PLANT HEMAGGLUTININS WITH SPECIAL REFERENCE

TO A PREPARATION FROM THE-NAVY BEAN.*

BY VERZ R. GODDARD AND LAFAYETTE B. MENDEL.
(From the Laboratory of Physiological Chemistry, Yale University,
New Haven.)

(Received for publication, February 21, 1929.)

Since the discovery of ricin by Kobert in 1887, considerable

interest has been shown in the hemagglutinins obtainable from
plants. The study of them has been somewhat overshadowed by
the more recently discovered serum agglutinins, which they so
remarkably resemble in action. They have, however, played an
important part in immunological research throughout its history.
Ricin, the extremely toxic hemagglutinin from the castor bean
(Ricinus communis) was used by Ehrlich (1897) in his first in
vitro studies of the reactions between antigen and antib0dy.l
The living organism was no longer to be regarded as an indispens-
able participant in the agglutinin-antiagglutinin reaction.
Much remains to be done before the distribution of the hemag-
glutinins in the plant world can be estimated. From the observa-
tions made by Landsteiner and Raubitschek (1908), Mendel
(1909), Eisler and Portheim (1909, 1926), Wienhaus (1909), and
others, the substances appear to lack wide distribution.2 The
early studies were confined to the toxic agglutinins: ricin, robin,
cretin, and abrin. Later, non-toxic extracts were prepared in

* The data reported in this paper are taken from the dissertation pre-
sented by Verz R. Goddard in partial fulfilment of the requirements for the
degree of Doctor of Philosophy, Yale University, 1927.
r He showed that ricin neutralized by antiricin would not agglutinate
rabbit corpuscles in a test-tube. From quantitative work, he concluded
that this neutralization followed the law of multiple proportions.
2 A detailed summary of the principal plant sources which have been
investigated with reference to the occurrence of hemagglutinins and of the
animal species furnishing the blood corpuscles tested is included in the
dissertation of Verz R. Goddard, Yale University, 1927.
417

This is an Open Access article under the CC BY license.

448 Plant Hemagglutinins

various laboratories. The seeds of leguminous plants are es-

pecially rich in potent products.
As blood cell clumping substances, phytohemagglutinins ap-
parently have no relationship to the activity of the plant producing
them. This is analogous to the well known fact that plants pro-
duce enzymes having no obvious bearing upon the activities of
the structures that yield them. The presence of pancreatic se-
cretin in spinach and of urease in beans are obvious examples.
A possible physiological relationship between the agglutinin and
the plant producing it has been indicated by Eisler and Portheim
(1911, 1912, 1926) and by Schneider (1912). The hemagglutinin
appears to be associated with the storage material of plants.
Upon germination the seed loses its agglutinin which may pass
over to the embryo (Russ and Oesterlin, 1921). It is interesting
to note that certain plants which have reserve material in their
stems, possess an agglutinating milk-sap. This is true of the
Euphorbia studied by Eisler and Portheim. More recently, Mar-
cusson-Begun (1926) has reported an agglutinin in the potato
and has suggested the possibility of the occurrence of agglutinating
substances in other root nodules.
The classic agglutinin-containing seed, that of the castor bean
(Ricinus communis) was studied chemically by Osborne, Mendel,
and Harris (1905), who made a separation of its proteins. Both
the toxic and the hemagglutinative qualities were found to be as-
sociated with the albumin, ricin, a name earlier indicated by
Kobert for the toxic principle. Their method for the purification
of ricin may be used for its preparation on a large scale (Karrer,
Smirnoff, Ehrensperger, van Slooten, and Keller, 1924).
For many purposes a non-toxic agglutinin is of value. Such
a substance might well prove of considerable practical importance
as an aid in the preparation of antisera. In fact, a crude extract
of the pea-bean (Phaseolus communis) is being used in the prepa-
ration of certain of the anti hog cholera sera on a commercial
scale (Dorset and Henley, 1916). It has been shown that where a
clear, corpuscle-free serum is desired, the yield obtainable from
a given volume of blood can be notably increased by the use of
this agglutinin which causes the corpuscles to pack and makes
possible a nearly perfect separation of cells and serum.
The present study involves the preparation of a non-toxic
 V. R. Goddard and L. B. Mendel 449
hemagglutinin in a highly purified form and an investigation of
the mechanism of its action in the hope of making the material
more useful in research as well as for practical, serological work.
The applicability of such a substance is, of course, not limited to
immunological research. It can serve in various capacities in the
study of the physical characteristics of erythrocytes and possibly
of tissue cells in suspension. Wienhaus (1909) has shown that
a bean extract would cause agglutination of pus, liver, and kid-
ney cells.
Preparation of Hemagglutinin and Other Experimental Procedures.
For the preparation of the water-soluble protein fraction of the
bean (Phaseolus communis) the method employed by Osborne,
Mendel, and Harris in the preparation of ricin was closely fol-
lowed. The influence of alterations in the integrity of the protein
upon the hemagglutinating properties was also investigated.
After extraction of the defatted bean meal with sodium chloride
solution (usually 3 per cent), removal of the undesired globulins
by dialysis of the extract, repeated salting out of the desired pro-
tein with ammonium sulfate, and dialysis of the precipitate to
remove inorganic contaminants, a clear, straw-colored filtrate was
obtained which, was evaporated in a current of air to dryness at
3540” on glass plates. The protein was then scraped from the
plates, further desiccated over sulfuric acid, and stored in the
form of a nearly white powder in glass bottles. Upon analysis
the samples, with corrections for moisture and ash, varied in
nitrogen content from 14.5 to 15.5 per cent. The material re-
sponded positively to all the common protein color and precipita-
tion tests. This substance had a high hemagglutinating potency.
A sample which has been stored for over 2 years still shows
(August, 1928) no appreciable loss in activity. Injections of as
much as 8 mg. per kilo into rabbits and 600 per kilo into mice in-
dicated that the material is non-toxic, in contrast to the com-
parable ricin preparation from the castor bean for which a lethal
dose as low as 0.002 mg. has been observed.
Macroscopic Agglutinatipn Method.-Many-macroscopic methods
for detecting agglutination of bacteria have been published and
a plea has been made for standardization of reports of agglutina-
tion tests (Hadley, 1916). Most of these procedures are based
450 Plant Hemagglutinins

upon the Kolmer technique (Kolmer, 1923). Gardner’s gelatin

standard method (Gardner, 1919) and Gates’ capillary tube
method (Gates, 1921) represent special modifications. The diffi-
culty in detecting agglutination appears to arise from the fact
that it is a two phase reaction, the binding of the agglutinating
agent occurring previous to flocculation. Gates (1922) has sug-
gested a method for eliminating this time element in the agglutina-
tion of bacteria. He hastened the process by centrifuging the
organisms in contact with the agglutinin. A similar technique
was recommended by Mudd (1927). The centrifuge method was
found, in the experience of the writers, to give reasonably good
results with erythrocytes also. After centrifugation agglutinated
cells would remain in the bottom of an upturned tube or if ag-
glutination were slight, would stream very slowly in comparison
to the controls.
In an attempt to devise a procedure, however, which would
give completely consistent results in the quantitative study of
hemagglutination, factors known to be significant in clumping
and sedimentation were taken into consideration. Temperature,
diameter of tube and length of liquid column, angle or slope of
tube were regulated to give the best results obtainable. A chance
observation led to the development of a method which proved
invaluable in determination of agglutination titer.
It was found in agreement with Berczeller and Wastl (1923)
that slanting the tube increased the rate of sedimentation. But
it was noted, also, that after erythrocytes had been left in contact
with agglutinin in chemically clean glass tubes, sloped at an angle
of 40” and at a temperature of 40-44”, for 12 hours, the cells
became firmly attached to the glass so that upon returning the
tube to the vertical position they remained unmoved. This pe-
culiar adherence of cells never occurred in the absence of hemag-
glutinin nor when the latter was too dilute to bring about clump-
ing detectable upon microscopic examination.
The ease by which hemagglutination is detected by this method
is indicated in Fig. 1. Note the appearance of tubes in which
the same volumes of rabbit erythrocytes, washed and suspended
in physiological salt solution, were treated with increasing quan-
tities of hemagglutinin. In the tube furthest left and where no
agglutination occurred the corpuscles settled; the other tubes show
 V. R. Goddard and L. B. Mendel 451
clearly the adherence of the red cells characteristic of agglutina-
tion upon the glass walls.
After various modifications had been investigated, the follow-
ing procedure was finally selected as most effective. Rabbit
blood freshly removed from the ear vein and defibrinated was
centrifuged until the serum could be removed. The corpuscles
washed twice with 0.89 per cent sodium chloride solution were
suspended therein. 2.5 cc. of packed corpuscles were suspended
in a total volume of 100 cc. of the isotonic sodium chloride solu-

FIG. 1. Hemagglutination test; one negative (left), four positive agglu-

tinations (right). Tubes contained (left to right): 0.0005, 0.0006, 0.0007,
0.0008, and 0.0009 mg. of hemagglutinin with 0.5 cc. of 2.5 per cent suspen-
sion of rabbit erythrocytes in sodium chloride solution. (Each tube was
made up to a total volume of 1 cc.)

tion. Aliquots of this suspension were used for the individual

tests. As a rule 0.5 cc. of the corpuscle suspensionwas added to
0.5 cc. of 0.89 per cent sodium chloride solution containing varying
amounts of the agglutinin preparation to be tested. The reaction
was carried out in chemically clean glasstubes, 75 X 8 mm., placed
in a rack which would keep them at an angle of 40” and incubated
at 4044”, for 12 hours, Table I indicates the uniformity of
results secured by this method of “agglutination titer” when tests
were repeated on different days, with fresh suspensionsof eryth-
452 Plant Hemagglutinins

TAI3LlG I.
Measure of Agglutinating Activity.

Date of experiment. Minimum effective quantity of hemagglutinin.

1926 VW.
Mar. 14 0.0004
“ 15 0.0006
(‘ 18 0.0006
“ 24 0.0006
“ 31 0.0006
Apr. 9 0.0005
“ 9 0.0006
“ 12 0.0005

The hemagglutinin was added to 0.5 cc. portions of a 2.5 per cent suspen-
sion of washed, rabbit erythrocytes in a total volume of 1 cc.

TABLE II.
Agglutinability of Erythrocytes of Different Species.

Date of experiment. Kind of erythrocyte. Minimum effective concentra-

tion of agglutinin.

1923 mg.
Mar. 15 Rabbit. 0.0006
“ 28 Man. 0.0007
“ 30 “ 0.0008
“ 31 I‘ 0.0007
Apr. 13 ‘I 0.0007
Mar. 20 Duck. 0.0008
Apr. 15 Dog. 0.0008
8‘ 15 “ 0.0008
“ 29 “ 0.0009
“ 29 Mouse. 0.002
May 4 “ 0.002
(‘ 9 Rat. 0.003
Mar. 20 Chicken. 0.006
“
Apr. 17 0.006
“ 17 ‘< 0.006

To 0.5 cc. portions of 2.5 per cent suspensions of the washed erythrocytes
in isotonic sodium chloride were added 0.5 cc. portions of isotonic sodium
chloride containing the quantity of hcmagglutinin to be tested.
 V. R. Goddard and L. B. Mendel 453

rocytes. In Table II the results of comparing the agglutination

titers of different species of animals are shown.
From Tables I and II it can be seen that the dilution at which
the agglutinin is still active is, for rabbit erythrocytes, 1 to
6,000,OOO or by analogy with ordinary immunological terminology
this figure could be called its titer, since clumping is effected in a
total volume of 1 cc. of solution containing only 0.0006 mg. of
agglutinin by dry weight. It is active in lower concentration
than the purified bean agglutinin “phasin” as reported by Wien-
haus in 1909. His product was, however, only partially soluble,
having suffered considerable denaturation during the process
of preparation. It seems probable that the soluble fraction of
his material was highly active. In contrast to Wienhaus’ phasin,
the bean agglutinin reported here gave remarkably similar results
with the washed corpuscles of the various species tested-es-
pecially with those of man, rabbit, dog, and duck. The eryth-
rocytes of the hen, more difficult to clump than the other species
tried, required only 10 times as much agglutinin as was necessary
for rabbit.

In&hence of Medium upon Hemagglutination.

In&Siting Factors.-Not only artificial media, but the natural
medium of the erythrocytes, i.e. blood serum, shows a strong in-
fluence upon agglutination of the corpuscles. This was noted by
Landsteiner and Raubitschek in their early work. Raubitschek
(Kraus and Levaditi, 1911) was of the opinion that the inhibiting
influence of serum is due to the presence of its proteins. He
noted the selective affinity of erythrocytes for certain substances,
such as peptones and albumoses. Agglutinated cells shaken and
incubated with peptone solution became unagglutinated. Our
own observation supports Raubitschek’s conclusion regarding the
retarding influence of protein material upon the agglutination re-
action. Table III indicates the effect of egg white upon the action
of the agglutinin preparation. In these experiments the standard
method already described was not employed. Whole defibrinated
blood in 0.1 cc. portions was treated with solutions of bean hemag-
glutinin dissolved in 0.89 per cent sodium chloride in a total
volume of 1 cc. Column 1 shows the effect of the hemagglutinin
alone; the results in Column 2 were secured when egg white was
454 Plant Hemagglutinins

introduced just after the addition of the hemagglutinin, whereas

in experiments in Column 3 the egg white was added just prior
to the addition of hemagglutinin. When the corpuscles were first
treated with egg white they failed to agglutinate in the presence
TABLE III.
InJEuence of Egg White upon Agglutination of Whole, DeJibrinated Blood.

Effect upon hemagglutination.

Blood +&;magglu- Blood + hemagglu- Blood + 1 cc. egg white

tinin + 1 cc. egg white. + hemagglutinin.
(1) (2) (3)

mg.
0.05 - - -
0.06 + - -

0.07 -
+ +
0.08 + + -
0.09 + + -

TABLE IV.
Inhibitory Influence of Serum upon Hemagglutination.
- 7-
I Winimum effec-
Kind of
Suspension. erythrocytes. 1,ive quantity of
agglutinin.
_-
vol. per cent cc. w.
2.5 Rabbit. None. 0.0006
2.5 “ Rabbit. 0.012 0.002
2.5 “ Turtle. 0.012 0.006
5.0 “ Rabbit. 0.025 0.003
5.0 Duck. None. 0.0008
5.0 “ Duck. 0.025 0.002
5.0 “ Hen. 0.025 0.006
- -
0.5 cc. portions of a suspension of washed corpuscles were used in a total
volume of 1 cc. with and without additions of equal volumes of serum, the
suspension being diluted with isotonic saline solution to a total volume of
1 cc. Before dilution to final volume, serum was added in a proportion
equivalent to the actual volume of the corpuscles.

of far greater quantities of hemagglutinin than were readily effec-

tive without the egg white. When the egg white was added after
the corpuscles and the agglutinin had had even a brief opportunity
to act the influence was less marked.
 V. R. Goddard and L. B. Mendel 455

The effect of adding traces of ‘blood serum to washed corpuscles

can be seen from Table IV, a summary of a series of experiments
in which washed corpuscles suspended in saline were compared
with those to which serum of the same or foreign species had been
added. Serum of the turtle and the hen were particularly in-
hibitory. For rabbit serum the ratio between the requisite

Injluence of Sodium Chloride upon Hemagglutination.

-
Date of experiment. Erythrocyte suspen- Agglutinin added. Minimum effective
sion. weight of N&l.

199s vol. per cent VW. ml.

Apr. 10 5.0 0.002 >0.7
“ 10 5.0 0.005 0.4
“ 10 10.0 0.005 0.4
May 15 2.5 0.02 0.3
“ 17 2.5 0.03 0.2
“ 17 2.5 0.04 0.2
“ 16 2.5 0.05 0.2
Apr. 9 2.5 0.05 0.5?
May 17 2.5 0.08 0.2
“ 17 2.5 0.08 0.2*
“ 16 2.5 0.1 0.2
“ 17 2.5 0.1 0.2
“ 16 2.5 0.15 0.2

Summary of a series of experiments in each of which the amount of

hemagglutinin was kept constant and sodium chloride was varied in differ-
ent test samples until agglutination was obtained. Samples consisted of
0.5 cc. portions of a suspension of washed corpuscles in isotonic sucrose,
diluted to a total volume of 1 cc. with an isotonic mixture containing known
weight of hemagglutinin and the quantity of sodium chloride to be tested.
*After the corpuscles of this series had been allowed to react with agglu-
tinin for 4 hour, they were washed with sucrose solution to remove excess
agglutinin, resuspended in sucrose solution, and titrated, with the result
indicated.

amounts of agglutinin for clumping equivalent volumes of eryth-

rocytes in whole, diluted blood and in sodium chloride solution
was approximately 5 : 3.
Favoring InJluence.-Electrolytes are of course factors in the
medium essentialfor hemagglutination. Rona and Gyorgy (1920)
concluded from their work on the influence of electrolytes upon
456 Plant Hemagglutinins
ricin agglutination that a definite cation series could be estab-
lished by arranging the salts in the order of their values. With
the hemagglutinin reported here, it was found that when the
concentration of electrolyte was greatly lowered, excessively large
amounts of agglutinin were required. Thus, corpuscles washed
three times in 0.25 M sucrose solution required 10 times as much
hemagglutinin as those washed and suspended in isotonic sodium
chloride solution. After washing the cells twelve times in isotonic
sucrose solution, it was no longer possible to cause agglutination
with any concentration of protein used.
In order to study the relationship between concentration of
hemagglutinin and quantity of electrolyte, a series of media was
prepared containing mixtures of varying concentrations of sodium
chloride and sucrose selected to keep them isotonic with rabbit
erythrocytes. These were used in a series of “titration experi-
ments” summarized in Table V. To 0.5 cc. portions of a 2.5 per
cent suspension of rabbit erythrocytes in isotonic sucrose were
added 0.5 cc. portions of sucrose solution containing the amount
of agglutinin indicated with varying quantities of sodium chloride.
Table V records the minimum weight of sodium chloride in the
presence of which agglutination is brought about in each case.
The “titration” was not carried far enough to determine the
concentration of sodium chloride necessary to permit agglutina-
tion of the corpuscles with 0.002 mg. of protein. It was greater
than 0.72 mg. of sodium chloride. As the weight of hemag-
glutinin increased, the requirement for sodium chloride decreased
until the value of hemagglutinin reached a certain level, i.e. 0.02 mg.
Beyond this value, the quantity of sodium chloride required was
shown to be constant-O.2 mg. Isotonic salt solution would con-
tain in an equal volume about 10 times this concentration of
sodium chloride.

Chemical Alteration of the Protein.

The question as to whether the hemagglutinating property is
dependent upon the integrity of the protein has been approached
by a study of the effects of chemical changes upon the latter.
Heat Coagulation.-The preparation studied could be denatured
by heat when in solution. Below 68” the protein did not separate
from aqueous solution. With slow heating, coagulation would
 V. R. Goddard and L. B. Mendel 457

begin at 68”, but the greater part of the coagulable protein sepa-
rated out between 75” and 81”. The filtrate from the coagulum
at the higher temperature, showed a greatly lowered potency to
agglutinate rabbit erythrocytes. At 100” coagulation became so
complete that no agglutinating power remained. Heating the
dry preparation to the same temperature was without influence
upon solubility or potency of the material unless the heating

TABLE VI.

InfEuence of Peptic Digestion upon Activity of Hemagglutinating Protein.

Minimum effective
quantityt;;iemagglu-

mg.
Untreated protein..................................... 0.0008
0.0008
Dissolved in pepsin-HCl and neutralized at once.. . .
i 0.0007
After 1 hr. digestion.. ... .. ... ... ... ... .. .. 0.002
‘L
14 hrs. “ . .. . . . . . . . . . . . . . . . . . ... ... ... ... . 0.004
“ 3 days “ . .. . . . . . . . . . . . . . . . . . ... ... .... ... 0.007
“ 6 “ “ . .. . . . . . . . . . . . . . . . . . ... ... .... ... Activity lost.

To 0.1 gm. portions of the protein weighed out exactly and placed in
test-tubes were added 2 cc. of 0.1 N HCI solution and 2 cc. of 1 per cent
pepsin solution with 2 cc. of water. The tubes were stoppered and incu-
bated in a constant temperature water bath at 37.5” for the periods of time
indicated in the summary. At the end of the incubation period, the solu-
tion was exactly neutralized by adding the calculated quantity of sodium
hydroxide. The sodium chloride content was increased to make the solu-
tion exactly isotonic to rabbit erythrocytes after dilution to 20 cc. with
water. Further dilutions were made with isotonic sodium chloride and the
agglutinating potency of the preparations with rabbit erythrocytes were
determined by the method already outlined. The minimum effective
weight of hemagglutinin was expressed in terms of dry weight of the protein
before digestion.

process was continued for a number of hours. Then the change

was only slight. After 4 days of heating at 100” the agglutinating
potency was lowered to one-thirtieth that of the original protein.
Wienhaus has reported a similar finding with his phasin; heating
the dry preparation for 2$ hours at 130” caused only a partial
destruction of potency.
Acid Hydrolysis.-Short exposures to weak acid produced no
458 Plant Hemagglutinins

apparent effect upon the protein. When 0.1 gm. portions were
treated with 1 cc. of 0.1 N hydrochloric acid and neutralized within
5 minutes, solubility and agglutinating potency were unaffected.
Upon longer exposures, the protein gradually separated out in an
insoluble form with an accompanying loss in agglutinating value.
Thus after 3 days exposure to 0.1 N hydrochloric acid, the requi-
site weight of protein for clumping a given volume of erythrocyte
suspension was increased to 5 times the amount required before
the exposure to acid.
Peptic Digestion.-This also destroys the agglutinating po-
tency. Hemagglutinating protein was dissolved in pepsin-HCI.
When the solution was immediately neutralized no change in the
minimum effective hemagglutinating dose of the protein could be
detected. With the progress of digestion, however, the agglutinat-
ing activit,y of the solution was lowered; and at the end of a
considerable period of incubation, it seemed to be entirely lost.
The preceding summary of protocols (Table VI) illustrates these
observations.

Mode of Action of Hemagglutinin.

There are indications that the hemagglutinins of plant origin
resemble those of the serum. Comparable reactions appear to
affect both groups of substances similarly. Both serum and plant
hemagglutinins require electrolyte for their action; in both cases,
a “fixation” reaction occurs between the agglutinin and the oor-
puscle prior to the flocculation which is dependent upon some
physicochemical action of salts. By referring to Table V, the
reader will note that rabbit erythrocytes may be washed with
sucrose solution after exposure to hemagglutinin and still retain
enough of the “fixed” material to clump upon addition of sodium
chloride. The electrolyte is needed to complete the reaction.
Wells (1925) has spoken of agglutination as a process of “salting
out” in which electrolytes are of great importance. He points
out further that one might think of the antigen-antibody (or
agglutinin-corpuscle) reaction as resulting in the formation of an
electrically amphoteric, colloidal suspension. The ions of elec-
trolyte, then, cause precipitation by discharging the particles un-
equally. Evidence bearing upon this theory has been given by
the work of Coulter (1920) who determined the isoelectric point
 V. R. Goddard and L. B. Mendel 459
TABLE VII.

Variations in Amount of Hemag MI; utinin Fixed by Corpuscles.

dinimum effective quantity of agglutinin:

Date of Kind of
experiment. erytbrooyte. For 3
successive
portions.

sol. per cent mg. mg. m7.

Mar. 15 Rabbit. 2.5 0.0006 0.006 0.008

“ 18 0.0006 0.006 0.006
“ 24 0.0006 0.002*
Apr. 9 0.0006 0.006 0.008
“ 13 0.0005 0.006 0.01
Mar. 12 5.0 0.0007 0.009 0.01
rlpr. 3 0.0005 0.003 0.003
“ 12 0.0008 0.006 0.01
Mar. 25 10.0 0.0009 0.003
“ 18 20.0 0.005 0.008

“ 28 Man. 2.5 0.0007 0.007

-4pr. 13 5.0 0.002 0.02 0.05t

Mar. 22 Chicken. 2.5 0.006 0.01

Apr. 17 0.006 0.03 0.08
0.006 0.04

Mar. 20 Duck. 2.5 0.0006 0.006 0.006

20.0 0.005 0.03f

Apr. 15 Dog. 2.5 0.0008 0.006 0.009

0.0008 0.007
5.0 0.003 0.009 0.03t

To 0.5 cc. portions of erythrocytes of suspensions indicated in isotonic

sodium chloride were added increasing quantities of hemagglutinin; and
the volume was made up to 1 cc. with isotonic sodium chloride. After
the corpuscles had clumped and sedimented, 0.5 cc. of the supernatant
medium was removed and tested for agglutinating potency with a fresh
suspension of corpuscles. Thus the amount of protein required in the
initial dose for clumping two successive and three successive portions of
corpuscles was determined.
*This value was obtained by using a 1.25 per cent suspension of
corpuscles.
t This value was obtained by using a 2.5 per cent suspension of
corpuscles.
1 This value was obtained by using a 10.0 per cent suspension of
corpuscles.
460 Plant Hemagglutinins

for normal sheep erythrocytes and showed that by the process

of sensitization, the isoelectric point was moved in the direction
of neutrality. Thus flocculation was made possible nearer neu-
trality, since particles in suspension are most easily aggregated
and sedimented at their isoelectric points.
The two types of agglutinin also have similar fixation processes.
According to the well established rule of Eisenberg and Volk
(foreseen by Bordet) “for the same mass of cells, the absolute quan-
tity fixed is directly proportional to the concentration of antibody;
the relative quant,ity inversely” (Wells, 1925). In this connection,
we found it interesting to calculate from data collected the maxi-
mum quantity of our agglutinin held by the cells of a given volume
of suspension and to compare this with the minimum effective
quantity of agglutinin for this same cell mass. With a uniform
suspension of corpuscles in a series of tubes, the quantity of ag-
glutinin was varied from the minimum requirement upward. By
the standard procedure, not until 0.006 mg. or 10 times the mini-
mum effective concentration had been used was there sufficient
agglutinin free in the supernatant medium to cause clumping of
a second unit of corpuscles (see Table VII). A liberal allowance
for incomplete removal of unbound agglutinin being made, the
bound fraction was at least 8 times the minimum required for
clumping the mass of cells used. Corpuscles, then, do not combine
with one and only one given quantity of a plant hemagglutinin.
The amount “fixed” depends upon the quantity available in
the medium.

Chemical Nature of Hemagglutinin.

The interpretation given by Eisler and Portheim (1926) to
results which they obtained from a study of the hemagglutinin
of Phaseolus multiJEorus was that the active substance was not
itself protein but was adsorbed onto the albumin fraction of the
bean. They compared the increase in agglutinating value of the
maturing seed with the increase in quantity of the various protein
fractions and found that the former greatly exceeded the increase
in total protein or in the albumin fraction itself (which contained
agglutinin in higher concentration than any of the other fractions).
In contrast to this conclusion, all the evidence afforded by the
work reported here points toward the protein nature of the
 V. R. Goddard and L. B. Mendel

hemagglutinin. We observed that the various phases of the

denaturation process affecting the albumin preparation were ac-
companied by diminution of the agglutinating potency. Thus,
the slow, denaturing effect of acids would in time destroy the
agglutinin as completely as would the rapid process of heat coagu-
lation. Neither was it possible to digest the protein by enzyme
action without a concomitant loss of agglutinating potency. One
would not necessarily expect this to happen were the agglutinin
merely adsorbed onto the protein since it might then be liberated
and be made available through the process of digestion. Peptic
digestion changed the effectiveness of the agglutinin along with
the elastic influence upon the protein molecule. By a peptic
hydrolysis of 6 days duration, the agglutinin was rendered inert.
Since from proteins with an agglutinating quality an individual
hemagglutinating fraction has never been separated, and since
chemical changes in the protein itself are accompanied by changes
inthe agglutinating potency, there is no reason for assuming two
closely associated entities rather than one endowed with an unusual
property. Moreover, the protein itself is reactive in such low
concentration that if the agglutinin were a separate entity oc-
curring as a contaminant, its effective concentration would be
astonishingly minute.

Practical Considerations.

The outcome of this study suggests that hemagglutinating

preparations of the sort that we have investigated may have a
larger field of usefulness in the preparation of therapeutic serums
than has hitherto been accorded to them. It should prove com-
paratively simple and inexpensive to prepare in adequate quanti-
ties readily soluble specimens of the bean albumin that can be
duly sterilized and preserved in dry form for long periods ready
for instantaneous use. The stoichiometric relations studied indi-
cate that the quantities required for corpuscular agglutination
are comparatively small. Furthermore, through preliminary tests
on the species of blood to be agglutinated, it should be possible
to estimate for large lots the dosage so selected that an excess nf
unfixed hemagglutinating protein will not remain in the serum.
Danger of anaphylactic reactions through the retention of a
 Plant Hemagglutinins

foreign protein like the bean albumin in a therapeutic serum ought

thus to be successfully avoided.
SUMMARY.

A non-toxic, highly potent, soluble, hemagglutinating protein

having the characteristics of an albumin was prepared in dry
form from navy beans (Phaseolus communis). A quantitative,
macroscopic method for measuring hemagglutination was devised
and used for studying the variables affecting the reaction. In-
dispensability of electrolytes and the inhibiting influence of certain
proteins, notably those of egg albumin and the serum proteins
were demonstrated. Chemical changes in the protein which led
to denaturation or hydrolytic cleavage were shown to be ac-
companied by a lessened hemagglutinative potency. The mode
of action, the chemical nature, and some practical aspects relating
to the application of the procedures to the production of thera-
peutic serums are discussed.
BIBLIOGRAPHY.

Berczeller, L., and Wastl, H., Biochem. Z., 142, 524 (1923).
Coulter, C. B., J. Gen. Physiol., 3,309 (1920).
Dorset, M., andHenley, R. R., J. Agric. Research, 6, 333 (1916).
Ehrlich, P., Fortschr. Med., 15,41 (1897).
Eisler, M., and Portheim, L., 2. Immunitiitsforsch., Orig., 1, 151 (1909).
Eisler, M., and Portheim, L., Ber. deutsch. bot. Ges., 29,419 (1911).
Eisler, M., and Portheim, L., Centr. Bakt., 1. AU., Orig., 66, 309 (1912).
Eisler, M., and Portheim, L., 2. Immunitiitsforsch., 47,59 (1926).
Gardner, A. D., Lancet, 1,21 (1919).
Gates, F. L., J. Am. Med. Assn., 77,2054 (1921).
Gates, F. L., J. Ezp. Med., 35, 63 (1922).
Hadley, P., J. Immunol., 2, 463 (1916).
Karrer, P., Smirnoff, A. P., Ehrensperger, H., van Slooten, J., and Keller,
M., 2. physiol. Chem., 135, 129 (1924).
Kobert, R., Lehrbuch der Intoxikationen, Stuttgart (1902).
Kolmer, J. A., Infection, immunity and biological therapy, Philadelphia
and London (1923).
Kraus, R., and Levaditi, C., Handbuch der Technik und Methodik der
Immunitatsforschung, Jena (1911).
Landsteiner, K., and Raubitschek, H., Centr. Bakt., f. AU., Orig., 46,
660 (1908).
Marcusson-Begun, H., 2. Immunittitsforsch., 46,49 (1926).
Mend& L. B., Arch. Jisiol., 7, 168 (1909).
Mudd, S., J. Immunol., 13, 113 (1927).
 V. R. Goddard and L. B. Mendel 463
Osborne, T. B., Mendel, L. B., and Harris, I. F., Am. J. Physiol., 14, 259
(1905).
Rona, P., and Gyiirgy, P., Biochem. Z., 106,120 (1920).
Russ, V. K., and Oesterlin, E., Biochem. Z., 114,258 (1921).
Schneider, E. C., J. Biol. Chem., 11, 47 (1912).
Wells, H. G., The chemical aspects of immunity, New York (1925).
Wienhaus, O., Biochem. Z., 18,228 (1909).