Kurze Originalmitteilungen
102
as a source of Co Q in view of t h e r e c e n t al:tempts to p r o d u c e
Co Q b y bacterial f e r m e n t a t i o n of c e r t a i n s y n t h e t i c mediaS).
T h e detailed p a p e r o n t h e s e a n d related a s p e c t s wilt be p u b l i s h e d elsewhere,
W e wish to t h a n k Dr. T. RAMASARMA a n d Dr. S.C. PILLAI
for t h e i r i n t e r e s t a n d advice.
Department o~ Biochemistry, Indian Institute of Science,
BangMore 12, India
C. V, VISWANATHAN, J. JAYARAMAN, (Miss) ]3. MEERA BAI
a n d P . V . R . SUBRAHMANYAM
Eingegangen am 3t. Oktober 1960
1) GREEN, D.E., and R.L. LESTER: Fed. Proc. 18, 987 (t959). - z) MORTON, R.A.: Nature [London] 182, ~764 ( 1 9 5 8 ) . - a) LESTER,
R.L., and F.L. CRANE: J. Biol. Chem. 234 (8), 2t69 ( 1 9 5 9 ) . *) KoNI~SZY, F.R., P.M. GALE, A.C. PAGE Jr. and K. FOLKERS:
Arch. Bioehem. Biophys. 87, 298 (1960). - - ~) OLsot% R.E., and
G.H. DIALAr*rEH:Bioehem. Biophys. Res. Comm. 2 (2), , 98 (~960}.-~)VlswANA~HAN, C.V., and S.C. PILLAI: Indian Inst. of Sci.
Golden Jubilee Res. Volume, 119 ( 1 9 5 9 ) . - ~) VlSWANAT~AN, C.V.,
and S.C. PILLAI: Naturwissensehaften 46, 324 ( 1 9 5 9 ) . - s)PAGE Jr.,
A.C., P.M. GALE, M. WALLICH, R.B. WALTON, L.E. MCDANIEL,
~u
WoODRUFF and K. FOLKERS: Arch. Bioehem. Biophys. 89,
318 (t960).
Coenzyme Q (Ubiqutnone) in Avian Egg and Embryo
C o n s i s t e n t w i t h t h e view of WARBuRG 1) t h a t a n e m b r y o is
a n aerobic o r g a n i s m several o x i d a t i v e enzymes~),~), ~) a n d
r e s p i r a t o r y pigmentsS) h a v e been s h o w n to be p r e s e n t a n d to
increase in q u a n t i t y d u r i n g d e v e l o p m e n t in chick e m b r y o .
F u r t h e r , p a r t i c u l a t e m a t e r i a l isolated f r o m chick e m b r y o n i c
liver h o m o g e n a t e s h a s b e e n s h o w n to c o n t a i n c o e n z y m e Q
(Co Q) a n d evidence h a s b e e n d e d u c e d for its p a r t i c i p a t i o n
in electron t r a n s p o r t activities similar to its role in a n i m a l
t i s s u e m i t o c h o n d r i a 5 ) . T h e chick e m b r y o inside t h e shell,
b e i n g a " c l o s e d u n i t " a n d free f r o m microbial c o n t a m i n a t i o n ,
s h o u l d h a v e alI its s u p p l y of Co Q d e p o s i t e d in t h e egg b y t h e
h e n or s h o u l d s y n t h e s i z e it d u r i n g d e v e l o p m e n t , if a b s e n t in
t h e egg. T h e d a t a p r e s e n t e d here s h o w t h a t Co Q is p r e s e n t
in eggs a n d it is p r o g r e s s i v e l y i n c o r p o r a t e d into t h e e m b r y o
during development,
I n eggs, Co Q w a s f o u n d entirely in yolks a n d t h e a m o u n t s
p r e s e n t in t h r e e b a t c h e s are g i v e n in t h e Table. T h e m e a n v a l u e s
Table. Coenzyme O content o] Hens' Eggs and Chick Embryos.
Wet weight (in gin.) and Co Q (whole)
Yolks from fresh eggs*)
Batch 1: wet weight .
18"0
16"5
t7.0 18.5 117-5 18.0
Co Q . . . .
0"13t
0',62
0.,20 0',801 0.172 0.,66
Batch 2: wet weight . 14'0
~5'0
Co Q . . . .
0"138
0"~43
0",,91 0"163 0'133
Batch 3: wet weight . 17-0
,6'0 16.0
'6'5
Co Q . . . .
0"088
0"088 0"I04
0"087
1,55 '20.01
Embryos
!6--7th 1 3 - - , 4 t h l , 5 t h J 1 6 t h 17-,8thday
wet w e i g h t . .
0-66
,0.00
,2-50 a6-00 21.00
Co Q . . . .
0'064
0"124
0'139 0"i64 i 0',52
*) Because of the high lipid content of yolks, a high concentration of alkali (0'5 gm. NaOH per gm. wet weight) was used and the
saponification time was reduced to 20 min. to minimize destruction
of Co Q.
of Co Q c o n t e n t in t h e t h r e e b a t c h e s are 0"155 (-4-0.024),
0'147 (~:0.038) a n d 0.092 (j=0.008) ~xmole p e r egg yolk. T h e
v a r i a t i o n in b a t c h 3 s u g g e s t s t h a t Co Q c o n t e n t of eggs m i g h t
d e p e n d on t h e h e n ' s feed as in t h e case of v i t a m i n s A a n d E~).
P r o g r e s s i v e l y i n c r e a s i n g a m o u n t s of Co Q were f o u n d in t h e
d e v e i o p i n g e m b r y o a n d b y a b o u t t h e ~ 5 t h d a y of d e v e l o p m e n t
negligible a m o u n t s of Co Q were left in t h e r e s i d u a l m a t e r i a l
in t h e egg.
T h e p r o c e d u r e u s e d in t h e isolation of Co Q f r o m eggs a n d
e m b r y o s of W h i t e L e g h o r n v a r i e t y h e n s c o n s i s t e d of saponific a t i o n in presence of pyrogallol, e x t r a c t i o n of t h e u n s a p o n i fiable m a t t e r w i t h h e x a n e , r e m o v a l of c a r o t e n o i d s b y w a s h i n g
h e x a n e l a y e r w i t h 80% ethanol, freezing o u t m o s t of sterols
f r o m p e t r o l e u m e t h e r (40 to 60 ~ a t 0% a n d f r a c t i o n a t i o n o n
silicic acid c o l u m n . T h e 50% CHC18 in p e t r o l e u m e t h e r fract i o n showed s m a l l b u t definite p e a k s a t 275 mix a n d on reduction w i t h N a B H 4 m a x i m u m decrease in a b s o r p t i o n w a s f o u n d
a t 275 mix, i n d i c a t i n g t h e p r e s e n c e of Co Q, f r o m w h i c h t h e
Die Naturwissensehaffen
a m o u n t of Co Q w a s calculated ~). T h e r e d u c e d spectra, however, s h o w e d a b s o r p t i o n p e a k s a t 280 m~z for egg f r a c t i o n s a n d
272 a n d 280 m~z for e m b r y o ~ractions. I n all cases t h e p r e s e n c e
of Co Q w a s c o n f i r m e d b y reverse p h a s e p a p e r c h r o m a t o g r a p h y s) a n d Q10 w a s t h e o n l y f o r m found. H o w e v e r , egg y o l k
unsaponifiable matter contained another compound having
R! 0'90 in 80% n - p r o p a n o l s y s t e m , giving positive t e s t w i t h
leucomethylene blue s p r a y a n d s h o w i n g a n a b s o r p t i o n p e a k
at 265 mtz w h i c h w a s n o t r e d u c e d w i t h N a B H 4 . [The leueom e t h y l e n e b l u e s p r a y d e v e l o p e d b y FOLKER'S g r o u p 4) for
d e t e c t i o n of q u i n o n e s was, however," f o u n d to be n o t specific
for q u i n o n e s since s o m e ring ketones, p a r t i c u l a r l y u n s a t u r a t e d
ones, also g a v e a p o s i t i v e test]. F u r t h e r , infrared d a t a (peak
a t 5"82 Ix) s u g g e s t e d t h a t t h i s c o m p o u n d p o s s i b l y is a s a t u r a t e d
ring k e t o n e or a m-diketone, b u t n o t a q u i n o n e .
T h e u n s a p o n i f i a b l e m a t t e r of egg y o l k a n d chick e m b r y o
also c o n t a i n e d r e d u c i n g c o m p o u n d s (~,~'-dipyridyl-FeC1 a
positive) h a v i n g R / 0 - 9 0 in 80% n - p r o p a n o t s y s t e m w h i c h are
different f r o m u b i c h r o m e n o l (R/, 0.42) a n d ~-toeopherol
(R!, 0'85) b u t a p p e a r to be s i m i l a r to t h e c o m p o u n d described
b y MAHLERh). A p r e p a r a t i o n of t h i s c o m p o u n d f r o m egg yolk
s h o w e d a b s o r p t i o n p e a k s a t 272 a n d 3 t 0 mix.
F u r t h e r w o r k following t h e c h a n g e s in Co Q, v i t a m i n E
and this new non-vitamin E reducing compound during emb r y o n i c d e v e l o p m e n t is in progress.
W e are g r a t e f u l to Drs. D . K . BAN~RJEE a n d B . H . IVGR
of t h e O r g a n i c C h e m i s t r y D e p a r t m e n t for t h e infrared analysis.
Department o] Biochemistry, Indian Institute of Science,
Bangalore 12, India
T. RAMASARMA*), J. JAYARAMAN a n d P.S. SARMA
~ingegangen am t5. November t960
*) C. S. I. R. Scientists' Pool Officer.
t) WARBURG O.: Biochem. Biophys. Acta 25, 429 (,957). - ~) ALBAU~, H. G., A.B. NOVIKOFF and M. OOUR: J. Biol. Chem.
165, ,25 (1946). - - ~) SCELVOLA,M.E., and A.D. DE BARBIERI: Bull.
Soe. Chim. biol. 39, 1305 ( 1 9 5 7 ) . - ~) BRAND, L., and H. R. MAHLER:
J. Biol. Chem. 234, t615 (1959). - - 5)BRANO, L., C. DA~L and
K. R. MAHLER: J. Biol. Chem. 23~, 2456 ( 1 9 6 0 ) . - 6) DEnL jr. H. J. :
The Lipids, vol. II, pp. 327, 503. London: Interscience Publishers
1955. - - 7)LINN, B.O., A.C. PaGE jr., E.L. WONG, P.R. GALE,
C.H. SHUNK and K. FOLKERS: J. Amer. Chem. Soc. 81, 4007 (1959).-a) LESTER, R.L., and T. RA~ASAR~*: J. Biol. Chem. 234, 672 ('959).
The Localization of Copper in Agar Gel
Electrophoretie Patterns of Crustacean Blood
R e c e n t l y ~vVHITTACK~R1) WaS able to d e m o n s t r a t e t h a t t h e
t w o p r o t e i n b a n d s , o b t a i n e d b y s t a r c h gel electrophoresis a f t e r
SMI~mXs 2) f r o m blood of two O r c o n e c t e s species, b o t h c o n t a i n
copper. T h e r e f o r e h e s e c t i o n e d t h e gels h o r i z o n t a l l y a n d while
one half w a s s t a i n e d for p r o t e i n w i t h a m i d o b l a c k 1 0 B dye,
t h e o t h e r half w a s placed for 24 h o u r s in a s o l u t i o n of 50 m l
of t 0 % a q u e o u s s o d i u m a c e t a t e a n d 3 m l of alcoholic 0 . t %
r u b e a n i c acid. I n t h e s e c i r c u m s t a n c e s t h e t w o p r o t e i n b a n d s
of Orconectes blood s t a i n e d a l i g h t greenish-black.
W i t h t h i s m e t h o d however, we were u n a b l e to d e m o n s t r a t e
copper in e l e c t r o p h e r o g r a m s of c r u s t a c e a n blood o b t a i n e d b y
micro a g a r gel electrophoresis after WIEM~a). Moreover t h e
t i m e r e q u i r e d in t h e m e t h o d of WHITTACKGR is tOO long bec a u s e diffusion p h e n o m e n a are v e r y i m p o r t a n t in a g a r gels
a n d b r i n g a b o u t a b r o a d e n i n g of t h e p r o t e i n b a n d s , p r e v e n t i n g
a s h a r p s e p a r a t i o n . T h e r e f o r e t h e p r o t e i n m i x t u r e is applied
as a n a r r o w b a n d (4 m m long a n d 1/4 m m wide) a n d electrop h o r e s i s is c o m p l e t e d in a v e r y s h o r t t i m e , t h e p r o t e i n f r a c t i o n s
b e i n g fixed i m m e d i a t e l y a f t e r w a r d s .
I n order to o v e r c o m e all t h e s e difficulties we e s t a b l i s h e d a
m o r e sensitive m e t h o d for copper 19calization b y c o m p I e x a t i o n
w i t h r u b e a n i c acid. T h e b e s t r e s u l t s were o b t a i n e d b y inc u b a t i n g t h e gels, a f t e r electrophoresis, in a m i x t u r e of: glacial acetic acid 5 mI; alcohol 94% 7 0 m l ; alcoholic 0-2%
r u b e a n i c acid 25 m h
I n t h e s e c i r c u m s t a n c e s t h e p r o t e i n s are f i x e d b y t h e
acetic acid-Mcohol m i x t u r e a n d t h e Cu2+-ions set free in t h i s
m e d i u m are i m m e d i a t e l y p r e c i p i t a t e d in situ b y t h e r u b e a n i c
acid. A g r e e n i s h colour develops w i t h i n 20 m i n u t e s a n d after 1
h o u r t h e p o s i t i v e f r a c t i o n t u r n s d a r k green. Copper e s t i m a t i o n
s h o w e d t h a t t h e m i n i m u m a m o u n t of copper, d e m o n s t r a b l e
w i t h o u r m e t h o d is 0 . t 3 2 txg Cu 2+, p u t into t h e n a r r o w slit on
t h e a p p l i c a t i o n line.
I n o u r e x p e r i m e n t s e l e c t r o p h o r e s i s w a s carried o u t in
0"9 p e r c e n t special agar-Noble(Difco)gels of p H = 8-4. T h e
t e n s i o n b e t w e e n t h e electrodes is k e p t a t 130 volts, t h e c u r r e n t