Equine and bovine serum proteins have recently been fractionated by means of a physical method utilizing an electrophoretic adaptation of the principles of the Clusius column (l-4), first described and tested by Kirkwood (5) and Nielsen (6). The method of electrophoresis-convection has now been applied to the fractionation of human γ-globulin. The γ-globulin was prepared by ethanol fractionation (7) from the plasma of individuals hyperimmunized to Hemophilus pertussis organisms. The resulting fractions of γ-globulin have been characterized electrophoretically, and the protective antibody activity and agglutinin titer have been measured

Brown, Raymond A.

Cann, John R.

Hink, John H., Jr.

Kirkwood, John G.

English

Caltech Authors - Main

FRACTIONATION OF HUMAN IMMUNE r-GLOBULIN 
BY JOHN R. CANN, RAYMOND A. BROWN,* AND JOHN G. 
KIRKWOOD 
(From the Gates and CrelEin Laboratories of Chemistry,? California Institute of 
Technology, Pasadena) 
AND JOHN H. HINK, JR. 
(From the Cutter Laboratories, Berkeley, California) 
(Received for publication, February 18, 1950) 
Equine and bovine serum proteins have recently been fractionated by 
means of a physical method utilizing an electrophoretic adaptation of the 
principles of the Clusius column (l-4), first described and tested by Kirk- 
wood (5) and Nielsen (6). The method of electrophoresis-convection has 
now been applied to the fractionation of human r-globulin. The r-glob- 
ulin was prepared by ethanol fractionation (7) from the plasma of indi- 
viduals hyperimmunized to Hemophilus pertussis organisms. The result- 
ing fractions of r-globulin have been characterized electrophoretically, and 
the protective antibody activity and agglutinin titer have been measured. 
The material used in this investigation was Fraction II of pooled immune 
human plasma prepared by the Cutter Laboratories from hyperimmunized 
donors. The apparatus and experimental procedures used in the fraction- 
ations reported here were essentially the same as those described for the 
fractionation of bovine r-globulin (3, 4). Fractionations were carried out 
in phosphate buffer, ionic strength 0.1, at a field strength of 1.7 to 2.0 
volt per cm. for 48 to 71 hours. The initial protein concentration was 2.0 
to 2.8 gm. of protein per 100 ml. Fractions possessing different mean 
mobilities and isoelectric points were obtained by proper choice of the 
operating pH. 
The agglutinin titers of the fractions were obtained by the Flosdorf 
method (8) wivith two different antigen strains. Protective activities of 
the fractions were determined by mouse protection tests. In these tests, 
the determinations of the virulence of the challenge and the protective 
activity of the fractions were carried out simultaneously in order to rule 
out any change in virulence of the culture. Each of the fractions was 
injected subcutaneously at doses of 10, 2, and 0.4 mg., with fifteen mice at 
each level. 6 hours following immunization, a challenge dose of virulent 
H. pertussis organisms was administered to each mouse by intracerebral 
injection. The virulence of the challenge was determined on forty-eight 
* Postdoctorate Fellow of the United States Public Health Service, National 
Institutes of Health. 
t Contribution No. 1394. 
663 
664 FRACTIONATION OF Y-GLOBULIN 
mice, twelve mice being tested for each of four dilutions. Death and 
survivals were recorded after 10 days, those mice substantially paralyzed 
on the 10th day being considered as dead. The results were analyzed 
by the method of Reed and Muench (9). 
A sample of r-globulin obtained from the plasma of donors hyperim- 
munized to H. pertussis organisms was carried through three stages of 
fractionation. The scheme used in this fractionation may be briefly des- 
cribed as follows. In the first stage the y-globulin was fractionated at 
pH 8.10. The fractions withdrawn from the top and bottom reservoirs of 
TABLE I 
Fractionation of Fraction II, Sample 1 
Fraction Perocfent 
original 
r-globulin 
Unfractionated r-glo- 
bulin. .............. 
Top 1.. .............. 24 
(( 2 ................ 27 
(’ 3 ................ 17 
Bottom 3 ............. 32 
1.27 
0.88 
1.23 
1.60 
1.44 
7.55 0.78 
,7.86 1.17 
7.47 1.00 
6.96 0.55 
7.28 1.32 
- 
Protective 
activity,t 
IDzs 
per mg. N 
1.4 (1.8: 
5.0 (2.7; 
1.3 
2.1 
1.9 
Agglutinin titeri 
against 
1:1280 1:1280 
1: 1280 1: 1280 
1:640 1:1280 
1:640 13340 
1:640 lxx0 
* The mean mobilities were determined in barbital buffer, pH 8.6, ionic strength 
0.1, and protein concentration 1 per cent. 
t The mean isoelectric points were determined in cacodylate-chloride buffer, 
ionic strength 0.1, and protein concentration 0.5 per cent. 
$ The challenge dose was 100,000 organisms or 250 LD~o. The values in paren- 
theses are results of check assays with 100,000 organisms or 910 LD60, 
8 The titers are expressed as the titers of 2 per cent solutions of the fractions. 
the electrophoresis-convection apparatus are designated as Top 1 and 
Bottom 1, respectively. Bottom 1 was then refractionated in the second 
stage at pH 7.78, the resulting fractions being designated as Top 2 and 
Bottom 2. In the last stage of fractionation, carried out at pH 6.50, 
Bottom 2 was separated into two fractions, Top 3 and Bottom 3. The 
electrophoretic and immunological data obtained on the resulting fractions 
are presented in Table I, where fi is the mean mobility at pH 8.6 and 
Ahii/ApH the slope of the mobility-pH curve in the neighborhood of the 
mean isoelectric point. This fractionation yielded four electrophoretically 
unique fractions of r-globulin. These fractions possess a mean mobility 
spectrum ranging from -0.88 X lOA6 to -1.60 X lo+ cm.2 sec.-l volt? 
at pH 8.6 in barbital buffer and a mean isoelectric point spectrum from 
7.86 to 6.96 in cacodylate-chloride buffer. 
CANN, BROWN, KIRKWOOD, AND HINK 065 
The results of the assays for protective antibody show that, although 
all of the fractions possess protective antibody, more protective activity 
is associated with the fraction of lowest mobility, Top 1, than with the 
other fractions and the unfractionated y-globulin. The conclusion that 
Top 1 possesses more protective activity than the unfractionated material 
TABLE II 
Fractionation of Fraction II, Sample 9; Electrophoretic and Immunological Properties 
of Fractions 
FIdiOIl 
Unfractionated r-globulin. . 
Top 1.. .................... 
Bottom 1.. ................. 
Top4.. .................... 
u 5 ...................... 
‘( 6. ..................... 
u 7 ...................... 
Bottom 7 ................... 
- 
-_ 
- 
10 
21 
23 
17 
29 
MeWi Mean 
mobility,* isoelectric 
-10’ x a pointt 
cm.~ sec.- 
OOll’ 
1.26 
0.96 
1.37 
1.28 
1.02 
0.96 
0.87 
0.91 
_- 
- 
fJa 
6.90 
7.15 
7.28 
7.36 
7.46 
-7 - 
- 
- 
101 x l3t 
0.53 
Protective 
activity,0 
IDso 
mg. E 
1.3 
2.1 
<0.9 
1.6 
2.0 
2.8 
2.1 
* Mean mobilities were determined in barbital buffer, pH 8.7, ionic strength 0.1, 
and protein concentration 1 per cent. 
t The. mean isoelectric points were determined in tris(hydroxymethyl)amino- 
methane buffers, ionic strength 0.1 and protein concentration 0.5 per cent. These 
buffers were 0.02 N in the hydrochloride of the amine and 0.08 N in sodium chloride. 
Discrepancies of the order of 0.6 pH unit between isoelectric points determined in 
cacodylate and tris(hydroxymethyl)aminomethane buffers have been found. These 
discrepancies are at least in part due to the different temperature coefficients of the 
pH of these buffers. pH values were measured at 25”, while the isoelectric points 
were determined at 2”. 
$ The standard deviations of the mobility distributions were determined from 
reversible boundary spreading experiments. These experiments were carried out 
in tris(hydroxymethyl)aminomethane buffer at the mean isoelectric points of the 
proteins. 
0 The challenge dose was 100,000 organisms or 690 LDbo. 
is supported by the results of two independent assays for protective anti- 
body. All four fractions gave nearly the same agglutinin titer against 
two different antigen strains. 
In order to accomplish still further enrichment of protective antibody, 
a five stage fractionation of a different sample of r-globulin from hyper- 
immunized donors was carried out. The results of the electrophoretic and 
immunological characterization of the various fractions are presented in 
Table II, where ii is the mean mobility at pH 8.7 and ,13 the standard 
666 FRACTIONATION OF ‘y-GLOBULIN 
deviation of the mobility distribution in the neighborhood of the mean 
isoelectric point. 
The first stage of fractionation was carried out under the same experi- 
mental conditions as were employed in the initial stage of the first, frac- 
tionation. A composite of the top fractions obtained in ten separate 
experiments was made. Electrophoretically, this composite resembles Top 
1 obtained in the first fractionation as closely as can be expected, consid- 
ering possible differences in the two samples of unfractionated r-globulin.* 
Accordingly, this composite has also been designated as Top 1. The im- 
munological assay of the composite Top 1 confirms the previous finding 
that more protective activity is associated with the low mobility fraction 
of y-globulin than with the unfractionated material. 
Subsequent stages of fractionation were designed to separate Top 1 into 
subfractions. The composite Top 1 was fractionated at pH 6.38, the 
resulting fractions being designated as Top 4 and Bottom 4. Bottom 4 
was then fractionated at pH 7.10 to yield Top 5 and Bottom 5. Bottom 
5 in turn was fractionated at pH 7.63 to yield Top 6 and Bottom 6. Fi- 
nally, Bottom 6 was separated into two fractions, Top 7 and Bottom 7, 
by fractionation at pH 8.00. The mobilities of the resulting subfractions 
range from -0.87 X 10V5 to 1.28 X low5 cm.2 sec.-’ volt-l at pH 8.7 in 
barbital buffer.2 In agreement with the results of the first fractionation, 
the immunological characterization of these subfractions revealed that 
greater protective activity is associated with low mobility fractions than 
with high mobility fractions. 
The rather broad distribution of protective antibody among the fractions 
may in part be due to the fact that the r-globulin was prepared from pooled 
plasma. Of course, there is some question as to whether the protective 
antibody, if derived from a single donor, might not also exhibit a broad 
distribution. Several factors might give rise to such a heterogeneity. The 
first of these is the fact that H. pertussis immunity is probably given by 
several different strains of bacteria. Also, it is conceivable that both 
bacterial- and toxin-produced antibodies are responsible for immunity. 
1 The fact that the two samples of r-globulin possessed the same mean mobility 
at pH 8.6 does not of course imply that they possessed the same distribution 3f mo- 
bilities. 
* It is of interest that little or no apparent separation was accomplished in the 
last two stages of fractionation, despite the fact that the operating pH of successive 
stages differed by at least 0.4 pH unit. This is somewhat difficult to understand, 
especially in view of the large amount of transport obtained in these runs and the 
fact that the standard deviation of the mobility distribution of Top 6 was found to 
be the same as that of the unfractionated y-globulin. 
CANN, BROWN, KIRKWOOD, AND RINK 667 
SUMMARY 
A sample of human antipertussis r-globulin has been separated into four 
electrophoretically unique fractions by electrophoresis-convection. These 
fractions possess a mean mobility spectrum ranging from -0.88 X 10m5 
to -1.60 X 10m5 cm.z sec.-l volt-’ at pH 8.6 in barbital buffer and a mean 
isoelectric point spectrum from 7.86 to 6.96 in cacodylate-chloride buffer. 
All of the fractions gave nearly the same agglutinin titer against two dif- 
ferent antigen strains. Although all fractions showed protective activity, 
the greatest activity was associated with the fraction of lowest mobility. 
This was confirmed by the fractionation of a second sample of immune 
r-globulin and subsequent subfractionation of the low mobility material. 
The authors are indebted to Dr. Dan Campbell for helpful discussion of 
the immunological significance of the results. 
Addendum-Since preparation of this manuscript, Hink and Johnson (10) have re- 
ported hheir studies on the distribution of antibodies among plasma fractions pre- 
pared by ethanol fractionation (7) from the plasma of individuals hyperimmunired 
to H. pertussis organisms. Their results indicate that the protective activity in 
antipertussis hyperimmunized human plasma occurs almost entirely in the purified 
-yz-globulin, Fraction II. Less than half of the H. pertussis agglutinins was recovered 
in Fraction II, which contained 90 per cent of the rl-globulin of the plasma. The 
remaining agglutinin activity was found in Fraction III-l. Their Fraction II was 
the material used in this investigation. 
BIBLIOGRAPHY 
1. Cann, J. R., Kirkwood, J. G., Brown, R. A., and Plescia, 0. J., J. Am. Chem 
Sot., 71, 1603 (1949). 
2. Cann, J. R., Brown, R. A., and Kirkwood, J. G., J. Am. Chem. Sot., 71, 1609 
(1949). 
3. Cann, J. R., Brown, R. A., and Kirkwood, J. G., J. Am. Chem. Sot., 71, 2687 
(1949). 
4. Cann, J. R., Brown, R. A., and Kirkwood, J. G., J. Biol. Chem., 181,161 (1949). 
5. Kirkwood, J. G., J. Chem. Phys., 9, 878 (1941). 
6. Nielsen, L. E., and Kirkwood, J. G., J. Am. Chem. Sot., 68,181 (1946). 
7. Oncley, J. L., Melin, M., Richert, D. -4., Cameron, J. W., and Gross, P. M., Jr., 
J. Am. Chem. Sot., 71, 541 (1949). 
8. Flosdorf, E. W., McGuinness, A. C., Kimball, A. C., and Armstrong, J. G., J. 
Pediat., 19, 638 (1941). 
9. Reed, L. J., and Muench, H., Am. J. Hyg., 27,493 (1933). 
10. Hink, J. H., Jr., and Johnson, F. F., J. Immunol., 84, 39 (1950). 


Fractionation of human immune γ-globulin

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Fractionation of human immune γ-globulin

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