B E N Z Y L T H I O G L UCOFURANOSI DE
O
O
J 3 - E T H Y L T H I OGLUCOFWRANOS I DE
O
O
C - E T H Y L T H I OGL UCOPYRA NOS I DE
0
O
J S - E T H Y L T H I O G L U C O P Y R A N OS I DE
O
O
^ - P H E N Y L T H I OGL UCOPYRANOS I DE
O
O
O
O
O
O
O
O
O
O
P H E N Y L A C E T O T H I OHYDROXAM I C AC I D S - j B - D - 1 - G L U C O P Y R A N O S I DE
P H E N Y L A C E T O T H I OHYDR OXAMI C
ACID-
S-^-D -I-X Y LpP Y R A N O S ID E
P H E N Y L A C E T O T H I OHYDR OXAMI C
AC I D -
S - J B - D - I - G A L A C T O P Y R A N O S I DE
P H E N Y L A C E T O T H I O H Y D R O X A M I C AC I D S - J S - D - I - M A N N O P Y R A N O S I DE
T
he
WITH
mustard
2
MG.
th io g l u c o s id a s e
OF
EACH
p r e p a r a tio n
INDIVIDUAL
,
COMPOUND AT
0.2
37°
m l
.,
C.
was
incubated
-6 3 substrate
molecule
.
Table
II
Vl
g ives
the
effects
of
the
enzyme
on
these
SUBSTRATES.
T
THE
THAT
he
results
shown
GLYCONE M O I E T Y
THIS
OF T HE
PREF EREN CE
OF T H E
OF MANY
^ - G L U C O S I DASES
he
MO L ECU L E
results
O IL
GLYCOSIDES.
SULFATASE
I
FACTOR
A
IN
THEIR
DEFINITE
MUSTARD AND THE
RESEMBLE
ONE ANOTHER
STRATES.
In
SUBSTRATE
OF ALMOND
ENZYME A P P E A R S TO
GLUCOSIDIC
IN
HOWEVER,
EFFECT,
NAT UR AL
TO
THAT
THE
T HE
M OIETY,
OUT
CLEAVED
BY THE
in o r g a n ic
NA T U R A L
BY
ASIDE
MUSTARD
RATE
A C T IV IT Y
OF THE
OF THE
PREVIOUS
VERY
EXPERI­
CLOSELY
SYSTEM
FROM T H E
T HE TWO ENZYME
S IM ILA R ITY ,
ON T H E I R
OF T H I S
RATHER
NAT UR AL
AMYGDALIN,
SUBSTRATE
BY T H E
IN
VIEW
NATURAL
BY THE
MUSTARD
OF A M Y G D A L I N ,
TH I O G L U C O S I D A S E .
S UB ­
T HE
EMULSI N ^-G LU CO S I DAS E .
PORTION
OF
SYSTEMS A L S O
SHOWN TO BE HY D RO L Y Z E D
GLYCONE
SUGAR
SPEC IFIC ITY
that
M Y R O S I NASE
GENERAL
T HE
THE
BY THE
THIS
GENT I O B I O S E ,
T HE
HYDROLYSIS
A C TIV ITY
OF
OBSERVED
59).
AND
OVERALL
THE A C T I O N
BE
for
FUNCTIONS.
OF THE A L M O N D .
HYDROLYSIS
MAY
TWO FACTORS ARE
BETWEEN T H E
HAS BEEN
s p e c if ic it y
W ITHIN
show
THEIR
PARALLEL
BOND A RE
SYNTHETIC
WAS BORNE
THAT
GLYCONE
IT
(58,
table
AFFECTED
W H I CH
EXISTS
E M U L S I N,
THE
SYST EMS
PHYSIOLOGIC AL
FOR T HE
IN
GLYCOSIDES.
pr eced in g
OF T HE
P O S S IB ILIT Y
ADDITION
SYSTEM.
BOND
APPEARS,
d e f in it e
CORRELATES WIT H
PLANT
the
E M U L S IN, SYSTEM
P RE F E RE N CE
J 3- 1----- > 6
in
SIM ILA R ITY
SPECIFIC
MYROSINASE
IN
FROM A L L
THIS
F U R T H E R S T HE
CONJ U GAT ED
C L OS E L Y
FOUND
a
CONFIGURATION
MAY BE C O N S I D E R A B L Y
TH I O G L U C O S I D A S E .
MENT S,
t
in d ic a t e
O IL
DEFINITE
VERY
observed
S U L F A T E WAS L I B E R A T E D
Vll l
MUSTARD
FOR A
PORTION
T
Table
in
T HE
AND THE
OF T H I S
-6 4 Table V l l l
T
he
Hy d r o
ly s is
Mu s t a r d
of
O
Gl y c o
il
s id e s
My r o s i n a s e
by
I
Substrate
M icrograms
Sugar
4
GLUC OT ROPAEOLAT E
L
R ed uc in g
HRS.
24
M ic r o g r am s
I
ib e r a t e d
4
HRS.
M icrograms
SO4
n o r g an ic
HRS.
T
h iourea
4
HRS.
O
O
47
O
O
O
30
O
O
O
50
O
O
O
55
O
657
C .H .3
420
500
630
C.H.
399
509
57
77
TETRAACETATE
S-yS-D-l - T E T R A A C E T Y L G A L A C T O - _
PYRANOSYL-PATHA-O-SULFONATE^
S-J3-D-1 - T
ETRAA C ET Y L M A N N O -
PYRANOSYL-PATHA- 0 -S U L F O N A T E
S -B -D -I - T R I A C E T Y L X Y L O PYRANOSYL-PATHA-0 -S U L F O N A T E
Glucotropaeolate
(
s y n t h e t ic
Gl u c o t r o p a e o l a t e
(
natural
)
)
.
S -B -D -I - G A L A C T O P Y R A N O S Y L 15
PATHA-O-SULFONATE
90
S -B -D -I - M A N N O P Y R A N O S Y L PATHA-0 -S U L F O N A T E
0
O
50
295
192
O
S-B-D-I-XYLOPYRANOSYL-
77
PATHA- 0 -S U L F O N A T E
^All
substrates
MYROS I NASE
were
FRACTION,
run
as
0.2
the
M L.,
TETRAMETHYLAMMONlUM
WAS
II
N C U B A T ED W I T H
2
SALTS.
MG.
200
T HE P U R I F I E D
S U B S T R A T E AT
C.
2
3
PATHA
-
PHENYLACETOTHlOHYDROXAMlC
C. H.
-
INDICATES
PRESE NT
IN
T HE
CO M P L E T E
INCUBATION
AC I D
HYDROLYSIS
MIXTURE.
OF T H E
QUANTITY
OF
SUBSTRATE
37°
-6 5 result
,
AS WELL
H Y D RO L Y Z E D
AS T HE
BY BOTH
I . E .,
PREVIOUSLY
I
n
a
A
SEVERAL
SYSTEM
LINE
GLYCOLYTIC
ALMOND
T HE
THE
p u r if ie d
OTHER
O ILS .
IT
^ - G A L A C T O S I DASE
ZYME
RESULTS
EXPERIMENTS
EM ULSIN ,
IN ALL
however,
HAVE
INSTANCES
almond
OF T H I S
BEEN
e m u l s i n .w a s
OF MERCA P T OPUR I N E S .
the
m yr o s in a s e
NECESSARY,
system
THEREFORE,
,
c o n ta in s
TO
PURIFY
FROM THE
BlOCHEMICALS
NUTRITIONAL
MATERIAL
OF T HE
FOR THE
EMULSIN
jb- g l u c o s i d a s e
,
OF T H I S
TO BE
ENZYME
HERE T H A T
HAVE
BEEN
THE
CRYSTAL­
CORP­
PU R IFIC ATIO N .
TABLE
SYSTEM.
although
WAS THOUGHT
EMULSIN
OF
(60).
REMAINED.
EF F E C T
ARE
EF F EC T
OF THE , B - G L U C O S I DASE
BE M E N T I O N E D
OF ALMOND
GLUCOSIDES
not
PURE
e n t ir e ly
free
ENOUGH TO
ON T HE
OBTAIN
GLYCOSIDES
THE ^ - G L U C O S I D A S E
SHOWN TO BE A
of
OF THE
AND THE
SINGLE
EN­
(62).
T
he
p u r if ie d
T ROPAEOL I N ,
IT
to
STARTING
ENZYMES,
MUST
GLYCOSIDES.
(61),
I T WAS
PURCHASED
ON T HE
O IL
CO NJ U GA T E S
AC TIV ITY
em u lsin
RESULTS
Al .
PURIFICATIO N
GLYCOLYTIC
REASONABLE
Er
ENZYMES.
EMULSIN,
NAT URAL
N EC ESSARY TO CHECK T H E
NEGATIVE
contrast
WAS USED AS T HE
he
MUSTARD
in
ONLY T HE
ILLUSTRATES
T
,
SEEMED
OTHER
OF TH I O G L Y C O S I DES W I T H
W ITH
GLUCOSE
SEVERAL
MUSTARD
Goodman
by
e m u ls in
UNTIL
ON THE
O UT ,
HY D R O L Y Z E
ORATION,
IX
paper
lmond
IT
HYDROLYSIS
CARRIED
recent
FOUND TO
T HE
THAT
SYSTEMS,
E M U L S I N J3- G L U C O S I D ASE
NATURE,
FACT
g lu c o s id a s e
A S WELL A S W I T H
HAD NO E F F E C T
INCUBATED W IT H
CONSIDERABLE
^ -
ON T H E S E
S A LIC IN
IN H IBITIO N
IN
OF
T HE
was
in c u b a t e d
SYNTHETIC
COMPOUNDS.
THE
MUSTARD
s in ig r in
OIL
and
gluco-
GLYCOSIDES,
BUT
WHEN A L M O N D j g - G L U C O S I DASE WAS
PRESENCE
AC TIV ITY
w ith
WAS
OF T H E
MUSTARD O I L
OBSERVED.
G L U C O S I DES,
I N F URT HE R
PURSUANCE
Table
T
En zym e
Pu
he
r if ic a t io n
Fr a c t io n
u l s in
Red uc in g
^-GALACTOSIDASE
^-G
luc o sidas e
Sugar
I
L
ib e r a te d
O . - M A N N 0 S I DASE
2
&-GALACT0S1DASE
1320
560
900
30
on
1970
801
615
19
Enzyme
(NH4 ) 2 S0 4
Fr
DEAE
(1
st
column
)
5280
2210
170
<10
DEAE
(2
nd
column
)
C .H .3
2610
15
I
Substrates
act
for
i
Em
lmond
M ic r o g r am s
^-G LU CO SlDA SE
Cr u d e
A
of
IX
the
enzymes
M E L L I B I OSE R E S P E C T I V E L Y .
THE
g
I
SUBSTRATE
n cu batio n
S lD ASE
3
C. H.
AT
p e r io d s
REACTIONS
-
37°
INDICATES
(5
ML.
mg
OF
.)
s a l ic in
EACH
,
ENZYME
lactose
,
qi - M e -
F R A C T I O N WERE
D - mannosi
de
and
I NCU BATED W I T H
C.
for
WERE
were
0.2
all
enzymes
I NCU BATED
COMPLETE
FOR
except
12
HYDROLYSIS
ol- m a n n o s i d a s e
were
20
HOURS.
OF T HE
S UB S T RA T E
PRESENT.
m in
.;
a-MANNO-
-6 7 OF T H I S
WITH
PHENOMENON,
THE
EXCEPTION
IN H IBITIO N
TRATIO N
I NG
IN H IBITIO N
SOMEWHAT
TION
ON T H E
I
REVER SE
THE
n
GLUCOSIDES,
IT
YIELDING
AFTER
INCUBATION
BEEN
MI CROGRAMS
REPORT ED
WITH
IS
ENZYMES,
IN
COLOR W I T H
IS
C.
FOR
ABOUT
THE
T HAT
V A L UE
GALACTOSE,
OF
SHOWN TO
DAYS
THE
FOR
DEPEND­
X.
NO
THE
IN H IB I­
OF T H l O G L U C O S I DASE
HYD ROL YZ E MANY
2 .0
MG.,
13$
ON
OF T HESE
WHEN
INCUB­
HYDROLYSIS,
P H E N Y L - S - ^ - D - G L U C O S IDE,
(L IT .
SAME C O N D I T I O N S
THUS
FAR ARE
S P E C IFIC ITY
BOUND
60),
YIELDED
OF
GLYCOPROTEINS
TOWARD THE
CONTAINED
ITS
EXTREME
CARBOHYDRATE.
ABOUT
3 $ , GAVE THE
IN
(63),
GLYCONE
T HE
PORTION
ENZYME
S IM ILA R ITY
(64).
TO
T HE CARBOHYDRATE
CHARACTERISTIC
INDICATING
GLUCOSE.
THE
0.95
ACC O R D I NG
TO GURI N AND HOOD
OR A M I X T U R E
CONCEN­
HYDROLYSIS.
BEMUSE
W H I CH
CONSIDERABLE
SEE T A B L E
BY M Y R O S I N A S E
CARBOHYDRAT E
ENZ YME,
GLYCOSIDES,
APPARENTLY
SHOWED ABOUT
LIKEW ISE
FOUND
OF MU S T A R D ,
PURIFIED
EFFECT
UNDER T H E S E
6$
ABOUT
SHOWED P R A C T I C A L L Y
TO BE A F F E C T E D
ENZYME
WAS CHECKED
10
O IL
I C T ^ MOLAR
INHIBITOR;
PHENYL-^-D-GLUCOSIDE,
DUE TO THE
CARBAZOLE,
GAVE A
INDICATE
THE
THE
BEEN
BROUGHT
FROM 4 0 - 6 0 $ ,
EXPECTED,
HAS
MUSTARD
A T ABOUT
OF T HE
GLUCOSE.
NOT
SOME B E L I E F
SUBST RAT E
CONTAINED
E 4 2 Q,
OF
OF G L U C O S E ,
TH I OGLUCOSI DASE
THESE
50°
VARY
I.E .,
OF T HE J B - G L U C O S l D A S E S
AND T HERE
OF THE
AT
MI CROGRAMS
HAS
ll
ENZYME
WAS FOUND T H A T
WHICH
A
THE
MYROSINASE
170
BE
SITUATION,
OF THE
A C T IV IT Y .
PORTION
AS M I G H T
ALL
DERIVATIVE,
FOUND TO
EMULS I N ENZ Y M E .
ATED W IT H
he
WAS
GLYCONE
DER IVATIVE,
COMPOUNDS.
T
MANNOSE
OF T HE
NORMAL
70
OF T HE
OF T HE ^ - G L U C O S I D A S E
THE
MANNOSE
I T WAS FOUND T H A T
ABSORPTION
OF GL UCOSE AND MANNOSE,
RATIO,
An
PINK
E ^
SHOULD
A T T EMPT
q
!
- 6 8 -
Table
n h ib it io n
Substrate
o f
'Em
u ls in
^-G
Sy s te m ^
l u c o s .i b a s e
Sa
l ic in
+
S
Sa
l ic in
+
Gl u c o t r o p a e o l in
in ig r in
Sa l i c i n + S - 6 - D - 1 - g a l a c t o PYRANOSYL-PATHA-0-SU LFON ATE
4* S-^—D-1
l ic in
+
l ic in
S-B-D-I-
xylo
Gl u c o s e
iber ated
Percent
I
n h ib it io n
O
3.28
10-5 M.
56.2
M.
1. 3 2
59.8
X
10-3 M.
I .97
40.0
1.1
X
10-3 M.
3 .2 6
0
I .0
X
10-3 M.
I .60
I .2
X
I .0
X
I .0
c o n ta in e d
5
mg
.
s a l ic in
P H E N Y L A C E T O T H I O HYDROXAMl C
.
ACID
CO
-
L
Gl y c o s i d e s
B
systems
Mg .
il
-
PYRANOSYL-PATHA-0-SU LFO N ATE
PATHA
s id e
O
—m a n n o —
PYRANOSYL-PATHA- 0 -S U L F O N A T E
^All
of
Gl y c o
il
Mu s t a r d
5
l ic in
Sa
O
NONE
Sa
Sa
by
Co n c en tr atio n
Mu s t a r d
I
I
Ul
he
O
T
X
-6 9 WAS MADE TO
YSATE
FOR
HY D RO L Y Z E
SUGARS.
C O N C L U S I O N S AS
T HE
PAPER
TO THE
ENZYME W I T H
TRYPSIN
CHROMATOGRAPHY
IDENTITY
OF THE
AND TO A N A L Y Z E
OF T H I S
MIXTURE
SUGARS C O N T A I N E D
GAVE
IN
T HE
T HE
HYD RO L ­
NO D E F I N I T E
PROTEIN
MATER I A L •
T
hat
SIDASE
the
A C TIV ITY
A C T IV IT IE S
T HE
th io g lu c o s id a s e
RELATIVE
MYROS I NASE
A C T IV IT Y
ITY.
TO T H A T
OF T H E
AND
Table
OF
ARE T HE
EACH T Y P E
SYSTEM .
S lN IG R IN
OF
that
REMAINED
T H I OGLUCOSIDASE.
the
and
HYDROLYSIS
factor
of
the
EVEN AT
BY
DU RI NG
CHECK THE
SUBSTRATE
r a t io
CONSTANT
the
ILLUSTRATED
WAS USED TO
WAS USED AS T H E
in d ic a te s
SA LIC IN
mustard
ENZYME WAS
TO
S ALIC IN
Xl
SAME
of
show ing
COMPARI NG
THE
PU R IFICATIO N
OF
TH I OGLUCOS I D A SE
FOR J g - G L U C O S I D A S E
h ydr o lysis
T HE
J3-g l u c o -
MAXI MUM
of
ACTIV­
s in i g r in
PUR IFICATION
-7 0 Table
Co m p a r a tiv e
Enzyme
T
h io g lu c o s id a s e
System
S
Cr u d e
Fr a c tio n
DEAE
Fr
Ea c h
system
a c t i on
REACTION
WAS
c o n ta in e d
INCUBATED
2
Sa
L
Ac
o r My r o s i n a s e
t iv it y
Ra
ib e r a te d
Sa
l ic in
l i c i n/
tio
S
40
22
.550
220
120
.545
190
105
.552
mg
AT
c o s idase
Gl u c o s e
in ig r in
Enzyme
(NH4 )2SO4
J3- G l u
and
M icro g ram s
Xl
.
of
37°
substrate
C.
FOR
20
and
0.2
MINUTES.
m l
.
of
in ig r in
enzyme
.
T
he
-7 1 C.
T HE R E S U L T S
CREPANCIES
IN
OF T H E
THE
ST RONGLY
SUPPORT S
ENZYME.
THE
POSAL
Neuberg
of
SPECULATION
TO
HYDROXAMIC
REMOVAL
P R E V I O U S WORK
THE
is
T HE MUSTARD
ACTUALLY
(Li t .
HAVE TO
14)
OXIMES
A CT ER
I N T HE
OF
(L
i t
.
EFFECT
TO
ANY
BE
THE
16)
and
SIM ILAR
T HE
THE
under
TO T H A T
OF
by
IS
EVIDENCE
OBTAINED
SYSTEM
THE
IN
T HE
OF
LACHMANN
THE
FREE
PROPOSED
THIS
(L
AFTER
by
PRO­
the
SIMILAR
ENZYMATIC
INFLUENCE.
theory
(65),
,
would
i t
NUCLEOPHILIC
ACIDS
.
Us h iyam a
NE G L E C T E D
COMPOUNDS,
15),
E .G .,
FOR
CHAR­
VERY
DO NOT
but
UNDERGO
rather
AND W A T E R .
(L
AND
i t
OR AT
DOES
PROCEEDS
OBSERVED
MECHANI SM
SUL FUR
REASONING
REACTION
on
A MANNER
BY E T T L I N G E R
and
ESSENTIALLY
T WO - E NZ Y M E
UNDERGOES AN ORGANI C
EXTREME
THIOHYDROXAMIC
Na g a s h i m a
T HE
T YPE
MAKES T H I S
N ITRILES,
T HE MUSTARD
ENZYMATIC
only
D IS­
LOSSEN REARRANGEMENT
c ir cum stances
OF T H E S E
ACIDS,
T HE
ACIDS.
any
MO L ECU L E
OF
IN
p r im a r ily
BEHAV E
es ta b lis h e d
HOWEVER,
O XYGEN .
KNOWN R E A C T I O N S
THIOHYDROXAMIC
OF
MECHANI SM
ATOM
THE
based
INDEPENDENT
SHOWN T H A T
FORMATION
SUL FUR
be in g
BETWEEN
STRONG
corroborated
,
RESIDUAL
been
( 6 6 ),
HAS BEEN
THEORETICAL
OF T H E
OF T HE
OF T HE
SUL FUR
MANY
A S S U M P T I O N WOULD MEAN T H A T
has
OF
rearrangement
DECOMPOSE W I T H
IN
w h ic h
INDICATE
CONTRADICTING
GLUCOSIDES
BE A CO MPROMI SE
DIVALENT
L ossen
O IL
APPARENTLY
P RESE NCE
IT
12)
d u bio u s
AND A REARRANGEMENT
IMPROBABLE.
a
IS
OUT
OF A TWO^COMPONENT
GLUCOSE M O I E T Y ,
rearrangement,
h is
THIS
CARRI - ED
ON M Y R O S I N A S E •
(L lT .
somewhat
REARRANGEMENT W H I C H
T
CONCEPT
ACIDS.
OF T H E
is c u s s io n
EXPERIMENTATION
REPORTED WORK
THAT
D
37),
LEAST
NOT
IN
.
LUNDEEN
the
ASSUMED
CORR EL AT E W I T H
T HE
ENTIRELY
ALKYLATION
OPPOSITE
TO
72THAT
OF T H E
WHICH
HY D RO X A M I C
T HESE
BY AN
ENZYME
PROPOSED M E C H A N I S M ,
YIELDED
I
THEIR
INVOLVED
T
he
IN
IMPLY
DEFINITELY
OF T H E
MUSTARD
TO THE
SUL FUR
THE
BY
IN
FORMATION
ENZYMATIC
EXPLAIN ED
m e n tio n e d
RATHER
AS
EASILY
work
OTHER
MANY
r if ic a t io n
T HE
of
the
IN
ENZYME
AC TIV ITY
T HAN
BROUGHT
BOTH THE
OUT
IN
INVOLVES
RNCS
THAN
CAN
TO T H E I R
THE
PREDOMI NANT
OR WEAK A C I D
SOLUTION
AS
T HAT
THE
UNLIKELY
OF THE
MUSTARD
BY T HE
and
V
PROPOSED
OF CRUC I F ERAE
T HE
NITROGEN
OF THE
THE
FROM
REARRANGE­
(L
irtanen
THESE
MIGRATION
THAT
O ILS
PROPOSED.
i t
.
31)
WORKERS
HYDROLYSIS
R GROUP
AT OM.
,S-GLUCOSE
R-C
system
AND T HUS
AND T H E
A
--------------- >
F O R M A T I ON
CONSIDER
PREVIOUSLY
GLUCOSE AND THE
apparently
IMPLIES
POSSIB LE
ONE MUST
THE
THAT
QUITE
e lin
SPECIES
T H A N TO THE
enzyme
COORDINATING
SYSTEM,
DRASTIC
UNDER
RSCN
xiNOSO3-
ISOTHIOCYANATE,
ENZYMATIC
Gm
of
S-G lucose
Pu
APPEARS
/
----------- >
(68)
AQUEOUS
T HE
GLUCOSIDES
CONDITIONS
ISOTHIOCYANATE.
/O iQ S O 3 "
R-C
L u NDEEN
IT
IN
THE
CONTRADICTION
CIRCUMSTANCES
SHOWN T H A T
ATOM
I N DIRECT
GLUCOSIDES
A MECHANI SM
O IL
ALSO
OBSERVED ARE MUCH MORE
OBSERVED
NOT
CAN BE
p r e v io u s ly
WOULD A L S O
WAS
AND
6 6 j6 7 ).
SYSTEM.
O IL
OF T H E S E
G L U C O S I DES
MENT.
HAVE
N ITRILE
VIEW
M E C H A N I SM
IT
OF MUSTARD
T HE
n
(llT .
REARRANGEMENT S ARE
BE T O L E R A T E D
HYDROLYSIS
AC I DS
A
SULFATE
DEFINITE
OF T HE
M E C H A N I SM
SEV E R A L
DISCUSSED
causes
CONNECTION
MUSTARD
BASED
OF T HE
BEING
only
of
BETWEEN THE
O IL.
ON T HE
O BSERVED TWO-
OBJECTIONABLE
MECHANISM.
M O IETIES
pro ductio n
THE
SITUATIONS
PROBABILITY
SIMULTANEOUSLY
OF
HYDROLYZED
-7 3 INVOLVES
T HE
AGAIN
T HE
P O S S IB ILITY
ENZ YME,
BY T H E
SIDE
BRINGS
OF
TO
OF T HE
CLEAVAGE
SULFATE
OCCURS
MUST
VERY
BE
ENTIRELY
BOTH
It
HAND,
T
Ush iy a m a
(69)
WHICH
ARE
Heavy
metal
SIDER
AT
OBTAIN
work
and
ENZYME
BETWEEN
THIS
a
OBTAINED
BY
CHARGES.
T
-
GROUP R E L E A S E D
YIELD
BEEN
A GL YCO ­
SHOWN BY
REACTION
THE
T HE A P P E A R A N C E
FIR S T.
RESULTS
RESPECTIVE
E LIM INATIO N
AS T H E
TO
G L UC OS E ,
THIS
OF T H E S E
CL EAVAGE
OF
THE
INORGANIC
NEXT
POSTULATION
INDICATE
DIGRESS,
THE
_e t
AlL
(L
i t
.
56)
in h ib it io n
WITH
As
of
A T T A C HM E NT
T HERE
HAS
BEEN
and
STEP
SEEMS
NECESSITY
ENZYME.
THESE
GROUPS
I T MAY
TO
THE
THE
of
Na g a s h i m a
NO P R E V I O U S
BE R E C A L L E D
T HAT
THE
POSSESSED
O P P OS I NG
and
reagents
as
in h ib i
­
CON N E C T I O N
ENZ Y M E S ,
IN
EXISTING
GROUPS.
c o n fir m e d
THAN
PO SITIVELY
by
SULFHYDRYL
were
AT
OF
OTHER
SEPARATION
WOULD MEAN T H A T
also
SUBJECT
m yr o sin ase
GROUPS AND G L Y C O L Y T I C
INVOLVING
FROM THE
UNDERSTANDING
chloromercuri benzoate
ELECTROPHORETIC
h is
SAME T I M E
TIME
strong
SULFHYDRYL
TO T HE
SINCE
THE
HAVE
ENZYME.
ITS
FIRST
GROUP WOULD
IN ITIA L
A MORE THOROUGH
SYSTEM.
P O S S IB ILIT IE S
SUBST RAT E
p
THE
CONSIDERING
HYDROLYSIS.
Reese
of
in d ic a t e d
salts
P RO BABL E
THE
IS
COMPOUNDS
BY THE
EXPERIMENTAL
NECESSARY AT
he
SULFATE
SUCH
ACID.
EACH GROUP BY
EITHER,
C O RR E L A T E D W I T H
AS T H E
OF
FOLLOWS T H A T
NORMAL LY A S S O C I A T E D
OF T HE
REPORT ED
IT
FOR T O T A L
ORDER TO
.
AND
MOST
ESSENTIALLY
ENZYMES
s it u a t io n
tors
MOIETY.
FEASIBLE,
IN
IF
OF T HE
ACID,
AS T HE
CLOSELY
SEEMS
CL E A V A G E
NOT TO BE A F F E C T E D
LEAVES
GLUCOSE
OF A T H I O H Y D R O X A M I C
QUESTION WHICH,
OF A T H I O H Y D R O X A M I C
PROSPECT I VES
THE
INDEPENDENT
HYDROLYSIS.
EXPERIMENTATION
OF
REARRANGEMENT
WE MUST
THE
CON­
BINDING
OF
TWO FACTORS
ELECTROSTATIC
CHARGED FACTOR
HAS AN
74I S O E L ECTR I C P O I N T
FACTOR,
AN
ISOELECTRIC
TO R E T A I N
MOST
ISTS
T HESE
T HAT
COENZ YME,
IN
OF
ITS
T HE
OF THE
OF
SULFHYDRYL
ENZYMES
WITH
GROUPS,
ASE
NOT
IS
R eturning
MUSTARD
CU L E
O IL
to
ENZYME.
NOT
ASSOCIATED WITH
the
the
THE
FACTOR
substrate
BINDING
OF T HE
O PHILIC
CHARACTER
THEREBY
INCREASE THE
HAS
BEEN
CARRIED
OF
OUT
is
OF T H E
m ec h an ism
SHOWN
P O S S IB ILIT Y
TO
AN
EX­
ENZYME AND
ENCOURAGE T HE
GROUPS
AND T H E
NECESSITY
F ACT
OR COFACTORS
SUCH AN OCCURRENCE
in vo lved
in
THIS
FACTOR
only
to
BEYOND THE
IN
T HAT
BOUND
T HE M Y R O S I N -
ENZYME
SUL FUR
ATOM
TIME
T HE
one
factor
or
IS
OF T H I S
COULD
IN
GLYCOSIDIC
AND
CAN
EVIDENCE
DEAL
OF
UNDETECTED.
both
d ur in g
LINKAGE
IN
ANY
t h is
THE
REDUCE THE
N U CL E ­
AND
CASE,
THE
MORE C O M P L I C A T E D THAN
CONSIDERABLE
BE
MO L E­
APPARENTLY
DISCUSSIO N.
EF F EC T
the
SURFACE
YET
to
of
RESIDUAL
BOUND TO T HE
OF A R E AR RANG EMENT .
M E C H A N I SM
THAT
THAT
AND R E M A I N S AS
OF THE
BY P R E V I O U S WORKERS,
EXACT
hydr o lysis
REARRANGEMENT
SCOPE
BY T HE
the
SPECULATE
SEEMS TO BE A GREAT
THIS
HAS BEEN
E X P L A N A T I O N WOULD
CONTENTION
REARRANGEMENT W H I L E
P LA U S IB ILIT Y
BEF ORE T HE
AT
OF
NEGATIVE
U N F O UN DE D.
bound
IS
HYDROLYSIS
WORTH M E N T I O N I N G
AND A L S O
PROSTHETIC
PROPOSAL
THE
SIM ILAR
Tm S
SYSTEM.
AND T HE
M Y R O S I NASE
56),
(LIT .
ON T H I S
SULFATASE
SUBSTRATE
SUGGESTED
BA S E D
CONTROLLING
T HE
PROPOSED REARRANGEMENT
MECHANI SM
OCCURRI NG
WE MAY F URTHER
DIRECTED
THE
Wh e t h e r
T HE
G L U C 0 S I DES,
UNDERGOES A
8.5
PH
As
PH°
8.5,
PH
BOUND T O G E T H E R ,
VARIABLE
ENTIRELY
now
SYSTEM,
OBSERVED R E A C T I O N S
THROUGH T H I O L
SYSTEM
AT
BE
GROUPS.
EXIST
OF T HE
BELOW T H I S
A C TIV ITY
NATURALLY
MANY
MANY
POINT
F A CT OR S MAY
SATISFY
FREE
PH
ABOVE T HE
DETERMINED.
EXISTS
WORK MUST
IT
INDICATING
BE
MAY BE
THAT
SULF-
-7 5 ATASES
ASES
ARE
NOT
(70).
T
HYDROLYTIC
h is
ENZYME-SULFATE
,
IN
BUT ARE A C T U A L L Y
would
INTERMEDIATE,
AND
n ece s s ita te
COULD
the
SULFATE
TRANSFER­
fo r m atio n
HAVE A D E F I N I T E
of
BEARING
an
ON THE
FOR M Y R O S I N A S E .
a tt e n t io n
OBTAINED
sults
course,
of
PROPOSED M E C H A N I S M
Gr e a t e r
ENZYMES,
THE
has
been
focused
DETERMINATION
at
t h is
OF T HE
tim e
on
S P E C IFIC ITY
the
unusual
OF T H E
re
­
MUSTARD
THlO G LW CO SID ASE.
T
FINED
he
absolute
TO T HE
CARBON ATOM
WITHOUT
s p e c if ic it y
NUMBER
ONE
IS
S P E C IFIC ITY ,
TWO MUST
IN
THE
PLACE.
is
known
SPEC IFIC ,
CARBON ATOM
THE
IF
OF THE
HOWEVER,
about
HELFERIC H
METHYL-PHENYL-^-D-GLUCOSIDE
the
AND LANG
WAS NOT
HY D RO L Y Z E D
LEDGE
BEEN
SUBSTRATE
EXAMINED
S P EC IFIC ITY
AT
DEPENDING
AS A
POSITION
VARIABLE
PLANT
SYSTEMS T H E ^ S - G L U C O S I DASE
CONFIGURATION
ON THE
ABOUT
THIS
SOURCE
DOES
at
POSSESSES
CARBONS
ONE AND
TWO A L S O
carbon
IS
TO
RENDERS
IS
three
BY , S - G L U C O S I D A S E .
Ep I -
FOR THE
RATHER
EXHIBIT
CARBON AT OM,
the
3-
WHICH
AS
TO MY1 KNOW­
ENZYME.
DUBIOUS,
OF T HE J ? - G L U C O S I D A S E .
NOT
of
HAVE REPORT ED T H A T
T HE j S - A L L O S I D E ,
CARBON ATOM FOUR
TO BE
FOR T HE
ABOUT
BY THE j S - G L U C O S I DASE
AT
(71)
YIELD
T HE
GROUPS AT
s p e c if ic it y
CARBON T H R E E WOULD
NOT
CONFIGURATION
NUMBER TWO A L S O
HYDROLYSIS
HYDROXYL
coN?
apparently
AS j S - G L U C O S I DASES ARE
HYDROXYL
MER I Z A T I ON AT
HAS
THE
is
UNHYDROLYZABLE.
l it t l e
SUGAR M O I E T Y ,
T HAT
POSITION
ESTERIFICATION
GLYCOSIDE
Very
A T RA NS
IN
systems
MOLECULE.
E X T REMEL Y
ON O t - G L U C O S I D E S .
AN A B S O L U T E
TAKE
g lu c o s id a s e
SWGARUPORTI ON OF T H E
ACTION
BE
of
AN A B S O L U T E
AND APPEARS
I N MANY
PREFERENCE
BOTH ^ - G L U C O S I D E S
AND
-7 6 ^ - G A L A C T O S I DES ARE
H Y D RO L Y Z E D
ENZYMES W H I CH W I L L
NOT
SITUATION
DOUBTFUL.
DOES
NOT
SOMEWHAT
SEEM TO
HYDROLYSIS
Ca r b o n
DELBRUCK
OF A
SUL F U R
SIDES,
T
ARE
he
CHANGES
I
THESE
n
(L
NOT
NOT
IT
F ACT
i t
.
48)
T HAT
changes
DETERMINING
THE
SYSTEMS .
AS A
AN A B S O L U T E
T
he
FOR
(73),
ON THE
BUT
, S - G L U C O S I DASE
THUS
LEAVING
HYDROXYL
DOES A F F E C T
OF
THE
CARBON
T HE
RATE
FOUR
OF
show
no
absolute
- X Y L O S I DES ARE
s p e c if ic it y
HYDROLYZ ED
,
as
may
BY MOST
be
KNOWN
igman
IN
(L
i t
.
reported
that
THE A N O M E R I C P O S I T I O N ,
of
c o n fig u r a tio n
ARE
S P EC IFIC ITY
s u b s t it u t io n
I . E .,
THIOGLUCO-
THE
TO THE
GALACTOS I D E .
SUBSTRATEj
SP E C IFIC ITY
HOWEVER,
OIL
MENTIONED
IN
TH?
effect
SPEC IFIC ITY
CO NJ U GAT E
DECREASE
ONE WOULD
CHANGE AT
of
these
MATERIAL
DIFFICULTY
IN
OF THE
SECTION,
AS MOST
O F j G - D - M A N N O S E WAS
I N T HE
EXPECT
CARBON
PORTION
EXPERIMENTAL
THE Ct-G LU C OS lD E
TOWARD T H I S
CONFIGURATIONAL
the
X II.
GLYCONE
SAME A B S O L U T E
MUSTARD
as
IN TABLE
WAS D E S C R I B E D
HAVE T H E
well
OF T HE MUSTARD J 3 - T H I 0 G L U C 0 S I D A S E ,
WERE A P P L I E D
As
as
SUMMARIZED
AND T HE RE WAS A D E F I N I T E
PREVIOUSLY
60)
BY , S - G L U C O S I DASE S Y S T E M S .
DERIVATIVES.
X Y L O S I D E AND
TRIED
P
and
ON ENZYME A C T I V I T Y
HYDROLYZED,
OBTAINED
s ix
OXYGEN
ENZYME WAS FOUND TO
FOR T H E
NOT
and
HYD RO L YZ ED
v a r io u s
JS-GLUCOSIDASE
HOWEVER,
(74).
ATOM FOR
O IL
SUBSTITUTION
ENZYME A C T I V I T Y ,
SAME M O D I F I C A T I O N S
MUSTARD
T HE
f iv e
BY T HE
J 3 - G L U C 0 S I DASES
ARE,
58).
atoms
DEMONSTRATED
THERE
H Y D R O L Y Z E ^ - G A L A C T O S I DES
IN H IBIT
(llT .
(72).
HYDROLYSIS
OF THE
THE
BECAUSE
MUSTARD
ENZYME TO
OF T H E
OIL
T HE
WAS
EXHIB­
RE S U L T
NUMBER TWO.
ALKYLATIN G
RAT E
CARBONYL
Table
T
he
Ef f e c t
Gl y c o n e
of
Mo
Gl y c o n e
Va
X.1T
r ia t io n
on
A
RAPID
c t iv it y
IR e f e r e n c e
72
HYDROLYSIS
to
9
S
Cf-D-GLUCOSE
E P I M E R I Z A T I ON ON CARBON ONE
NO H Y D R O L Y S I S
72
(S - D - m a n n o s e
EP I MER I Z AT I ON ON CARBON TWO
NO H Y D R O L Y S I S
72
EP I MER I Z AT I ON ON CARBON T HREE
EFFECT
f3 - D - G A L A CT OS E^
NUMBER
ONE
EP I MER I Z AT I ON ON CARBON FOUR
NO H Y D R O L Y S I S
UNKNOWN
DECREASED' H Y D R O L Y S I S
72
RATE
H
/3-D -X Y L O S E
FOR
CHgOH
ON CARBON F I V E
Dec rease d
h ydr o lysis
73
RAT E
I
C T - L - A R A B I NOSE
H
FOR
CHgOH
ON CARBON F I V E
OF
jg-D-GALACTOSE
I
Gl y c o
s id e s
of
THESE
SUGARS ARE
DECREASED
HYDROLYSIS
RAT E
NOT
HYDROLYZ ED
BY A L L j S - G L U C O S I DASE
59
S YST EMS.
-7 7 -
-D-ALLOSE
ON CARBON
O
j g -D-G LU CO TH lO SE
FOR
O
- G l .u c o s i d a s e
Enzyme A
d i f i c a t i on
NONE
- D - glucose
l m o n d j3
-7 8 OF
oxygen
MUSTARD
HY D RO X A M I C
OIL
GLUCOSIDE
WOULD
BE
OF
WOULD
EXPECT
T HE
EXTREME
CU B A T E D W I T H
THIS
SOME
NOT
ENZ Y M E ,
GLYCONE
CONFIGURATION
l ic in
HYD RO L YZ ED
,
BUT
G L U C O S I DE WHEN
AS
H Y D RO L Y Z E D
TO T HE
DAS E •
T
HYDROLYSIS.
BY T HE
EXPERIMENTAL
E .G .,
AS TO THE
SUBSTRATES
HAVING
M A T E R I A L S WERE
AN A P P R E C I A B L E
50°
C.
I HESE M A T E R I A L S ,
( L IT .
56),
FOR
BRI NG
RESULTS
INDICATING
PHENYL-S^g-D-G LUCO SIDE
HYDROLYZED.
T HE
SAME
ITS
NO A B S O L U T E
LINKAGE.
CONDITIONS
THUS
IT
SP E C IFIC ITY
IF
IT
SIM ILAR
APPEARS
THAT
TO
THE
AS A G L U C O S I DASE
S P E C IFIC ITY
SUBSTRATES
THE
SAME
g l u c o s i de
were
Ph ENYL-JB-DDAYS AL S O
REPORTEDLY
ABOUT
FOR
WERE F E A S I B L E
TO
OIL
NO A C T I V I T Y
WAS FOUND T H A T
UNDER
OF
ARE
SPECULATION
BETWEEN A ^ - G L U C O S I DASE AND A ^ - T H I OGLUCOS I -
ZLING
ENZYME
AS
SEVERAL
W H I CH
MUSTARD
T HE
-^-D -
RATE.
OTHER T HAN T HE
IT
AND
HYDROLYZED.
p- n it r o p h e n y l
ENZYME AT
IN­
S P EC IFIC ITY
SYNTHETIC
and
ONE
PLACE.
SA LIC IN
SERVE
TH I O G L U C O S I D E S
UNTIL
RESULTS
TH I OGLUCOS I DASE WAS
GLUCOS I D E S ,
TH I OGLUCOS I DASE
DIFFERENCE
OBTAINED
OF THE
EXPERIMENT
GLUCOSIDES
p h lo r id z in
THE
THIS
NAT UR AL
NAT URAL
INCUBATED W IT H
EXACT
he
SEV E R A L
OXYGEN ANALOGUE
COMPOUND TO T A K E
CONFUSION
SEV E R A L
AS T HE
,
OF THE
OF T H I S
TH I OGLUCOS I DASE AT
SHOWED C O N S I D E R A B L E
NOT
DID
ALSO
VIEW
OCCURRI NG
CONSIDERABLE
a m y g d a lin
BY T HE
IN
THE
ENZ YME.
O B T A I N E D WHEN T HE
NATURALLY
FOR T HE
AND
FOR T H E
HYDROLYSIS
RESULTS
ONLY
CHECKING
SUBSTRATE
INTEREST,
I N T RO DUC E D
ENZYME.
Sa
AS A
CONSIDERABLE
SURPRISIN G
AMYGDAL I N ,
AC I DS P R E V E N T E D
COMPOUNDS WERE
ITS
FOR T HE
VARY T HE
EXTREMELY
WHEN
PUZ­
INCUBATED WITH
OXYGEN ANAL OGUE WAS AL S O
TH I O G L U C O S I D A S E
THE ATOM
ON S Y N T H E T I C
HAS
ESSENTIALLY
GLYCONE M O I E T Y ,
INVOLVED
IN THE
CONDITIONS
OF
BUT
EXHI B - ,
GLYCOSIDIC
HYDROLYSIS
FOR
—7 9 —
EACH
SUBSTRATE,
MATERIALS"
ONE MAY
AFFECTED
MYROSINASE,
AND AT
TYPE
A
THOROUGH
STITUTED
t
WAS
f
AROMATIC
CONE GROUP,
IT
RAT E
T
h is
SOLELY
EFFECTS
CAN
BE
AND
EXTREME
S T A B ILITY
SERVE S AS AN
TO
INDUCTIVE
HAS T H E
T HAT
DISTANCE
I MP OR T A N T
Perhaps
the
EFFECTS
GROUPS,
OF
OF T H E
MAY
OVER A W I D E
IDEAL
PH RANGE,
ENZYME
FOR T H I S
MUSTARD T H I O G L U C O S I D A S E
PARTICULARLY
PERH A P S
THOSE W I T H
R ESO L VE T H I S
IN
T HE
INCREASE
OF
that
the
EFFECTS
EFFECT
OF T HE
ORTHO,
T HE
SUBSTITUTED
PARA
RATE
AND META
OF
SUB­
LEAST
NATURE
ALL
AGLY-
POSITIONS,
PO SITIONS
OF T H E
OF THE
CORRESPOND­
GROUP
(75).
BE G RE A T E S T
IN
CONSEQUENCE WHEN
IN
POSI­
of
the
SINCE
GROUP
THE
su b s tit u t e d
RATE,
FROM THE
PARA
groups
C O NJ U GAT ED
SUBSTITUTION
HYDROLYSIS
SUBSTITUTED
IN
OF THE
T R A N S F E R R E D THROUGH THE
ON THE
G L U C O S I DE.
GROUP A P P E A R S TO
effects
MOREOVER,
A
ON AN A R O M A T I C
HYDROLYSIS
ON T HE
SUBSTITUTED
LINKAGE.
MOST
GROUPS
COURSE,
HAVE THE
SUBSTITUTIONS
ON
PHENOMENON.
UNSUBSTITUTED AROMATIC
VARIOUS
SHOWN T H A T
OF A
in d ic a t e s
GLYCOSIDIC
T HE
MAY
EFFECT
POSITION
A L S O AN
QUESTION.
T HAN WAS THE
DEPENDING,
ORTHO P O S I T I O N
TO T HE
"uNHYDROLYZABLE
P - N I T R O P H E N Y L - ^ - D - G L U C O S I DE WAS HY D RO L Y Z E D AT
THE
NUCLEUS,
T HE R E L A T I V E
NOT
ITS
OF T HE
AGLYCONE
FASTER
G L U C O S I DE
TIO N .
OF
IN
TEMPERATURES,
MONOSUBSTITUTIONS
AROMATIC
I NG
ENZYME
SO C A L L E D
AND T H l O G L U C O S t D E S ,
OBSERVED T H A T
WE C O N S I D E R
I . E .,
ST UDY
GLUCOSIDES
CONSIDERABLY
I
HIGH
MANY OF THE
STUDY.
SYNTHETIC
I
BY T HE
BECAUSE
RELATIVELY
OF
FIND
IT
IN
THE
APPEARS
GLYCOSIDIC
THE
are
SYSTEM
ORTHO
LIKELY
BOND
IS
those
in
FACTOR.
most
im p o r t a n t
of
these
group
effects
may
be
-
-8 0 VOLVED W I T H
POSSESSING
CAUSE
T HE
ADSORPTION
STRONG
IONIC
A DECREASE
FOR T HE
IN
THE
ENZYME A C T U A L L Y
OF T HE AGLYCONE
CHARACTER
OVERALL
F A V OR S
BIND
HYDROLYSIS,
ENZYME-SUBSTRATE
COMPLEX TO
ENZYME
ON T HE
APPEARS
I NG
SUBST RAT E
MUCH OF T HE
WHICH
EFFECT
MAY
DATA
OBTAINED
HAVE T H E
WEAKLY,
IF
AT
ENERGY,
AS
HEAT,
I NG AN
SUBSTRATE
EFFECTS,
i t
.
UNDER
INTO
61)
INTRODUCTION
' SHOULD
IS
SYSTEM
OF A
THE
BINDING
GLYCOSIDIC
FOUND T H A T
BOND.
DROLYSIS
T
he
OF T H I S
MATERIAL
present
concept
P - G L U C O S I DES
LINKAGE.
IF
NO A B S O L U T E
THEREIN.
THE
S
DOES NOT
IN
of
S P E C IFIC ITY
the
a
INVOLVE
WE ASSUME T H I S
in c e
VIEW
ENZYME WOULD A T T A C K
BINDING
T HE
is
ON T H E
FAVOR
T HE
THIS
TO
SUBSTRATE
FROM T H E
IN
DETECTING
ENZYME
VERY
ADDITIONAL
OF FORM­
OF T H I S
OF T H I S
INDUCTIVE
HI S F ELL OW WORKERS
CONDITIONS'
HYDROLYZE
CONSIDERABLE
HY­
SITUATIO NS.
enzym atic
one
INTERPRET­
POSITION
hydr o lysis
ATOM
OF T H E
BE R E A S O N A B L E ,
apparently
IN
PROBABILITY
EXPECT
LINKAGE
OF THE
REACTION.
AND THROUGH
THROUGH THE
DISSOCIATION
PUTTIN G
PARA
DISCUSSED
for
AND THUS
EFFECT
HYDROLYSIS
NORMAL
ONE WOULD
M E C H A N I SM
m ec h an ism
UNDER
m ec han ism
INVOLVING
ON A G I V E N
T E NDE N CY
OF T H E
LIES
CONDITIONS.
IN
T HE
DIFFICULTY
OF WORK
GROUPS
I NCR EASED A F F I N I T Y
T HAT
GOODMAN AND
M Y R O S I N A S E WOULD
2 , 4 - D I N ITROPHENYL-SyB-D-GLUCOS ID E .
EX T E NT
ENHANCE THE
GROUP
ENZYME
DECREASES T H E
THE
AND T H E R E B Y
SURFACE.
T HE
THIS
ADSORBED
SHOULD
NITRO
WITH
BUT
INFLUENCE
REACTION
ENZYME
RATE.
TYPE
PROBABLY
COMPLEX
INCREASE
WEAKEN T HE
FROM T H I S
NORMAL
THE
ENZYME-SUBSTRATE
MATERIAL.
(L
ALL,
SUCH AN
LESSENED.
G RE A T E S T
PHENYL-S^B-D-GLUCOSIDE
TIGHTLY
HYDROLYSIS
OF T H E
ON THE
OR THE
THERE
ATOM
OF
DOUBLE
BACK
SIDE,
of
GLUCOSIDIC
SHOULD BE
CONTAINED
DISPLACEMENT,
I . E .,
THE
SIDE
- 8 1—
OPPOSITE
THE
PARTICULAR
STRAINING
Un
LITTLE
T HE
GLUCOSIDlC
EMPHASIS
OF T HE
t il
a
CAN BE
SAID
HYDROLYSIS
SET
T HE
REV E R S E
ALTHOUGH
CANNOT
BE
BEEN
ENCOU NT: ERE d ( 7 6 ) .
ATIC
HYDROLYSIS
Successful
Ev id e n c e
IS
A
VERY
OTHER WORKERS
/ 3 - G L U C O S I DASE
been
IN
as
THE
OF
CO RR E L A T E W I T H
i t
.
T HAT
61).
have
WE CANNOT
OTHER
is
THAN
PLA CE
PERHAPS
out,
c a r r ie d
NATURE ARE
T
THE
THE
CERTAIN
hus,
THIS
APPEAR
TIM E,
BY
AS MANY
HYDROLYZ ED
BY
in
does
t h is
FEW
EXPECT
THIS
EXCEPTIONS
EXHIBIT
LATTER
also
SULFUR
BEEN
HYDROLYZ ED
an
OF
AND
CHECKED FOR
AFFECTED.
IT
.
by
IN H IBITIO N
HYDROLYSIS.
e x e m p l if ie d
ENZYM­
h y d r o ly s is
but
G L U C O S I DES HAVE
OF A C I D
a g ain
,
BE
SURFACE.
guarantee
LITTLE
AND
OF T HE
ENZYME
laboratory
CANNOT
HAVE AL R E A D Y
I N S T A N C E S WHERE T H E
COMPOUNDS ARE
is
AC I D
ON THE
TH I OGLYCOS I DES ARE
there
ONE M I G H T
ENZ Y M E S .
not
LITTLE
ENZYM­
ENERGY
,
OF
BETWEEN AC I D AND
ACTIVATION
THIOGLUCOSID ES
RESULTS
HAVE A
BETWEEN A C I D
seen
THE
TO
HYDROLYSIS
DIFFERENCE
IDENTICAL
THIO
T HE A T T E M P T E D
SUBSTRATE
only
IN
OTHERWISE
COMPOUNDS
(L
IN THE
THAT
(77).
HYDROLYSIS,
s I
ATOM
effects
THIS
RESULTS
HOWEVER,
not
FIELD
ENZYMATIC
HOWEVER,
these
AFFECTED
OF THE
we
o b t a in e d
SYST EMS
SHOWN,
of
CORRELATION
ESSENTIAL
BY A D S O R P T I O N
OXYGEN ANAL O GU ES
base
LITTLE
RULE,
THE
,
T HE
A DIRECT
DECREASE
ad s o r p tio n
has
SITUATION,
LINKAGE
SITUATION,
BE ASSUMED AT
DOWN AS AN A B S O L U T E
ABOUT
THE
SYSTEM .
COMPOUNDS TO
BROUGHT
OF T HE
in v e s t ig a t io n
B Y j 3 -GLUCOSIDASE,
MORE F O U N D A T I O N .
STABLE
VOLUME
BEING
AS TO WHY G L U C O S I DES OF
CONSIDERING
n
THIS
BO ND.
s y s te m a t ic
T H I OGLUCOSIDES
A TIC
ON T HE
TH I O G L U C O S I DASE
I
BOND.
THESE
HAS BEEN
BY j S - G L U C O -
apparent
over-
”82—
LAPPING
T
hat
BE
OF T HE
the
mustard
EXPLAINED
l in k a g e
.
GREATLY
ENZYME
o il
BY T HE
He l f e r
SYST EMS
g lu c o s id e s
EF F E C T
ic h
DECREASE T HE
T
MUSTARD
h is
AND
does
not
INDEED
im p ly
IS
STILL
of
PRESENTLY
T HE
ENZ YMES.
BE
that
OTHER
groups
of
E S P E C I A L L Y WHEN
GROUPS ARE
BE
g l u c o s id ase
not
THlO GLYCOSIDES
IN
type
SEPARATION
ONLY
adsorbed
may
GLYCOSIDIC
t h is
OBSERVED
SE P A R A T E D
are
GROUPING.
CLOSE TO T H E
I T MAY
compounds
BE
ST ATED T HAT
THE
THERE
T
hat
T HESE
IMAGINE,
THE
(79)
THE
is
OUR PRESENT
APPARENT
on
T HAT
FOR
BY TWO A T OM S .
enzyme,
the
THEIR
KNOWLEDGE
CROSSING-OVER
OF
BUT
CERTAINLY
UNANSWERED
U N DO UB T E DL Y
DIFFERENCES
P O S S IB ILITY
c o n s id e r a b ly
RESULTS
FUTURE
PREDIC TED,
ARE
HOWEVER,
LIKELIHOOD
ENZYMES W I L L
REMAIN
MAY
WORK ON G L Y C O S I D A S E S .
S IM P LIFICATIO N ,
NOT
IT
RESULTS.
We i d e n h a g e n
ICATE
ENZYMES,
RES E M B L E
INCOMPLETE.
TWO E N Z Y M E S ;
RECENT
found
-
ACTION
more
ALTHOUGH
OBTAINED
LIM ITED
A CAREFULLY
ANSWER
im p r e s s iv e
MANY
THEORY
THIS
DIFFERENCES
EXPERIMENTATION
BE L E S S
MAY
EXECU TED
OF T H E
in
IS
EXISTING
STUDY
THE
of
THEORY
the
PERH APS AN
BETWEEN THE
IN
PRONOUNCED THAN
v ie w
LAB ORATORY
R ESO L VE T H I S
ENZYMATIC
L E A V E S MANY
OF A P P R O X I M A T I N G
THIS
IN
DIFFERENCES
MAY
OF T HESE
OF THE
OF THE j3 - G L U C O S I DASE AND T H E p - T H I OGLUCOSI DASE
UNEXPLAINABLE
WE CAN
GROUP
EXT REME.
these
b y j3
hydrolyzed
ACIDIC
T HESE
OF THE AGLYCONE
G L U C O S I DASE.
A C T IV IT IE S
THE
CLOSELY
CONCLUSION
SYST EMS
that
not
(78)
OF
I S NOT
G L U C O S I DES,
THEY
TOWARD T HE
IN
O IL
OF T HE
A C T IV IT Y
EFFECTS
were
S chnorr
and
FROM T HE G L U C O S I DE BOND
THE
FROM
OVER­
CERTAINLY
IND­
GLYCOLYTIC
RELATIONSHIP
OF T H E S E
QUESTIONS WHICH
CAN­
COMPOUNDS AND
TODAY MUST
-
83-
Pa r t
111
Summary
1.
Sy n
t h e t ic
mustard
Of-ACETOHALOGLYCOSE W IT H
ACID
AND
SULFONATION
GALACTOSE
IVE
AND
SYNTHETIC
TO T H I S
THE
T
he
MUSTARD
OIL
FRACTIONS,
THESE
RESULTANT
TH I O G L Y C O S I D E .
NATURALLY
SYNTHETIC
MUSTARD
DERIVATIVE
r e a c t in g
GLUCOSE,
GLYCONE M O I E T I E S
OCCURRI NG
by
an
P H E N Y L A CET OTH I OHYDROXAM I C
COMPOUNDS WERE
GLUCOSE
MUSTARD
T HE
G L U C O S I DES
OF T HE
CHARACTERIZED
G L U C O S I DES,
MANNOSE,
RESPECT­
BY
COMPARI­
GLUCOTRO-
WAS FOUND TO BE
T
he
MANY
FOUND
IN
RESPONSIBLE
SPEC I ES
FRACTIONATION,
CHROMATOGRAPHY
IDENTICAL
F OLLOWED
p u r if ic a t io n
SULFATASE
F ACT ORS
VERONAL
IN
c m v o l t
- 1
sec
THE
SYST EM.
CLEAVAGE
OF THE
RELATED
BY AMMONI UM
PLANTS,
SULFATE
ON N , N - D l E T H Y L A M I N O E T H Y L C E L L U L O S E .
t h is
POSSESSING
DETERMINING
OF CROC I F ERAE AND
results
ENZYME
IN
FOR THE
CHECKED
of
USED
THE MYROSINASE
ENZYME WAS A L S O
A GLYCOSIDIC
2.588 x 10*5
G L Y C O S I D E S WERE
ENZYME
IN
BY AL C OH OL
AND
O IL
THIOGLUCOSIDASE
MYROSINASE,
TROPHORESIS.
OF
OF T HE
THESE
FRACTIONATED
FACTOR
SALT
MATERIALS.
OF T HE
FRACTIONATION
A
POTASSIUM
THE
OF
prepared
T HE
USED AS
SYNTHETIC
WAS P U R I F I E D
SALT
were
GLUCOSIDE.
SP E C IFIC ITY
2.
g ly c o s id e s
X Y L O S E WERE
SON TO ONE OF T HE
PAEOL I N .
o il
FOR
PURITY
in d ic a t e d
I N D E N T I F I ED AS THE
A C T IV IT Y .
THE
two
PH 8.5,
.
-.86 3 x 10"^
AT
ELEC­
a c tiv e
THIOGLUCOSID ASE
ELECTROPHORETIC
BUFFER,
and
BY BOUNDARY
0.1
IONIC
CM.^
T HE
AMD
M O BILITIES
STRENGTH WERE
volt
~^
sec
RESPE CT I V E L Y .
3.
T
he
two
components
found
in
the
m yr o sin ase
system
were
in c u bated
—84"*
SEPARATELY W IT H
S IN IG R IN
FRACTION,
HOWEVER,
LIBERATED
BY
SULFATASE
FACTOR.
SI N I G R I N ,
CONSIDERABLE
WHILE
No
At
pH
WHEN T H E
3.0,
SIDASE
the
AC TIV ITY
5.
T HE
OR S U L F A T E
WAS
6.
AC TIV ITY
T
he
FRACTIONS
WERE
HYDROLYSIS
AS
factor
incubated
in
30$
RELEASED
41$,
G L UC OSE ,
REACTION.
ITS
no
a c t iv it y
ORIGINAL
buffer
WHEREAS T HE
,
AC TIV ITY .
PH 3 . 0 .
SYSTEM WAS RUN AT
phosphate
BY THE
INCUBATED WITH
e s s e n tia lly
OF
EACH
GLUCOSE WAS
YIELD IN G
OF T HE
shows
ABOUT
ABOUT
p u r if ie d
SHOWED NO H Y D R O L Y S I S
E ST ERS
SULFONATED
OF T H I S
the
th io g lu c o
SULFATASE
-
AC TIVITY
OF T HE
THESE
ETHEREAL
CONTAINED
O X I M E S WERE FOUND TO
t h i oglucosi dase
THAN T H A T
TYPE
OF
IN
SULFATES
THE MUSTARD
EXHIBIT
HYD RO L ­
FACTOR.
INVOLVED
IN
A C T IV IT IE S
w a ,s
THE
WERE
completely
HYDROLYSIS
free
of
g ly c o s id ic
OFjg-GLUCOSIDES
SHOWN TO BE A T T R I B U T E D
AND
TO THE
ENZ Y M E .
T HE MUSTARD T H I O G L U C O S I DASE
S P E C IFIC ITY
T HE
OCCURRED,
PRODUCTS
M AINTAINS
FACTOR
SEVERAL
j 3 - T H I OGLUCOSIDES.
SAME
C O M B I NE D
OTHER T HAN
PRESE NCE
OTHER
I . E .,
SULFATE
IN H IB ITE D ,
SULFATASE
GLUCOSI D E S •
A C TIV ITY ,
INORGANIC
sulfatase
was
WAS O B S E R V E D .
TH EORETICAL. MAXIMUM.
ESTERS,
IN THE
RESPECTIVE
WAS D E T E C T E D WHEN T H E
system
PROCEEDED TO THE
YSIS
the
HYDROLYSIS
AND
TOTAL
T HE T H I O G L U C O S I D A S E
Wh e n
ITS
ISOTHIOCYANATE
ISOTHIOCYANATE
O IL
SHOW
NO T O T A L
THE T H I O G L U C O S I D A S E
K H S O ^ AND A L L Y L
4.
DID
AND
AS ALMOND
CONFIGURATION
GLYcoNE
m o ie t y
.
ESSENTIALLY
EMULS I N J 3 - G L U C OS I D A SE .
OF T H E
Wh e n
POSSE S S E S
the
HYDROXYL
enzyme
GROUPS A T
was
THIS
CARBONS
in c u b a t e d
w ith
T HE
SAME A B S O L U T E
SPE C IFIC ITY
I NVOLVES
ONE AND TWO OF THE
the
s y n th e tic
mustard
-8 5 o il
GLYCOSIDES,
SIDE,
X Y L O S I DE AND
T
he
HOWEVER,
em u lsin
THESE
ENZ Y M E ,
AS
7.
T
T HE ATOM
he
BY T H I S
DERIVATIVE
GALACTOSIDE
enzyme
d id
MATERIALS
ILLUSTRATED
DID
ENZYME.
^ - D - G L U C O S I DE,
AS
POSSESS
not
GLYCOSIDIC
the
SYSTEMS WERE
VERY
show
INCUBATED
AT
an
C.
o il
THE
GLUCO
g l y c o s id e s
AFFINITY
absolute
;
FOR THE
s p e c if ic it y
AMYGDALIN ,
GLUCOSIDES,
HYD RO L YZ ED
50°
BUT
OF T H E - G L U C O S I DASE A C T I V I T Y
LINKAGE.
SYNTHETIC
SLOWLY
mustard
CONSIDERABLE
P H E N Y L - S - J 3 - D - G L U C 0 S I DE AND
WERE
HYDROLYZED,
HYDROLYZED.
IN H IBITIO N
d id
SEVERAL
WAS NOT
hydrolyze
BY T H E I R
I N T HE
AS WE L L
WERE
not
th io g lu c o s id a s e
INVOLVED
P H L O R I D Z I N,
T HE
T H E MANNOSE
ITS
WERE
SALIC IN
for
AND
HYDROLYZED
OXYGEN A N A L O G U E ,
PHENYL-
BY THE TH I O G L U C O S I DASE WHEN
- 8 6 -
IV
Pa r t
L
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