244 SNORT CO,~tMU~ICATXONS VOL.

34 (1959)

Fig. I is a r a d i o a u t o g r a p h of the ionogram. The free p h o s p h a t e b a n d has been

allowed to run off the anode end of the paper. The e x a c t correspondence of the radio-
active b a n d s from the three di-isopropyl p h o s p h o r y l - e n z y m e s indicates t h a t t h e
h y d r o l y s a t e s contain identical peptides of phosphoserine which are entirely different
from those d e r i v e d from ovalbumikx. The s t r u c t u r e of the different peptid~" b a n d s is
n o t certain at present b u t is u n d e r investigation, F r o m the n u m b e r present arid their
i d e n t i t y in the three e n z y m e s it is p r o b a b l y safe to conchtde t h a t elastase, like trypsitl
a n d c h y m o t r y p s i n contains the sequence G l y . A s p - S e r - ( ; l y - a r o u n d its r e a c t i v e
serinc residue.
B . S . HARTLEX"
Dvpartmcnt o/Biochemistry, ~.Miversity o/Cambridge M . A . NAL'¢;HTON
(Great Britah~) F. SA.~G~R"

a G. H . 13Ixo,~, D. L. KAUFr'.~1A,~ X.~D H . NEtJ~ATr~, J , .4m. Ch~,m. See,, 8 0 ( t 9 5 8 ) t26o.

e ] ; TURBA A~'D G. GUN1DLACH, l~iochem. Z., 327 ( I 9 5 5 ) i 8 0 .
a X . 1{. ~CHAFWER, L. SI.~IEL, G. |IARS~tM,%N. R . R . ]~NGLF, ,AND R. XV. I)RISKO, J . l~iol. Chem.,
225 (Iq57) ~97-
4 p . E . ](0SHLAh'D A,'iD 3I. J, ERXVIN, J..,J1i~, Chem. See., 79 (1957) 2057.
5 ]{, A. {)DSTERBAHN, H . S. JANSZ ASD J . A. COltEN, BiochDtl. Bie~phys. ,.Jcta, 20 (I930} 4o2.
6 j . ~.. GLAD,~I~R AND K , L.al~l, j r : I r a . Chem. See,, 80 (x958) 1204.
7 M . A. NAUGHTON AND F. SANGER, Diocllem. J . , 7 ° (1958) 4 P.
s U. J . Ll~xvis, D . E. *X*~:ILLIAXlSAND N. G . BRINK, J , Biol. Chron.. 222 (I(95(J) 705.
H . M z e n L , a lom, tsh. Chem., 8 : ( I 9 5 1 ! 489.
R e c e i v e d N o v e m b e r 27th, I958
* Member of the external staff of the Medical Itesearch Council.

A simplified spectrophotometric determination

of ester groups in lipids
Our l a b o r a t o r y ' s need for an e x t r e m e l y simple y e t highly sensitive a n d a c c u r a t e a s s a y
of lipid ester groupings in large n u m b e r s of ehromatogi-oDhic samples p r o m p t e d t h e
d e v e l o p m e n t of the m e t h o d r e p o r t e d here. It is b a s e d on a h y d r o x y l a m i n o l y s i s in
which an ester reacts with alkaline h y d r o x y l a m i n e to form a h y d r o x a m i e acid; t h e
latter forms a purple iron-chelate complex in the presence of acid ferric p e r c h l o r a t e .
This m e t h o d is a modification of more lahorlous procedures i - a a n d its range e x t e n d s
to 4.oo Fequiv. ester.
A s t a n d a r d c u r v e was d e t e r m i n e d on samples of tristearin, tripalmitin, m e t h y l
stearate, or m e t h y l p a l m i t a t e (California F o u n d a t i o n for Biochemical Research). The
weighed lipid was dissolved in Fisher reagent-grade chloroform. A l i q u o t s of t h e
s t a n d a r d s were p i p e t t e d into test t u b e s (85 >~ 15 ram), a n d the solvent r e m o v e d under
infrared lamps. A c e t o n e (about 0,5 ml) w a s r o u t i n e l y a d d e d to all samples at this
p o i n t to insure complete removM of the chloroform. The acetone was agai:x e v a p o r a t e d
u n d e r infrared lamps.
Reagents: Stock Ferric Pemldovate t. 5 g ferrid perchlorate (nonyellow) were dis-
solved ip zo ml 7o % HCtO4 a n d re ml water, then d i l u t e d to i o o ml with cold a b s o l u t e
e t h a n o I (stored in refrigerator); l~eagent Ferric Perchlorate. 4 ml s t o c k ferric per-
chlorz, te and 3 ml 7 ° % HC10 t were d i l u t e d to IOO ml with cold a b s o l u t e e t h a n o l ;
Alkali~e Hydroxylamine. E q u a l vols. of a 4 o~ ethanolic h y d r o x y l a m i n e solution (2.o g
v o L 34, (~959) " SHOR'r CO.M,~IUNICATIONS 245

d i s s o l v e d in 2.5 ml HeO, d i l u t e d to 50 ml w i t h a b s o l u t e ethanol) a n d of a n 8 %

e t h an o lic N a O H (,~ g dissoh, ed in z.5 mt I.,"~,J,°"¢mt, t c ......
u ' " ~ "~Lu.5o ml ~,,.,'""",.,,.,.,.~."~
..... ~. . . . . .,~d.,.,.,"
.......
were m i x e d in a s t o p p e r e d cylinder. T h e NaCI was s e p a r a t e d b y c e n t r i f u g a t i o n a n d
t h e s u p e r n a t a n t was d e c a n t e d for use.
Proaedure: (I) x ml alkaline h y d r o x y l a m i n e r e a g e n t (fresh daily) was a d d e d to
e a c h of th e d r y lipid samples. (2) T h e samples were placed in a w a t e r b a t h at 65 "~ for
2 rain. (3) T h e s am pl e s were r e m o v e d from the w a t e r b a t h a n d allowed to cool for
5 rain. (4) T h e ferric p e r c h l o r a t e r e a g e n t (~.5 ml, fresh daily) was a d d e d to t h e tubes,
m i x e d , ,~.d a f t e r 30 m i n tim purple color was react iu a I-cm c u v e t t e a g a i n s t a r e a g e n t
b l a n k in a B e c k m a n I3 s p e e t r o p h o t o m e t e r at 53o mt~- Tl,e color was stable for m o r e
t h a n I h.

:L J
F
O ...;'"
t~
>..
I--
o6
ILl
..'1
...I o4
¢D
7-- .j:
O.. O.,2
0

I I ) I | I I I !
1.0 2.D 3.0 ¢ko

MICROEQUiVALENTS OF ESTER
Fig. z.

T h e precizion a n d a c c u r a c y of this m e t h o d can readi l y be seen f r o m t h e c u r v e in

Fig. I, wh ich shows t h e r es ul t of d u p l i c a t e d e t e r m i n a t i o n s of est er iti t r i s t e a r i n samples
as m e a s u r e d o n 3 different days. E s t e r e q u i v a l e n t s of t r i p a l m i t i n , m e t h y l p a l m i t a t e ,
o r m e t h y l s t e a r a t e g a v e c u r v e s identical to t h a t o b t a i n e d w i t h tristearin. W e h a v e
also u s e d th is m e t h o d successfully w i t h lecithin samples. T he a c t u a l t i m e r e q u i r e d t o
d e v e l o p t h e p u r p l e color in 20 t o 30 s a m pl es is b e t w e e n I5 a n d 2o rain, T he m e t h o d
d e s c r i b e d a v o i d s t h e t i m e c o n s u m e d in t r e a t i n g each sample individv_ally2 a n d at the
s a m e t i m e p r o v i d e s a m e a n s of d e t e r m i n i n g e x t r e m c l y small q u a n t i t i e s of lipid esters
with excellent reproducibility,

This work was done u n d e r c o n t r a c t with the, U. S. A t om i c E n e r g y Commission.

3 l ¢ d i c a l Divisio~z, Oal¢ R i d g e In, stitnta o / N u c l a a r Studies, FRED ~ N YDI.~[~

Oak Ridge, 7"curt. ( U . S . A . ) ~-N]ELS O N ~TF I'I-IENS

L R . F . G o o D u , N, lY. 1,EBL,~XC a.~p C. M. XVuv'-ar, Amd. Chem., :7 (!055) z25t.

M. M. RAVPoRT Am~ N, Az,o.~zo, J . Biol. Chcol.. 2h ) ([055) lO3.
a A. R . THOXlPsoy, .-luslralian .]. Nci. Research, 3 (lo5 n) w2S.

R e c e i v e d D e c e m b e r z:.t. I958
246 SHORT COMMtINIC,VrlONS VOL. 34~ (1959)

The purification of ren|n by use of ion-exchange chromatography

R e c e n t advances in the preparation and uses of cellulose ion-exchange agents for t h e

purification of proteins 1.2 s , g g e s t c d their possible use i n tile purification of hog-
k i d n e y rents.
Tbe adsorbent chosen was I)EAE-cellulose prepared according to Pt*,TERSON
A,XI~ Sol~l'--Ra, with a c a p a c i t y of 0.76 mequiv./g. Columns w i t h dimensions of
1.2 ~: 55 cm and hold-up vol. of i i ml were prepared from 4 g of the ion exchanger.
Semiqmre renin was prepared from fresh hog kidneys a n d from desiccatect h o g - k i d n e y
powder b y a procedure described b y HAAS el ~l. a,a, These p r e p a r a t i o n s were carried
t h r o u g h five of the ten steps of their isolation procedure. The fraetionations were
performed in the cold room (6°), and the precipitate o b t a i n e d from Step 5 was dis-
solved in water an~t dialyzed. Alter dialysis the d r y weight of e n z y m e was o b t a i n e d
b y d r y i n g to c o n s t a n t weight by h e a t i n g to 1o5 ° and cooling in a desiccator over
P20~. Presser a c t i v R y of the preparation was m e a s u r e d b y injection of solutions of
suitable strength i n t r a v e n o u s l y into a dog anesthetized with N a P e n t o b a r b i t a l
(35 m~.k~,). A unit" of renin is the q u a n t i t y required to raise the m e a n femoral blood
pressure (,f a dog 3o m m ttg. The specific a c t i v i t y of the r e n t s i~ expres.~ed as t h e
mzmber of dog units/rag protein.
The equilibrated e n z y m e preparation was w a s h e d into a glass c o l u m n containing
the exchanger which h a d been a d j u s t e d to p H 7.o b y a d d i t i o n el N a H ~ P O , solutions
a n d packed with air pressure (~o lb./in. -~) until a c o n s t a n t column height was obtained.
The c o l u m n would n o t run d r y u n d e r g r a v i t y flow a n d m a i n t a i n e d an effluent flow
of two drops pet" m i n u t e when a t o t a l h y d r o s t a t i c head of e 7 in. was employed.
G r a d i e n t s of salt (NaH2PO4) a n d p H were e m p l o y e d for elution, w i t h the a d d i t i o n
of NaC1 in the later stages of the elution to raise the salt concentration. G r a d i e n t s
were established b y introducing from a s e p a r a t o r y fnnnel into a c o n s t a n t - v o l u m e
(5o ml) mixing c h a m b e r buffer h a v i n g the composition ot the g r a d i e n t limit. All
column operations were carried o u t in the cold room. I n d i v i d u a l tube collections of
a b o u t 6 ml were d e t e r m i n e d by a d r o p c o u n t e r b u t the v o l u m e of each tube was
m e a s u r e d directly.
T h e effluent fractions were e x a m i n e d for protein in a B e c k m a n DU spectre-
p h o t o m e t e r b y measuring the absorbance at z8o m ~ Paper-electrophoretie d e t e r m i n a -
tions were m a d e on xo ~[ of m a t e r i a l which was o b t a i n e d b y c o n c e n t r a t i n g the effluent
against polyvinytpyrrolidone. Gross sampling was necessm'y to provide sufficient
protein for these analyses which were m a d e with veronal buffer (/~, o.o75; p H . 8.6)
at re.gin t e m p e r a t u r e for z6 t~ at a c o n s t a n t current of 6 mA. The paper strips were
stained with b r o m o p h e n o t blue.
Fig. I shows the elution d i a g r a m obtained when the dialyzed semi-pure r e n t s
wa~s c h r o m a t o g r a p h e d on DEAE-eellulose. The s t a r t i n g buffeli was 0.005 M N a H a P O 4
at p H 7.0, after which decreasing p H a n d increasing salt g r a ~ e n t s were employed,
All effluent fractions wetc assayed for protein c o n t e n t and e n z y m e a c t i v i t y .
Fractions 4z-6o which contained a c t i v i t y were combined a n d c o n c e n t r a t e d against
polyvinylpyrrolidone. This active fraction was a p p r o x i m a t e l y ten times as pure as
the m a t e r i a l placed on the column i.e., 9 mg protein was recovered containing 85 %
of the original activity.
The original semi-pure cenin gave an absc~rptL~r., s p e e t r m n a t y p i c a l for a protein
VOL. 34 (I959) SHORT CO.'q.",iUh'IC.kT1OXS 247

i.e,, no m a x i m u m in t he rz:gion of zSo m/~ (tyrosine a n d t r y p t o p h a n ) , b u t did give

a s t r o n g a b s o r p t i o n b a n d b e t w e e n I8O and z3o m/z (CO--NH) c o r r e s p o n d i n g t o the
]3-form p r o p o s e d b y H.\.xs ~t aLL T h e s e a u t h o r s report t h a t these a':omatie am i no
acids a p p e a r in high c o n t e n t as c o m p a r e d with t h a t of o t h e r proteins. On t he o t h e r
h a n d , renin elu te d f r om t he col um n gave a t y p i c a l u.v. s p e c t r u m with a m a x i m u m
at ~8o m/~. T h e s e differences were f o u n d to be due to acet one c o n t a m i n a t i c m a n d
not t o differences in pr ot e i n configur;~tion, as p r o p o s e d by HAAS et ~d., since [ ml of
semi-pure e n z y m e c o n t a i n s 3.6 t~moles acet one. F u r t h e r m o r e , serum a l b u m i n t r e a t e d
in like m a n n e r can be m a d e to yield two different c~trvcs dep¢.nding u p o n w h e t h e r
a c e t o n e is p r e s e n t i~, t he p r e p a r a t i o n . This s t u d y then indicates th~tt t h e differences
o b s e r v e d with u.v. s p e c t r o s c o p y are dtt,. to the st rong :tbsorption of the a c e t o n e -
c a r b o n y l fflnetion in the region of 260 ny,.

11013.
f Z .... p,
t"

t-4
-<

r~

g
.~ Og ' -GO

=SG

-40

- 2 0 t~
~

0.q --[ ...... ,~ -- -10

oI ......................... X ~ . . . . . .

o ~0 ~0 ~0 ~0 ~0 ~o 70 8O
FRACTION TUBF" NU, M ~ i ~ P

F i g . i . F - f t l u e n t d i a g r a m c~f s e m i - p u r e I : c * g - k i d n e y r c n i u : 1t)¢~ m g d i a l y z e d p r o t e i n (43 ° mlit.~] i n

t o m l a p p l i e d t*, 4 g a d s , t h e n , ; e ' . ] h l e n t c o l l e c t e d in 5~ t~) t)-rnl f i n e , i f , u s . B u t l e r s : I, o . o o 5 .11
N a H ~ P O v p H 7 . o ; 1 1 , o . o 2 3 I N a H ~ P ~ ; , . p H * ) . o ; II I. ~.o5 31 X a H d ) U 4 ; iX', o . v z 31 X a t ' l - o . , ) 5 ,1;
NaHsPO,;V. o . o 5 A I ' ~ a C i - o . ¢ 3 5 . 1 1 N a H ~ l ' ( ) t ; V [ , o . t .II N a C I o . o 5 3 I N a H z [ ' ( ) l ; V I I , o._, .li
NaCl-o.o 531 Nai-lePO4; VIII, o. 5 .1! X a C t - o . o 5 .Xf N'al-4L, P O , ; I.ft., 1.o 31 N a C l o . o 5 A I
N a H z l t ' ( ) 4 : -K. t. 5 31 XaCI-c~.',) 5 .*tl N,',~l-tat'()a; X I . 2 . 0 .U N a t 1 t x o 5 3 ! N n l q ~ l ' ( ) , : X I t , ~qatd.
N a C l - o . 5 31 N z t t l ? ) O v M i x i n g c h a m b e r x'~)l., 5 o m.1. S h a d e d a r e a is t h e enzvmt~, d i s t r i b u t i n n .

T h e s e m i - p u r e renin p r e p a r a t i o n gave a positive n i t r o p r u s s i d e reaction for a

s u l f h y d r y l - e o n t a i n i n g c o m p o u n d while t h e c h r o m a t o g r a p h e d renin g a v e a n e g a t i v e
test. Tile e x t r a n e o u s proteins in the semi-pure preparatic,a m a y t h e r e f o r e be p r o t e i n s
h a v i n g s u l f l l y d r y l g r o u p s a n d s om e of the renin present m a y be in t h e d e n a t u r e d form.
P a p e r electrophor e s i s at p H 8.6 revealed the presence of t w o c o m p o n e n t s in t h e
e l u t e d renin w h e r e a s t h e semi-pure m a t e r i a l wa_n c o m p o s e d of at [east four bands.
T h e c o l u m n c h a r g e in these studies was t o o mg prot ei n for 4 g a d s o r b e n t . This
was d ecr eas ed to 3 g e x c h a n g e r a n d p a c k e d undec (~4-7 lb./in.") air pressure w i t h a
flow r ate of one d r o p per 2 min. U n d e r b o t h condi t i ons with different samples of
e n z y m e t h e r e p r o d u c i b i l i t y of t h e positions a n d m a g n i t u d e s of the c h r o m a t o g r a p h i c
p e a k s were good. I n a n o t h e r s t u d y the e n z y m e was n o t e q u i l i b r a t e d to p H 7.0 b u t
r u n a t p H 5 .r , t h e p H of s t e p 5- T h e e/fluent p a t t e r n of this was similar to t h a t
o b t a i n e d w i t h t h e e q u i l i b r a t e d samples.
24 8 SHORT C O a N I J N m . X ' n O N S vo~.. 34 (x959)

An ion-exchange ~:hromatographic technique dor the purification of reI~in has

been described, The st)ecific activity of the preparation which was applied to the
cotunm was 4.3- The eluted renin had a specific activity of 43.2. This constitutes a
Io-fold Imrification of renin hy use of cellulose ion-exchange chromatography, with a
recovery of 85 ",,, of the enzyme activity.

I wish to thank Dr. P~,.'DI~OI3ZAQUIER for performing the bioassays reported in this
manuscript, and Dr. O. M. HEL,~Z~R Of Eli Lilly and Co. for generous supplies of
desiccated hog-kidney powder. This worked was aided b y grants from tl;m National
Heart Institute, U.S. Dept. of Health, Education and Welfare (H-2578c) and the
Michigan Heart Association.

Hypcrtensio~, l~nit, Dcpart*~cnt o / I ~ t e r n a l ~llediai**e,

3Icdical School, ('niversit v o[ Alichiga~r,, G . T H O M A S P.XSSANANTI"
A ~t4t .4 rbor, 31ich. (U.S.A .)

I H . . k . Sc)TaER. I:. J. (;UTTER, ~I. 51. X,V%'CKOI-'F AND E . A. |'ETI~RSON, J . -IJ$2. (.7ht'lJL .~oc., 78
Iz950) 73t~.
2 H , A. 5~)m:R _~xl~ E , A. I~t.';TER%ON, ill {-',.(-'AI.MON AND "['. R . E, NRIiSNMAN, loll 1~.1"(]t(11t~£'Y5 iJl
()ry~ON6 ¢41ld l.~zc~rJlcs~iiMrJ,', l n t e r s c i e n c e PubliMxers, I n c . , N e w Y o r k , 1.~57.
:~ E . . \ . t'v:rv:~s(~.x A':t> H . A. S o m c a , .]..-tin. Chem. Nor., 78 (195~)) 75t.
4 l:.. It-*.~-~, H. I.~XMFI~oM AND i l . GOLDBLATT, Arch. t3iocl~em. I~iophy,~,, 42 (1953} 3(~S.
:' E . ttAAn, in S. P. COLOWICK AND N. (). I'~.AI'LAN, 31ethods in ER-7.y~stolog3h Vol. I[, A c a d e m i c
P r e s s , I n c . , N e w Y o r k , 1955.
6 f t . GOLI)liLATT, Y. J . tqXTZ, It. A. I.I':WIS AND E. I{tCHARI?SON, .], EX['tl. 3h'd., 77 (1943) 3°O.
E . ItAAs, 11. t..\MFII(I.XI AND iI. (JrOI.I)IILAIi, .'lvch. l~iochem. I~iophys.,-t4 (t9531 79.

Received November 24th, I058

" P r e s e n t a d d r e s s : I ) c p a r t m e n t of l ) c r m a t o l o g y , U n i v e r s i t y [)f .'Michigan M e d i c a l SchfJol, A n n
, \ r b ~ , Mich.

A simplified method for the purification of

mushroom polyphenol oxidase
Purification of nmshroom polyphenol oxidase has been sought it, this laboratory in
order to pursue structural studies on this enzymO and to explore the role o[ tyrosine
in the secondary and tertiary structure of proteins 2-~. While this work was in progress,
KEI~TF-SZ ,X.','l) Zrro 6 reported the preparation of homogeneous mushroom polyphenol
oxidase. Taking advantage of the excellent early steps of this procedure', we have
used chromatography on diethylaminoethyl-ceUulose (DEAE-cellulose) ? as the basis
for z ifighly simplified method to prepare the purified enzyme in good yield. A similar
chromatographic procedure for the p-eparation of a soluble mamlnali,'m tyrosinase
was also rerently reported by B R o w \ AND V~r.XRD~.
The yields and steps in the method are summarized in Table I and in the ex-
perimental section. After extraction and preliminary purification through Step 4,
the crude enzyme is prepared for direct chromatography by an extensive dialysis
" * W e a r c v e r y g r a t e f u l t o ]h'~ffcssor 1). ](I~RrESz for d e t a i l s of h i s m e t h o d in a d v a n c e o f ptLblica-
ticHl.