114
ROBERT
.\.
-4LBERl'Y
drops rapidly and reaches zero a t p H 12. The degree of hinding at fixed pH falls with increase in protein concentration. h comparison of the binding affinities of bovine albumin, p-lnctoglobulin, and bovine y-globulin indicates a decrease in t'lie order listed. Rl.:FF;RESC'I,-secoiidh-iltioiial Colloid S.yinposiuin, whicli WHY Iieltl under tlic nuspic,es of tlic Division of Colloid C,lic,inistr!. of the A 4 ~ n e i ~ i Chemical c~ii Society at' ('niiil Iritlgc , .R Inssa cl iiise ti s . ,Jriiic 23-25, 1 9-l-R .
VARIATION O F ISOELECTRIC POINT IVITH IONIC STRENGTH
1.15
norinn1 human 2nd y2-globiilins2aid crystallized human 2nd bovine a h miiis (4). The ?-globulins are responsible for the antibody fUllCtiol1 in the pl:lsmn, v.hile the albumin is responsible for approximately three-fourths of the c.oIIoitl osmotic pressitre of plasma, and t,hesc. prot'eins represent, respectively, the romponelits of highest 2nd loi\-est,isoelectric pointjs present in appreciable lu t i n t i tj- in the plasin:~. l'hc strtdy of proteins by electrophoresis offers the most direct method for the determination of the isoelectric point, which may be defined as the pH and composition of a, solution in which there is 110 net movement of protein in an electric field (10). The salt concentration and the nature of the specific salt may have a inthei. large effect upon the isoelectric point, so that it is essential to gi1.e complete information as to these variables. Itj is difficult t o find purified proteins i\-hich are sufficiently homogeneous so that all the molecules are isoelectric under the same conditions, and it is usually necessary t>ouse the terms "average" isoelect'ric point and isoelectric point distribution. The moving-boundary method of electrophoresis has the advantage that it yields information as to the electrical homogeneity of a protein preparation as well as the average isoelectric point, and may be used t'o determine the isoelect'ik point' distrihut'ion in certain cases ( I , 2). JIEI'HODR
'rile electrophoretic patterns in pH 8.6 buffer for the human 71- and r2-globulins used in these studies are given in figure 1. The proteins were prepared from pools collected by the American Red Cross by methods developed a t the University of Wisconsin (7, 8). As judged by the conventional methods for analyzing electrophoretic patterns, the r2-globulin contJainedless than 1 per cent of @-globulinand albumin and the rl-globulin contained less tlinn 3 per cent of other proteins. Crj+stallized bovine albumin (4) \\-as obtained from Arniour Laboratories, Chicago, Illinois (Cont,rol No. G4502), and the crystallized human albumin (4) was a gift of Dr. 15'. L. Hughes, Jr. (Department of Physical Chemistry, Harvard Medical School). Seit'her of these prot,eins sliowed any globulin components in electrophoresis in pH 8.6 and 0.1 ionic strength diethylbarbiturate buffer. It was found that accurate mobilit'ies could not be obtJainedwith the customary electrophoresis electrode vessels because of the contraction of the Neoprene rubber sleeves connect'ing the electrode vessels to the Tiselius cell, the separation of small air bitbbles in the parts of the apparatus which extended a little above t,he surface of tile t)herrnostat, and the difficulty in eliminating small leaks and current losses. T o uvercome these difficulties :in electrode vessel similar to that designed by Longsvorth (14:) for the incasurement of salt t,ransference numbers by the diff ereiitial moving-boundayy method was used. The const'ruction of the modified top section of the standard 11-ml. Tiselius 2 'J'he ~p~-glokniliii is ptxcticallj, itlent,ir:d $\.it11tlic: Hut11 of fractions 11-3 :tiid II-1,P o i Oi1cie.v ~1 u l . ( I S ) , n l i i l c -,-globulin is ii noli-fibrinogen globulin iiaviiig the mobility of fi1)riiiogeii i i i pH Y.(i atid 0.10 iotiic st rctigth tlietti~lbnrl)itur.ntel i i i f t ' t ~ i , (rJ. S u I T H . E . L , : . I . n i n l . Chcin. 171, 3555 (19471~.
116
ROBERT A . ALBERTT
FIG.1. Electrophoretic patterns a t pH 8.6. (a) Human 71-globulin in diethylbarbiturate-citrate buffer, F/2 = 0.088, after 120 min. a t a potential gradient of 10 volts/cm. (b) Human rz-globulin in diethylbarbiturate-citrate buffer, F/2 = 0.088, after 120 min. a t a potential gradient of 10 volts/cm.
n
F I G .2 . Llectrode vessel for mobility determinations. The right electrode v ~ s s c lnlay be patterned after the one on the left or the customary typc of electrode vessel may be connected.
VARIATION O F ISOELECTRIC POINT \VITH I O S I C STRENGTH
117
cell is illustrated in figure 2.3 The electrode vessel attached to the left sid.e of the top section is closed to the atmosphere, and the right electrode vessel may be patterned after the left, or the usual form of electrode vessel may be attached t o the right side. The silver-silver chloride electrode W, consisting of coils of I-mm. silver \\.ire, fits into the small test tube T. In setting up the apparatus, it, is important that a sharp boundary be formed between the concentrated salt solut,ion in the test tube and the buffer so that during the experiment salt will not overflo\v the test t,ube into the electrophoresis cell. This is accomplished in the left' electrode vessel as folloivs: The apparatus is set up filled with buffer and t,hen a strong salt solution is injected directly int'o the test tube contairiing the electrode b y means of a long hypodermic needle inserted through t'he threeway stopcock. Being heavier, the strong salt solution displaces buffer u p through the hole H just above the electrode. When the level of t,he salt)solut'ion in the test, tube reaches the hole H, the strong salt solut'ion rises up the tube lea.i.ing n "sharpened" boundary at level H. The boundaries are compensated into the cell by the addition or withdrawal of 1 ml. of salt solution, using a mechanicallv driven syringe attached to the three-\vay stopcock S. The elect'rode is attached to a silver wire in the glass tube which passes through the standard-tnper joint' and the wire is soldered through a st#ainless-steelcap C attached to the glass side arm \vith -4piezon IV \vas. ELECTROPHORETIC STUDIES O F HUMAN "(1-
AND 72-GLOBULINS
E.xperimcntn1 The elect'rophoretic mobilities of the normal human y-globulins have been determined over the pH range of 4.0 to 10.0 and a t two ionic strengt'hs, 0.01 and 0.10. I t is necessary to use a vaixiet'y of buffers over this pH range (see table l ) , and follo\ving the recommendation of Longs\vorth (13) only uniunivalent buffer salts n-ere used. Sodium chloride \\.as added to a number of the bu!Yers to make up part of the ionic strength, so that the ion atmosphere surrounding the protein molecules in solution would not change so markedly in passing from one buffer salt t o the other. The buffer capacities, p, Tvere cnlculated by tjhe method of T'an Slylte (25). The buffer capacit,y is defined as t,he number of equivalent's of st'rong alkali or acid taken up by 1 liter of buffer in changing t'he pH one unit, as calculated on the basis of a small addition. These Imffer capacities may be compared with that of the routinely used 0.1 ionic strengt8hcliethylbarbiturate buffer, pH 8.G, ,B = 0.033 equiv./l. The protein concentration of the electrophoresis samples \vas kept low (about 0.3 per cent) in order to reduce t)he conductivity anomalies. The solutions were dialyzed a t least, 2 days before electrophoresis. The specific conductivities of the equilibrium h f f e i , solutions measured a t 0°C. were used in the calculations of electrophoretic mobility. Esperiment>s showed that the difference in conduct'ivity betn.een the dialyzed protein solution and the buffer \vas negligible cornpared to ot'lier errors in the mobility determination. The pH of the buffer 3 The author is iiide1)tetl t.o Dr. C;. Kegeles for suggestions concerning the construction of t h i s eleat,rode vessel.
118
ROBERT -4.ALBERTY
was measured a t 25"C., iising a glass electrode. The mobilities were calculated from the boundary velocity in the descending limb of the cell, using the bisecting ordinate to locate the position of the boundaries since they were, in general, quite symmetrical. Calculations of the electrode correction for these experiments showed this correction t o be negligible (131. TABLE I Birfers uscd in delcrtninntions of electrophoretic ttkobilitu PH
'' ,
__
___
4.0 5.0 6.0 7.0 8.0
~
~
'
~
I
0.08 0.08 0.06 0.00 0.08 0.0s 0.04 0.09 0.08 0.06 0.0s 0.05 0.05
-
I
XaCl
~~~
4 .O 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 0.5 10.0
'
NORNALITY
- - . ...--
, ~
, ,
1
' ~
1
_ ~ pH _ ADJGSTEn
\\.ITK*
,
~
I
~
i '
9.0
13 ~.~ .
0.10 ionic strengt,li buffers .~
~
0.02 0.02 0.04 0.10 0.02 0.02 0.06 0.01 0.02 0.04 0.02 0.05 0.05
' ~
~
,
, '
I
,
I
HOAc IiOAc HOAc HOAc HCac HC:tc HCac HV HV IIV Glycine Glycine Glycine
0.01 ionic strength buffers I
,
,
NaOH
~ _ _ _ _ _ _ _ _
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
HOAc ,
' 1
'
I
,
HOAc HCac HCac HV Glycine Glycine Glycine
~~
__
~~
4.98 4.94 4.62 3.65 5.31 5.31 3.55 5.35 4.94 1 5.08 1 4.53 1 4.41 4.24
0.03s 0.029 0.031 0.032 0.029 0.016 0.021 0.017 0.021 0.018 0.030 0.041 0.039
~
'
~
I
~
1
__ ___
i ~
'
I' 1
!
0.019 0.008 0.015 0.004
0.010 0.015 0.008
~
~
~
i
10-3
~
'
1
-
..
-
0.43 0.41 0.40 0.38 0.40 0.40 0.42 0.41
9.5 10.0 1 0.008 _____ _ _I ~ _ _ _ * Acid ;Icetic acid, HOAc.. . , . . . . . . , . . , . . , , . . . . . . . . , . . , . . . . . . . . . , . , . . . . . . . Cacodylic acid (diinethylarsonic acid), HCac. . . . . . , , . . . . . . . . , . . . . . . , Verona1 (diethylbarbituric acid), HV, . . . , . . . . , . . . . . . , . . , . . . . . . , . . , . , Glycine , . . . . . , , . , . , . , . . . , . . . . . . . . , , , , . . . , . . . . , , . , . . . . . , . . , . . , , . . . , . . . . , . . . ~
x
~
pZi9s"
4.7 6.3 7.9 9.7
The electrophoretic mobilities of human y1- and 72-globulins a t ionic strengths of 0.01 and 0.10 are plotted in figures 3 and 4 as a function of pH. The boundaries for both of these proteins became diffuse rather rapidly because of electrical heterogeneity, and human 71-globulin is resolved into two components below pH 5. However, part of the deviations of the experimental values from a smooth curve is undoubtedly due t o variations in the effects of the various types of buffers on the electrophoretic mobility.
119
\.ARIATION O F ISOELECTRIC POINT WITH IONIC S T K E S G r H
Discussion I t is tjo bc emphasized that the isoelectric points obtained for y i - and y2globulins from tigures 3 and 4 represent average values, because botli of these proteins slinu. considerable reversible elec,trophoret'icspreading a t the isoclect,ric +€I
c7 +6
+5 +4
+3
+2 no
-
9I
X
>
I2 m
* -I
0
-2
-3 -4
-5
-6
-7 -8
4.0
5.0
6.0
7.0 PH
9.0
9.0
Frc. 3. 1,:lcctixqhoi,ctic iiiobility of n o l m d human 71-globulin as ionic s t r r n g t l i . 0 : 0.10 ionic st,rength; 0 , 0.01 iniiic sti,eiigtli.
it
10.0
fuiictJioii of pH atid
point (1, 2, 3 ) . 111 the rase of these particular proteins t'lie mobility distril)ution at the isoelectric point may be adequately represented hy n Gaussian function, and since t'he slope of the mobilit,y-pH curve is linear in the neighborhood of the isoelectric point,, the isoelectric point distribution may also be represented by a Gniissian t'unct'ion. The lieterogeiieity constant I) is defined as the st'andard
120
ROBERT .I. I L B E R T T
deviation of the mobility distribution. If the slope of the mobility-pH plot at the average isoelectric point, pl,,, is dzg/dpH, R yglobiilin moleciile with a mobility of h a t this pH mill have an isoelectric pH of PI,,:
t6 $5 +4
+3 +2
0 X
>
t
+I 0
2
-I
0 5
-2
rn
-3 -4
-5 -6 5.0
4.0
8.0
7.0
6.0
9.0
10.0
P" FIG.4. Electrophoretic niobility of norninl liuinaii y:-globulin aR a function of pH and 0.10 ionic strength; , 0.01 ionic sti,ength. ionic strength. 0, TABLE 2 Isoelechric point distTibution for normal humatr 71-and r.-ylobulins ul 0.10 ionic slrenglh
____
-I
yl-Globulin.. . . . . . . . . . I r,-Globulin. . . . . . , . . . . ~
cm.2 z'oii-1 sec.-l p H - I
5.7 7.3
-0.9 -1.2
X
x 10-5
1
1 ~
cnr.?
roi1-1 sec.-l
~
0.2G X (3) 1 0.50 x 10-5 (2)
1
0.29 0.42
The calculations of the standard deviations of the isoelectric point distributions, p l ~ ,- pl,,, for normal human 71- and rz-globulins a t 0.10 ionic strength are given in table 2 and the isoelectric point distribution curves are plotted in figure 5 .
121
VARLiTION O F ISOELECTRIC POINT WITH IONIC STRENGTH
There is only slight overlapping of t,he isoelectric point distributions for these two proteins. The rather broad isoelectric point distributions would indicate that the average isoelectric points might be expected to depend upon the fractionat'ion method used, because molecules of high or low isoelectric point might he selectively precipitated. The protein samples studied here represent' as large a. fraction of the antmibodyglobulins as we have been able to isolate from plasma wit'hout reworking residues (7, 8). The average isoelectric points of the normal human yglobulins are compared ivit'h those for animal 7-globulins in t,able 3. The buffer used for the isoelectric point determination is given in column 3, and in case the isoelectric point was obtained by interpolation between mobilities determined using two different buffer systems, both salts are listed. The isoelectric values for the normal human 1.5
T
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
PH
FIG.5. Isoelectric point distributions for nornial human ionic streiigth calculat,ed from the data in table 2.
71- and
r?-gloliuliris a t 0.10
and r2-globulins are similar to those obtained for the corresponding horse and cow proteins, but are considerably higher than the values obtained by Tiselius and Kabat (23) for animal antipneumococcus antibodies which were obtained from salt-dissociated specific precipitates. It is possible that the pneumococcus antibodies represent proteins at the lower end of the isoelectric point distribution or that the carbohydrate antigen had not been completely dissociated and lowered the isoelectric point of the protein-carbohydrate complex because of its acid groups. The change in isoelectric point with varying ionic strength in buffers containing univalent ions is somewhat larger than has been observed for other proteins (6, 21, 24), less than obsewed for aldolase (26), and in the same direction, that is, increasing the ionic strength lowers the isoelectric point.4 In studying ovalbumin by microcataphoi.esis experiments, Smith (21) found that although yl-
tlic
I n buffers containing multivalent cations, Smith (21) has observed the elevation of isoelectric point with increasing ionic strength.
122
ROBERT 9. ALBERTY
the isoelectric point obtained at, n given ionic strength depended upon the specific salt present, for each salt the plot of isoelectric pH versus ionic strength mas linear and the limit a t zero ionic strength was the same for all salts. ELECTROPHORETIC STUDY O F CRYSTALLIZED BOVINE AND H U M A N ALBUMINS IN
0.16 111
SODIUM CHLORIDE SOLUTIONS
Experimental Determinations of the isoelectric points of crystallized bovine and human albumins were carried out in 0.15 Ji sodium chloride solutions for comparison __
-
TABLE 3 Average isoelectric points
__
-____~REFERENCE
-
_-____
Human
71,
normal.,
i
.
I
Human yn, noriiial Horse
71,
liyperiiiiinuiie
I ---
. 5.8 .
'
0.10 6.6 0.01 7.3 0.10 8 . 2 1 0.01 5.6 0.10
Horsc y2, hyperiininune. . . . . i 7.6 Con. y , , hyperiinmune. , , . . . , 5.9 6.4 Cow y:, h y p e ~ i ~ n n i u n.e. . , . . 7 . 3 i 7.5 Horsc, H,iitipiieuniococcusS. , 4.4 Corv, :tntipiieumococcusS.. . . ' 4 . 8 Pig, antipnemnococcus~... . 5.1 Rabbit, aiitipneumococcusS. 5 . 8 Rabbit, norinals.. . . . . . . . . , . 5.S ~
~
~
.I ~
~
,
0.10 0.10 0.01 0.10 0.01 0.17 0.17 0.17 0.17 0.17
~
_
--
NaCac, NaOAc, NfiCI* NaCac NaCac, NaCI* NaV NaCac, KaOAc, ISaCI* NaV, NaC1* NaCac, NaOAc SaCI* YaCnc NaV, NaC1* NaV, XaCac NaCI, XaAct NnC1, NaAct NnC1, XnAct NaCI, NaAct NaCl, NaAct
_
~~- _ _ -
Alberty IDeutscii and Nichol (9)
J
i
Hess and Deutsch (11)
aid 1Tiselius Kabat
(23)
I
'+ Socliuin chloride inakes up 80 per cent of ionic strcngth.
7 Sodiuin chloride makes up 88 per cent of ionic strength. $ Salt-dissociated specific precipitate. Isolated electrophoretically.
with 1 lie osmotic pressure minimuin determined by Scat'chard, Batchelder, and Bro\\.n (191. It, \\.as desirable not to add other salts because of their specific hillding elyeuts ( l G ) , and so the only buffering capacity \vas that of the albumin. The loriflering capacity of solutions of bovine albumin in 0.15 df sodium chloride may be calcdated from the titration curve (19), and for a 2 per ceht protein solut'ioii the buffer capacity, p, is 0.0028 a t pH 5.5 and 0.0080 a t pH 4.5. This may be compared with the buffer capacity of a 0.01 ionic strength acetate buffer of pH 4.7, p = 0.012. The 2 per cent protein solution and 2 1. of 0.15 M sodium chloride solution were adjusted t o the desired pH by slow addition of 0.01 N hydrochloric acid or sodiuni hydroxide, and the protein solution was dialyzed for 4 days before electrophoresis. The pH values of the dialyzed protein solu-
\7.-\RIITIOS O F 1SOELE;CTBIC P O I S T WITH IOlcIC STRENGTH
123
tions and ot' samples of t'lic protei11 solutions remo\*etl from the electrophoresis cell aftw elcct'roplioresis \yere measured : i t 23"C., using n glass electrode. The t'wo pH's in 110 case differed appreciably. The fact, tlint the salt8soliltion above t'he boundary is not significantly buffered dues not seem to he important when the inobility is calculated from t8hedescending boundary, sinw the Irelocity of tthe cent8roid,zlordinate of this lioundary is det~ermiiietlby the mo1)ilit)y of {;heprotein molecules in the hod>. of the proteiii solution \rhiFli is hiiffcwtl. Discl1ssLo;l Crystallizccl huvine albumin is yesolved into three components in elec trophoresis in 0.15 JI sodium chloride at pK -4.15, as illri.;tratecl by figure 6. The components htt1.e been designated 1, 2, and 3 in orclcr of increasing isoelectric point. ('omponciit 1 ninlrcs up 37 per cent of the area, cnmponent 2 33 per cent,
-- I
DESCENDING
'
A S C E N 0I NG
FIG.6. Electropliui~eticpattern of a 2 per cent. solution of crystallized bovine albuiiiiri a t pH 4.15 in 0.15 ill sodium chloride after 185 iiiiii. electrophoresis n t a potential gradient of 4.26 volts,/mi. The .\w?icnl line a t thc eiid of the arroIvs indicates the initial houi1dar.j. posi t,ion.
a i d component 3 10 per cent .5 I t is possible to identify these components in the isoelectric region because of their differing amounts, and the mobilities obtained ase plotted in figure 7 . At pH 4.42 and 4.52 the resolution was het,ter in the limb of the U-tube in \rhich component 3 descended; and since the mobilities of component 1 and 2 were calculated from ascending boundaries, and are therefore somewhat high, the mobility curves are indicated by dashed lines. I n such experiment's there is frequently considerable deviation from enanti0graph.y bet\veen the ascending and descending patterns, as illustrated by figure G . Leutscher (17) has obsen-ed the resolut'ion of carbohydrate-free horse albumin and hunian albumin into tiro components in 0.02 ionic strength acet,ate buffer of pH 4.0. Crystallized boT-ine albumin also yields two moving boundaries in this buffer. Further evidence of the elect,rophoretic inhomogeneity of crystallized albumin is obtained in 0.01 d l sodiuni chloride solutions iit pH's allialine to the isoelectric point. In this case two moving boundnries are obtained in t'he ascending pattern. Resolution is not obtained on the descending side, A stimplr of cq.stallizet1 lmviiic
froin w l i i v l i fatty :wit1 lint1 Iieeti extracted t h e cold, siron-et1 tlir m i i w coinpotieiiis i i r pH 4.1s a n d 0.15 .lI .sodiuiii cliloi~irleivitli 19 ])PI' cent iiioi'c of conipoiirrit 3 . 5
dliiiiiiiii
1)y Dr. \\-. 1).Hughes, J r . , using iiietiintiol
it1
124
ROBERT 1. .iLBERTT
probnbly because of the rapid blurring of the boundary due to the conductivity effect. Hoch and nrlorris (12) have previously observed part ial resolution of human albumin a t pH 8.0 in 0.10 ionic strength phosphate buffer in the ascending limb in long experiments (up t o 2G hr.), and Svensson (22) had observed that the descending albumin boundary \vas aln-ays split into two in sodium fluoride +6
4-5 +4
+3 f 2 v)
2
+I
x
2 - 0
!z -1 ;
-I
0
I
-2 -3
-4 -5 -6
4.0
4.5
5.0
5.5
6.0
6.5
7.0
PH
Fro. i . Electrophoretic mobility of crystallized bovine albumin as a function of pH i n 0.15 ,?I sodium chloritle. The dashed lines indicate 1nol)ilities calculated from ascending boundaries.
buffers at pH i . 7 (total ionic strength, 0.20; ionic strength due to phosphate, 0.02), whereas i t was not split in buffers containing sodium chloride. The average isoelectric points of components 1, 2, and 3 are pH 4.23, 4.40, and 4.65, respectively. The average isoelectric point of the \vhole protein, interpolated from moliilities calculated from the centroidal ordinates (14) of the yefractive index gradient curves, is p H 4.40. This may be compared with the pH of minimum osmotic pressure, pH 4.55, determined by Sratchard ct al. (19)6 for hovine albumin. 6 Since tlie osmotic pressure measurements were carried o u t nt 25°C'. while tlie electrophoretic nirasui'rmeiits were carried out at 0°C. arid the pH iiieasuiecl :ti 2j0C'., :I sniall correct i o n for the temperature difference should be applied.
V;\RI.ITIOK
O F ISOELECTRIC" POINT IVITH IONIC STRESGTH
125
Longs\\-orth and .Jacobsen (15) have sliown that the isoelectric point of crystdlized boi.ine ~lburiiinis raised by substituting sodium acetate for part or all of the sodium chloride atj const,ant ionic strength. The isoelectric point is also higher a t 1ou.er sodium chloride concentrations. For example, in 0.01 JI soclium chloride, the average isoelectric point is approximately p H 4.8. As the salt concentration is decreased, the isoelectric point approaches the isoionic point at, zeixo ionic strength (20). The fact that increasing the sodium chloride concentrat ion lowers tlie isoelect'ric point' suggests that chloride ions are b o u ~ d more st,rongly by albumin than sodiiim ions in this pH i'nnge, and is in agreement with the fact that the pH of an electrodialyzed solution of a l h n i n is raised hy the addition of sodium chloride (19). The elect'rophoretic pattern of crystallized human albumin is sonie\\-hat different' from that of crystallized bovine albumin in the isoelectric region in 0.15 JI sodium chloride. The hiiman ulbumin is resolved into only tn.0 components, having the mobilities of component's 1 and 3 of bovine albumin. The average isoelectric point' of the crystallized human albumin in 0.15 J I sodium chloride is pH 4.40, in agreement, with t8hat of crystjallized hovine albumin. SUMMARY
The average electrophoretic mobilities of normal human 71- and y2-globulins have been determined a t 0.01 and 0.10 ionic strengths over the pH range 4-10. The isoelectric point distributions have been calculated from the heterogeneity constants determined from reversible electrophoretic spreading studies and du 'dpH. The average isoelectric points of these proteins are n marked function of ionic strength. Electrophoretic studies of crystallized bovine and human albumins have been carried out in 0.15 111 sodium chloride solutions. Although the electrophoretic patterns of these proteins are somewhat different in the isoelectric region, their average isoelectric points are both pH 4.40. The author is indebted t o Dr. ,J. TV. Williams for helpful suggestions and encouragement in this I\ orl; and to AIrs. R. McGilvery for technical assistance. Financial assistance was receii.etl from the National Research Council and the National Institute of Health. REFERENCES . ~ L B E R ~ ' YR, .
Cheiii. SOC.70, 1675 (1948). ALBERTT,R. A , , .\!iDF,RSOS, E. .i.,.ISD WILLIA~IIS, J. W.: J. P I ~ ~ Colloid s. Chein. 62, 217 (1918). ANDERSOS,E..&I.,.kSD . i r . B E R T T , R . -4.:J . Phys. Colloid Cheiil. 52, 1345 (19481. COHN,E. J., HITCHES, \T. L., JR.,.ISD WEARE,J. H.: J. Am. CIlieni. SOC. 69, l i s 3
.4.:,J.
.\in.
(194i).
E . , J . , S T R O S C i , L. E.. H I T G H E S , L., .JR., ~ I U L F OD. R D , . \ S H \ Y O R T t t , J. N . , ~ I E L I XAI., , . \ N D T A Y L OHR. ,L . : J . Ani. Chriii. SOC.68,160 (1943). Dai-ts, 13. D . , .ASD C(OHK. E . J . : .J. .\in. Cheni. SOC.61, 2002 (1039). DEVTG(~ H.HF., , GOHT~SG L., J . . A L B E R T Y ,R . A . , A S D W I L L I A R I.J. S , \V. : J . Biol. Clieiii. 164, 100 (1940). DISI.TSC,H, II. F . , .L\I.BERTT, R . A . , t s n C ~ S T I S C , L. J.: J. Biol. Clieiii. 166, 21 (1946). C'OHI\.,
S J . ,
12G
L . G . LONGSTVORTFI ;\ND C. F. JACOBSKT
-4K ELECTROPHORETIC S'L'CDT O F THE BISDISG OF SALT IONS BY 8-LACTOGLORTTLI?\' -\SD ROT'ISE SERUM .lT,RU1\IINL I,. Cr. I.OS('rSWORTII h l i o , ~ c t l o ii r s
(I,/'
7'he
.\SI)
C ' . F. .JACOBSEN'
ftialilrtlc ,foi, .lf(,dkxl h't'sw,.ch, .Tt?i/'I'orb, Nvrcr York
~ ~ ~ ) f , ~ , t , , ~ ' i ~ ~ ~ ( , l ,
R~'wic'c:t/.4 ' I ( yrtst 19, 2948
The iiiit,ial objective of this investigation was the nieasurement of the isoelectric pH ~ ~ ~ l iofi e$-lactoglobulin s as a function of the ionic strength of the buffer s o l ~ w iincl ~ t the nntiire of added salt, ions. I n contrast t o their experience with egg olhumin, Caniiaii, Palmer, and IZibrick (4) found that the pH of a p-IactogloLuliii solution is independent of t,he concentration of added potassium chloride :tiid is identicnl ivith the isoelectric pII value obtained by Pedersen (14). On the basis of these oi,ser\rat'ions they suggest,ed that t'he isoelectric pH values of this proteiu might prove to be independent of the ionic strength. Consequently we \\.ere sonie\dmt surprised to find that the isoelectric, pH of P-lactoglolnilin changed wit'li the concentration of t8heacetate buffers in much the same manner that, 'I'iselius and SI.ensson (21) had observed in the case of egg albumin. We then stiltlied l)o\,ine seruin albumin and found that this protein behaves similarly to egg albnniin and p-lactoglobulin. Finally, in an attempt t o learn
