323
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
NOTES
BBA 71 O14
The solubilization of human erythrocyte membranes by n-pentanol
During the past few years, several studies concerning the isolation and characterization of structural proteins from erythrocytes, have been reported 1-9. MADDY1,2
described a method for the solubilization of the proteins of ox erythrocytes by
n-butanol fractionation. We observed that mpentanol causes a solubilization of
erythrocyte ghosts, but in contrast to the butanol procedure, not only the proteins
but also the lipids were recovered in the water layer.
H u m a n erythrocyte ghosts were prepared according to the method of DODGE,
MITCHELL AND HANAHAN1°. The haemoglobin-free ghost suspensions were frozen
overnight at --25 o in hypotonic phosphate buffer (20 mosM, pH 7-4), thawed the
next day and washed 3 times with distilled water at 2 °. The ghost suspensions in
water were mixed with equal volumes of pentanol, butanol or mixtures of both
solvents at o ° and 25 rain later were centrifuged at 30000 x g for 15 rain. The upper
organic phase was removed and the aqueous phase under the insoluble interfacial
film was sucked out with a cold hypodermic syringe and dialysed against water for
20 h at 4 °.
With pentanol, 80-85 % of the proteins and over 80 % of the phospholipids
(and also cholesterol) of the original ghost were found to be solubilized in the aqueous
phase (Fig. I), whereas only 2-5 % of the phospholipids were recovered in the pentanol
phase. Using butanol, we found, in agreement with MADDY 2, that 85-9 ° % of tile
proteins and only 3-5 % of the phospholipids were present in the aqueous phase
(Fig. i), while 9o-95 % of the phospholipids were detected in the butanol phase. As
shown in Fig. I, the percentage recovery of phospholipid in the water phase is strongly
dependent upon the ratio of pentanol to butanol.
100-
0--0~0--0
0
~6o-~
a~
"~
~40m
g
c~20"
;e
~00°/o
PENIANOL
50/50
100°/o
BUTANOL
Fig. I. P r o t e i n a n d p h o s p h o l i p i d r e c o v e r y in t h e w a t e r p h a s e after t r e a t m e n t w i t h p e n t a n o l b u t a n o l m i x t u r e s of a q u e o u s g h o s t s u s p e n s i o n s . O - - O , p r o t e i n ; LX--&, phospholipid. D e t e r m i n a t i o n s of p r o t e i n n a n d p h o s p h o l i p i d 12 were carried o u t b y e s t a b l i s h e d procedures.
Biochim. Biophys. Acta, 15 ° (1968) 323-325
324
PRELIMINARY
NOTES
C o m p a r a t i v e analyses of the amino acid composition of the proteins from ghost
a n d the w a t e r layers after b u t a n o l a n d p e n t a n o l e x t r a c t i o n , respectively, gave the
following values: Asp, 8.3-8.o--7.9; Thr, 5 . 2 - 5 . o - 5 . I ; Ser, 6.3-6.2-6.1; Glu, 13.9-14.o13.8 ; Pro, 5.6-5.8-5.8; Gly, 6.8-7.o-7.o; Ala, 8.1-8.1-8.o; ½ Cys, o . 5 - o . 5 - o . 5 ; Val,
6 . 6 - 6 . 7 - 6 . 6 ; Met, 2.1-2.2-2.2; Ile, 4.8-5.o-5.3; Leu, I I . 6 - I I . 6 - I I . 7 ; Tyr, 2.4-2.3-2.3;
Phe, 5 . o - 4 . 9 - 4 . 9 ; Lys, 5.o--4.9-5.o; His, 2.7-2.6-2.6; Arg, 5 . I - 5 . 2 - 5 . 2 . (Expressed as
m o l e % ; analyses were m a d e with a B e c k m a n amino acid a n a l y s e r ; Thr, and Ser,
u n c o r r e c t e d values; Trp, not determined.) These results confirm t h a t the proteins
o b t a i n e d after b u t a n o l or p e n t a n o l e x t r a c t i o n are r e p r e s e n t a t i v e of the original ghost
proteins. Disc electrophoresis at p H 3.5 in 6 M urea using 7 % p o l y a c r y l a m i d e as a
s e p a r a t i n g gel, showed up to I3 p r o t e i n fractions b o t h after p e n t a n o l and b u t a n o l
solubilization. No significant differences in the electrophoretic p a t t e r n between the
two p r e p a r a t i o n s were observed.
B y d e n s i t y g r a d i e n t c e n t r i f u g a t i o n in sucrose gradients, a c o m p a r a t i v e s t u d y
was carried out between the aqueous solutions o b t a i n e d after p e n t a n o l (Fig. 2A) and
b u t a n o l (Fig. 2B) extractions. In b o t h cases two m a j o r fractions were o b t a i n e d " . W i t h
respect to F r a c t i o n I, a difference in d e n s i t y was a p p a r e n t (pentanol solubilized 1.16o
b u t a n o l solubilized L I S I ) b u t the densities of F r a c t i o n s I I were a b o u t equal (i.o9 o)
In a n o t h e r e x p e r i m e n t , the w a t e r phase o b t a i n e d after b u t a n o l t r e a t m e n t was m i x e d
prior to the c e n t r i f u g a t i o n procedure, with an aqueous lipid suspension p r e p a r e d b y
ultrasonic v i b r a t i o n of t o t a l e r y t h r o c y t e lipids. A s e p a r a t i o n into three fractions was
o b t a i n e d (Fig. 2C). In a control e x p e r i m e n t , it was found t h a t the lipid alone r e m a i n e d
floating at the t o p of the g r a d i e n t d u r i n g the centrifugation. The a d d i t i o n of lipid to
the p r o t e i n s solubilized b v b u t a n o l did not affect the d i s t r i b u t i o n of the proteins
over the g r a d i e n t (compare Figs. 2B a n d 2C) a n d the lipid was recovered m a i n l y at
the top. However, after c e n t r i f u g a t i o n of the solution o b t a i n e d after p e n t a n o l ext r a c t i o n , no d i s t i n c t lipid fraction was d e t e c t a b l e at the t o p of the gradient. A f t e r
e x t r a c t i o n of the fractions with c h l o r o f o r m - m e t h a n o l a n d e x a m i n a t i o n of the e x t r a c t s
C
B
g1
]I
II
I
1
m
/ ./\
1
C~l / /
\ j"
~[//
-
\
]
FRACIION NUMBER
FRACTION NUMBER
FRACTION NUMBER
Fig. 2. Density gradient patterns obtained after centrifugation for 3 h in sucrose gradient (I3-55 %)
in a Spinco Model L2-65, SW 39 rotor at 37 5oo rev./min. A. Aqueous phase after pentanol extraction of human erythrocyte ghosts. B. Aqueous phase after butanol extraction of human
erythrocyte ghosts. C. Protein solution obtained under B, mixed with micellar lipid suspension
in water (the ratio of protein to total erythrocyte lipid was 5:4). Samples (o.5 inl) containing
4 5 mg of protein were applied to the gradient and I I fractions were collected after puncture of
the bottom of the tube. - - - ,
absorbance at 28o re,u; . . . . .
, protein content according to
the method of Low~Y el al. 11 (A75omt~).
* Disc electrophoresis of Fractions i and II revealed 4 and 9 protein fractions, respectively,
which exhibited mobilities corresponding with the ~3 fractions detected in the starting material.
t3ioct~im, t 3 i o p h y s . A c t a ,
I5o (1968) 323-325
325
PRELIMINARY NOTES
by thin-layer chromatography, it appeared that both cholesterol and the various
classes of phospholipids were moving downwards into the gradient together with the
proteins. Preliminary analyses suggest that differences m a y exist in the lipid to
protein ratio among the fractions. Electron microscopic studies showed that the
material in the aqueous layer after pentanol treatment is homogeneous, in contrast
to the material in the water phase after butanol extraction mixed with the micellar
lipid suspension. These results demonstrate that soluble lipoproteins can be obtained by
treatment of aqueous ghost suspensions with pentanol. We hasten to emphasise that
it is not known at present whether some of the original lipid-protein associations
have been preserved.
The present investigations have been carried out under the auspices of the
Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from
tile Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The
authors are indebted to Mr. A. DE VRIES ROBBfi for his participation in the experiments on pentanol and butanol solubilization, and to Drs. G. H. I. T. VERVERGAERT
for electron microscopic studies.
Department of Biochemistry,
Laboratory of Organic Chemistry*,
Utrecht (The Netherlands)
1
2
3
4
5
6
7
8
9
IO
II
12
R. F. A. ZWAAL
L. L. M. VAN DEENEN
a . H. MADDY, Bioehim. Biophys. Acta, 88 (I964) 448.
A. H. MADDY, Biochim. Biophys. Acta, 117 (1966) 193.
M. D. POULIK AND P. K. LA~IF, Nature, 208 (1965) 874.
T. E. MORGAN AND D. J. HANAHAN, Biochemistry, 5 (1966) lO5O.
p- H. ZAHLER AND D. F. H. WALLACH, Biochim. Biophys. Acta, 135 (1967) 371.
S. BAKERMAN AND G. WASEMILLER, Biochemistry, 6 (1967) ilOO.
A. F. REGA, R. I. WEED, C. F. REED, G. G. BERG AND A. ROTHSTEIN, Bioehim. Biophys. Acta.
147 (1967) 297.
1~. A. AZEN, S. ORR AND O. SMITHIES, J. Lab. Clin. Med., 65 (1965) 44 o.
L. J. SCHNEIDERMAN, Biochem. Biophys. Res. Commun., 20 (1965) 763.
J. T. DODGE, C. MITCHELL AND D. J. HANAHAN, Arch. Biochem. Biophys., ioo (1963) 119.
O. H. LOWRY, N. V. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (I95 I}
265.
C. J. F. B6TTCHER, C. M. VAN GENT AND C. PRIES, Anal. Chim. dcta, 24 (1961) 203.
Received December i4th , 1967
* Postal address : Croesestraat 79, Utrecht, The Netherlands.
Biochim. Biophys. Acta, 15o (1968) 323-325