Physics. - The spreading af avalbumin. By E. GORTER, J. VAN ORMONDT
and F . J. P . DOM. (Communicated by Prof. P. EHRENFEST.)
(Communicated at the meeting of June 25, 1932) .
I t has been shown previously 1) by one of us, in collaboration with
Dr. F. GR ENDEL, that proteins can be made to spread in a mono-Iayer at
the air-water interphase of a Langmuir tray.
For casein the influence of the acidity or alkalinity of the water in the
tray has been carefully studied, with the result that at the isoelectric
point 4.7 and at a great distance from it, in a distinctly acid and a distinctly
a lkaline medium, maximal spreading was obtained , whereas at intermediat",
values of the hydrogen ion concentration at a pH 3 and at pH 8.5 minima
were seen. We have concluded that the protein itself and its salts spread
beautifully, but that protein-ions occupy a much smaller surface area per
molecule.
In the experiments to be related , we have studied the spreading of
ovalbumin. We have preferred this protein because it is easily soluble in
water without the addition of electrolytes.
Moreover it can easily be prepared in a very pure state. \Ve made use
of the HOPKIN s -PINK US method 2) of preparation as modified by
SÖRENSEN .
The ovalbumin was recrystallized 5 to 6 times and th en dialyzed against
distilled water in the icebox until the sulphate reaction of the water
remained negative after 48 hours standing in the dialyzer. Usually several
weeks were necessary to reach this state of purity.
The ovalbumin concentration was estimated in two ways:
1. the nitrogen content of the solution was estimated by the TER
ME ULEN and HESLINGA method 3). In this method hydrogen gas is passed
over a heated mixture of powdered nickel with the sample in which nitrogen
is to be estimated and the ammonia formed titrated directly in a receiving
tube with 0.01 n hydrochloric acid. The nitrogen found , was multiplied
by the factor 7.0 to get the ovalbumin.
2. the ovalbumin as such was determined by BANG's method for the
estimation of albumin in urine. The albumin is precipitated by boiling with
a mixture of dilute acetic acid and sodium acetate, filtered , washed with
I) E. GORTER and F. GRENDEL. On the Spreading of Proteins. Transactions of the
Faraday Society 71. 1926. p. i77 . Biochemische Zeitschrift 201. 1928, p. 391.
2) F . GOWLAND HOPKINS and S. N. PINKUS. Journalof Physlology. 23. 1898. p.130.
1) TER MEULEN and HESLINGA. Nieuwe methoden voor elementair analyse. Delft 1930.
Rec. Trav. Chem . • 9 (1930), p. 396.
839
alcohol and ether and dried to constant weight at 105° . The results of
the 2 methods were in good agreement with each other.
These estimations of the ovalbumin content had to be performed fairly
often as it appeared to be impossible to keep a solution for an indefinite
length of time. Always after some weeks and sometimes even after one
week a slight flocculation appeared which made it necessary to filtrate the
solution and estimate again. This flocculation had nothing to do with a
decay of the protein. but was caused by the instability of the colloidal
system when dia lysis is prolonged so far that most of the electrolyte is
taken away. The stock solutions were always kept in the icebox af ter
adding some preservative. for which thymol was usually taken; chloroform
and toluene seemed less satisfactory. as they rather increased the tendency
to flocculate. The concentration of the solution was always about 5 milligram per cc ; e.g . one of the stock solutions had at the outset a
concentration of 5.6 mg per cc and after three months when it had been
filtered several times. a concentration of 4.6 mg per cc.
sq. M.
per
rT
g.
O'A LBUMIN
Hl
0
y:;
0
09
06
0
O.~
0
0
0
\
0.2
~
\-lJ
a
1
3
5
Fig. 1. InHuence of pH.
ti'
pH.
840
The buffer solutions used were in the earlier measurements acetate
buffers as described by W AL PO LE 1 ).
They were diluted to a molarity of 1/3 00 and could be used for pH 3.6 to
pH 5.6. The more acid solutions were made with hydrochloric acid. The
later experiments were made on veronal-ace ta te buffersolutions as
described by MI CHA EL IS 2 ) , also diluted to 1/ 300 mol. This latter buffer
has several advantages :
1s t . it covers a large pH range , from pH 2 to pH 10 ;
2nd . it contains only monovalent ions ;
3 rd . its ionic streng th can be kept constant over the whole pH range.
The pH of all buffer solutions was controlled by colorimetrie and
electrometric mea surements.
The influence of changes of the acidity of the water on the spreading
of ovalbumin was the same as had been observed with casein. (See fig . 1.)
We have now studied the influence of the addition of different salts
to the wa ter in the tra y upon the spreading of ovalbumin .
It was easy to show that, at the isoelectric point, salts had no influence
on the surface occupied by the protein. But wh en studying the influence
of potassiumchloride on the sprea ding in acid medium , it was possible to
discover a very simple relation between the size of the area and the
concentration of Cl ions in the fluid . This relation is to be seen from Fig. 2,
where the pCI is plotted a gainst the surface area occupied by the protein.
ErrECT OF Cl- ION,
Sq . M.
OVALB ~MIN.
perm ~.
Hl
DH 2.4
I
01l
/
as
I
0.4
.-
V
L-/
0
~
O~
O.
tB
Sf4
fa
Log. mltLI aequlv.
Fig . 2
I) G. S. WALPOLE . Journ. Chem. Soc. lOS. (191-4) . p . 20001.
L. MICHAELIS. Bioch. Ztschr. 23 • . (1931) . p . 139.
2)
841
The part of the curve between pH 1.2 and pH 3, is explained by the presence
of Cl ion in the water, because HCl and KCI have exactly the same influence
on the spreading (fig. 3). We may conclude from this observation which can
be made as weIl with other salts (e.g . BaCI:! fig. 3a) that the addition of
INFLUENCE
a-
laNS.
sq.M.
per IT ~.
aVAL BUM,'N.
Hl
Qe
./
as
0.4
0.2
Q
I
r"
0
/
J
40
9 9 HCI without salt
--r
o
0
o0
0
+ KCI
pH 2.4 14 milli eq. HCI) + BaCI
pH 1.8 [16 milli eq. HCI)
2
• • • pH 2,4 [4 milli eq . HCI)
t 0 11 0
80
..
+ KCI
CL-
mllli aequlv .
Fig. 3
EFFECT OF
Sq. M .
cr
0 IALB UMIN
nU
perm ~ .
1D
EFF"EC T OF" 50;- ION.
~4
I
Qf3
V
_0.6
O~
Q~
0..
ION.
~
0
V
4
/
/
V
sq M.
per rr g.
1.0
Fig . 3a
nl-l
~4
ml
1/
· O.f:i
0.l4
O~
BaCl1
1$ . Ei.~
log. mllli aequlv.
o IALB JMIN
/
/
11
0
0
~
16
.
Ei 4
.
K 50"
log. mllli aequlv.
Fig . i
842
EFFECT OF K· ION.
EFFECT OF MTSK.---'ON.
sq. M.
per rr .g.
OVA
DH
10
BUM
,.
3.1
N.
0' ~LBlJ MIN.
Ui
//
OB
Sq. M.
perm! .
DU G.7
O.B
I
I
J
j/
OS
1/
O~
O~
0.
OB
0
OA
V
-V
./
-V
O~
1/ ~S6 J 6lt 1 16
. .
log .mllli aequlv .
L~
a.
0
i6
4
M.T S.K.
Fig. 5
sq. M.
[11-1 G7
sq. M.
per rr .g.
OB
as
OS
~
G..t
a
p 1~
•
./
.A'
a
Plg. 7
DH. ).7
O.l!
O~
I~
~ Be.Clt
Log . miUi ae qUIV.
OVAl BUMI N.
lO
na
04
6 ~ 2 6 K~O
..
Log. mlLlI aequlv.
EFFECT OF LUTEO··· ION.
OVA ~BU~ IN .
perm g
Hl
'/
Fig . 6
Ba·· ION.
EFFECT OF
/
O.
/
o
V
/
V
/
V
Lf 3 te 13 6~ 13
.
.
Log.mlUI aeqUiv. LUTEO COBALT.
L '3
Plg. 8
843
Cl ions has the tendency to increase the area of spread of a protein salt.
that is completely ionized. It has a disionizing effect. The relation between
pCI and the area is Iinear (See already Bioch. Zeitschr. 1. c. p. 404) . Now
if this interpretation is the right one. bi-valent and tri-valent negative
ions should have an identical effect in much lower concentration. This is
Indeed what had been observed .
We found that S04 ions have in much smaller concentration the same
discharging effect as the monovalent (See fig. 4). And the tri-valent
have an effect. that is identicaI. but can be observed when making use of
still lower concentrations. The tri-valent negative ion was methane tri-
/ -H
sulphonate
~,,_~g~
]).
(See fig. 5.)
'-- S03
lt was interesting to see what would happen on the alkaline side of the
iso-e1ectric point after addition of positive ions.
It is hardly necessary to say that KCI has also an influence on the
minimum spreading at the alkaline side (fig . 6). but that K 2 S0 4 and
the methane trisulphonate have no effect in the small concentrations used.
On the contrary the bi-valent positive ions Ba++ and Ca++ have the
same effect on the spreading at pH 8 as the S04-- at pH 3 (fig. 7) and
we were also able to observe a very strong influence of luteocobaltchloride
CO(NH3 ) 6CI 3 on the spreading at pH 8 (see fig . 8) . lt is obvious. that
this adds convincing arguments to the interpretation given before: at
the isoelectric point the neutral protein-molecule spreads in a thin layer:
the addition of H+ (and OH- ) ions diminishes the area occupied by the
protein. so much so that it occupies only 1/9 of the surface. Further
addition of negative ions at the acid side of the isoelectric point (from
pH 3 downwards) increases the surface by eliminating the charge of the
protein ions. The same holds good for the addition of positive ions in
alkaline medium. Valency has a very striking influence: tri-valent ions
acting more strongly than bi-valent and mono-valent ions having a much
smaller influence than bi-valent.
1) We made use of this salt following the advlse of Prof. BUNGENBERG OË JONG.
It was kindly given to us by Prof. BACKER.
Mathematics. - Zur generellen Feldtheorie. DIRACsche Gleichungen
und HAMIL TONsche Funktion. Van J. A. SCHOUTEN und
D. VAN DANTZIG. (Communicated by Prof. P. EHRENFEST).
(Communlcated at the meeting of May 28. 1932).
In der vorigen Mitteilung 1) haben wir u. a . gezeigt dass in der
1) J. A . SCHOUTEN und D. VAN DANTZIG. Zum Unifizierungsproblem der Physik.
Skizze einer generellen Feldtheorie. Proc. Kon . Akad. 35 (1932) 612 - 655. zitiert mit G. F . I.
55
Proceedings Roya! Acad. Amsterdam. Vol. XXXV. 1932.