Published January 1, 1960
The Electrophoretic Velocity of Human Red Cells,
of Their Ghosts and Mechanically Produced
Fragments, and of Certain Lipid Complexes
E R I C P O N D E R and R U T H V. P O N D E R
It is often thought, a l t h o u g h there is evidence to the contrary, t h a t properties
of the red cell ghost, such as its electrophoretic mobility a n d its m e c h a n i c a l
fragmentation, are the same as those of the red cell from which it is derived.
T h e electrophoretic velocity a n d the m e c h a n i c a l f r a g m e n t a t i o n of h u m a n red
cells a n d ghosts, however, have n o w been shown to d e p e n d on the w a y in
which the ghost is prepared, i.e. on the w h e t h e r the red cells are washed or
unwashed, as well as on the a n t i c o a g u l a n t into which the blood is d r a w n
( F u r c h g o t t a n d Ponder, 1941; P o n d e r a n d Ponder, 1955, 1959). Dervichian
(1955) has recently suggested t h a t the ghost has some of the features of a
precipitation artifact. As a result, a situation which was once t h o u g h t to be
simple has n o w become extremely complex, for if we c o m p a r e the red cell
From the Nassau Hospital, Mineola, Long Island.
This work wa~ done under a grant, No. H-1598, from the United States Public Health Service, and
Contract No. DA-49-007-MD-458 from the Department of the Army.
Received for Publication, May 15, 1959.
503
The Journal of General Physiology
Downloaded from on June 16, 2017
ABSTRACT Ghosts prepared in COs-saturated water from unwashed human
red cells can be fragmented mechanically, but ghosts from thrice washed cells
cannot. If the ghosts are prepared by freezing and thawing, this difference is not
observed.
The electrophoretic velocity varies also with the way in which the ghosts are
prepared. The pH-mobility dependence of washed red cells flatten off to a
plateau at pH 9, and the electrophorefic velocity is zero at about pH 2. Ghosts
prepared by freezing and thawing have almost the same pH-mobility de=
pendence, but if the ghosts are prepared in CO ~-saturated hyptonic saline, the
mobility at pH 9.4 is 0.75 times that of washed cells. Fragments of ghosts of unwashed red cells have a smaller mobility than that of the red cells. Trypsin reduces the mobility of washed red cells and of ghosts.
Sols of lipid complexes (lecithin, cephalin, and lipositol), at varying pH's,
have a mobility 1.2 times that of the washed red cell. The pH-mobility relation
is otherwise similar. These complexes can be coated with dextran and trypsin.
Published January 1, 1960
504
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
43 • t96o
w i t h its ghost we h a v e to consider w h e t h e r the ghosts are m a d e f r o m w a s h e d
o r u n w a s h e d r e d cells, the a n t i c o a g u l a n t used ( E D T A t, h e p a r i n , a n d possibly
A C D , citrate, a n d oxalate), as well as the m a n n e r in w h i c h the red cell is
c o n v e r t e d into a ghost, as b y freezing a n d thawing, b y lysis in a CO2-satur a t e d h y p o t o n i c m e d i u m , etc.
T h i s p a p e r is p r i n c i p a l l y c o n c e r n e d with u n w a s h e d a n d w a s h e d h e p a r i n i z e d
h u m a n red cells, with the ghosts p r e p a r e d f r o m t h e m b y freezing a n d t h a w i n g
a n d b y C O s - s a t u r a t e d h y p o t o n i c saline, w i t h the m e c h a n i c a l l y p r o d u c e d
f r a g m e n t s of ghosts ( " m y e l i n f o r m s " ) w h e n t h e y c a n be m a d e , a n d with
lipid complexes w h i c h m i g h t c o n c e i v a b l y h a v e the same e l e c t r o p h o r e t i c
velocity as t h a t of red cells.
Methods
PREPARATION OF GHOSTS These are prepared by freezing and thawing the red
cells of unwashed or washed blood, any intact cells which remain after three freczings
and thawings being centrifuged down. Alternatively, 1 ml. of the red cells of unwashed
or of washed blood (either collected in hcparin or in EDTA) is added to 50 ml. of
0.1 per cent NaC1 saturated with CO 2. After standing in the refrigerator for a few
hours, the flocculent masses of ghosts can be separated from the supcrnatant hemoglobin-containing fluid. Their form is observed by phase contrast.
FRAGMENTATION Mechanical fragmentation is carried out in a VirTis homogenizer running at 23,000 R.P.M. for 5 minutes. Fragmentation, if any, is observed by
phase contrast microscopy.
ELECTROPI-IORETIGVELOCITY This is measured with the vertical cell described
by Ponder and Ponder (1955), the red cells, ghosts, or fragments being suspended in
a large volume of Michaclis buffer at pH's between 9.6 and 4.2. The extent of dilution
of the cells, ghosts, or fragments in the Michaelis buffer is about 1 in 500. This ensures that only from ten to twenty cells appear in the microscopic field of the vertical
cell; the number of ghosts and fragments which appear may be even less. It is almost
essential that the cells, ghosts, and fragments be freshly prepared because if they arc
not, they become unstable in the vertical cell because of the rapid fall of clumps of
red cells, ghosts, or fragments. Under any circumstances, the electrophorctic velocity
of minute fragments is difficult to measure, particularly if it is slow, as it is at the
lower pH's.
a Disodium salt of ethylenediamine tetraacetic acid.
Downloaded from on June 16, 2017
PREPARATION OF RED GELLS The red cells of normal human blood are collected either in hcparin or in EDTA. T h e y are spun down and the plasma and burly
coat arc removed. The cells are then either left unwashed or are washed three times
with 1 per cent NaC1, and then are made up to the same volume concentration as
that of the unwashed cells.
Published January 1, 1960
ERIC PONDER A N D
RUTH V. PONDER Properties of Red Cell Ghosts
505
1. Mechanical Fragmentation
It has already been reported that human red ceils and ghosts prepared from
unwashed red ceils are easily fragmented~ but that under the same conditions
the ghosts of thrice washed human red cells are scarcely fragmented at all
(Ponder and Ponder, 1959). Table I shows the results of more recent experiments in which unwashed cells and washed cells from blood drawn into
EDTA, and unwashed cells and washed cells from blood drawn into heparin,
were converted into ghosts by addition to 0.1 per cent NaC1 saturated with
TABLE
Kind of ghost
Anticoagulant
I
Form of ghost
After fragmentation
EDTA
Circular or flat
Tiny fragments
Unwashed red cells in CO2saturated 0.1 per cent NaC1
Heparin
Circular or flat
Tiny
fragments,
casional ghost
Washed red cells in CO~saturated 0.1 per cent NaCI
EDTA
Circular or flat
Ghosts, folded and erehated : " b o d i e s "
Washed red cells in CO~saturated 0.1 per cent NaCI
Heparin
Circular or flat
Ghosts, distorted, folded,
and crenated: " b o d ies," a few fragments
Unwashed red cells freezing
and thawing
Heparin
Circular and flat,
some
myelin
~orms
Tiny fragments
Washed red cells freezing
and thawing
Heparin
Circular and flat,
some
myelin
forms
Tiny fragments
oc-
CO2, after which an attempt was made to fragment the ghosts mechanically
in the VirTis homogenizer.
As Table I shows, the ghosts of unwashed and washed red cells of blood
drawn into EDTA and the ghosts of unwashed red ceils and washed red ceils
of blood drawn into heparin, when prepared in hypotonic NaC1 saturated
with CO2, are similar in form; i.e., flat and circular. After 5 minutes in the
homogenizer, the ghosts of washed red cells of blood drawn into either EDTA
or heparin are not extensively fragmented, although they are folded and
crenated. On the other hand, after the same time in the homogenizer, the
ghosts of unwashed red cells are reduced to tiny fragments and myelin forms
regardless of whether they are derived from blood drawn into EDTA or
z Sometimes the fragmentation is so extreme that it may almost be referred to as homogenization.
Downloaded from on June 16, 2017
Unwashed red cells in COssaturated 0.1 per cent NaCI
Published January 1, 1960
506
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
43
" ~96o
2. Electrophoretic Velocity
The results of this section are shown on the left-hand side of Fig. 1, ~ V/X
being plotted on the ordinate and pH on the abscissa. V is the electrophoretic
mobility in #/see., r/the viscosity of the medium, and X the field strength.
It has already been found
(Ponder and Ponder, 1955) that the electrophoredc mobility of washed,
heparinized h u m a n red cells varies between -- 1.0 and - I. I at pH 9.4. If the
entire curve showing the relation between the electrophoretic mobility and
p H is plotted, the mobility flattens off to form a plateau in the neighborhood
of p H 9.4 and falls with a decrease in pH; by extrapolation, the curve reaches
a tentative point of zero mobility at about p H 9.8 In the case of pH's below
A. WASHED, H E P A R I N I Z E D HUMAN RED CELLS
s Until recently an operation, such as a change in pH, which resulted in the net positive and negative
charges on a surface becoming zero was considered in electrophoretic investigations to define the
"lsoelectric point" of the surface. A continuation of the same operation, such as a further change of
pH, should, according to the work of H a r d y (1905), result in an electrophoretic mobility with a
change of sign. As a matter of fact, no such change of sign has been reliably observed in the case of
red cells, ghosts, or their fragments when the p H is reduced below the value at which the electrophoretic mobility becomes zero (about p H 2), although it is also true that it has not been carefully
looked for. It is now recognized that there are several mechanisms other than those considered by
Hardy, A b r a m s o n (1934), and the early investigators in the field of electrophoresls which can result
in the electrophoretic mobility becoming zero. T h e term "lsoelectric point" when used in connection
with electrophoretic investigations of the red cell surface, etc. has in the m e a n t i m e a historical meaning
only.
Downloaded from on June 16, 2017
heparin. Unwashed or washed red ceils, regardless of the anticoagulant, are
also reduced to tiny fragments and myelin forms in the homogenizer.
The observation that the ghosts of unwashed red cells and those of washed
red cells, prepared by this method, behave differently as regards mechanical
fragmentation supports Dervichian's contention (1955) that the surface of
the red cell ghost is not the same as the surface of the red cell itself, but
depends on the way in which the ghost is prepared. The mechanical fragmentation of the ghosts of unwashed red cells and the relative absence of
fragmentation of the ghosts of washed red cells do not depend on the anticoagulant into which the blood is drawn, at least when the anticoagulant is heparin or EDTA. It depends, however, on the method used for making the
ghosts (Table I, lines 5 and 6). If freezing and thawing (three times) is used,
the ghosts which result from unwashed or from washed heparinized hum a n blood, are hemoglobinized to varying extents, and there are some myelin forms surrounding them. If these ghosts are placed in the homogenizer,
they are reduced to minute fragments; i.e., the difference in fragmentability between ghosts from unwashed cells and ghosts from washed cells, found
with ghosts prepared by the CO2-saturated hypotonic saline method, is not
observed. Two differently prepared kinds of ghosts apparently have different fragmentation properties.
Published January 1, 1960
ERIC PONDER AND R U T H V. PONDER
Properties of Red Cell Ghosts
507
about 3.8, extrapolation is necessary because red cells hemolyze. Nevertheless,
the curve is similar to that obtained by Furchgott and Ponder in 1941, and
there is no question but that the point of zero mobility is in the neighborhood
of p H 2.
B. BEHAVIOR OF GHOSTS Ghosts produced from washed h u m a n red
cells by freezing and thawing have almost the same electrophoretic pHmobility dependence as that of washed red cells (Ponder and Ponder, 1955).
T h e curve is not shown in Fig. 1, but it apparently proceeds to the same
point of zero mobility at about p H 2. Ghosts prepared from unwashed or
washed h u m a n red cells by the hypotonic saline and CO~ method, on the
other hand, have an electrophoretic mobility at pH 9.4 which is only about
0.75 times the electrophoretic velocity of washed red cells (Furchgott and
~v/x lipid
• oZ-'--.\
^o CO~-miur~ted \
\
-~,,,
',,~- J.y~i~
tz]l~
Xx ,~
\
-0.4
~
',, \
atlon
%% t~l|
10
o
6
4
~
p~
FIGURE 1. Abscissa, pH. Ordinate, electrophoretic mobility corrected for viscosity and
field strength. The figure is self-explanatory.
Ponder, 1941; Ponder and Ponder, 1955). These results are shown in Fig. 1
(crosses).
C. FRAGMENTSOR "MYELIN FORMS" The fragments resulting from the
homogenization of ghosts prepared from unwashed, heparinized red cells by
freezing and thawing have an electrophoretic velocity which is smaller at all
pH's than that of the red cells from which they are derived. In this respect,
they resemble the ghosts prepared by the COg.-saturated hypotonic NaC1
method.
D. EFFECTS OF TRYPSIN Just as the electrophoretic velocity of washed
h u m a n red cells is reduced by about 30 per cent if the surrounding m e d i u m
contains 0.5 per cent trypsin, so the electrophoretic velocity of ghosts resulting
from freezing and thawing is reduced, and to an even greater extent; it also
seems probable that their point of zero mobility corresponds to a slightly
higher pH.
Downloaded from on June 16, 2017
25"C. comtflexe~
Published January 1, 1960
508
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
43 • x96o
It is a pleasure to thank Mr. Joseph Eichberg, President of the American Lecithin Company, for
providing the lipid complexes and an analysis of them, and we take pleasure in thanking the American
National Red Cross for supplying us with a sample of Cohn's fraction V with a known molecular
dispersion.
REFERENCES
ABRAMSON, H. A., 1934, Electroldnetie Phenomena, Monograph 66, New York,
Chemical Catalog Company.
D~.RVIemAN, D-G., 1955, Probl~mes de structure, d'ultrastructure et de fonctions
cellulaires, Paris, Masson et Cie., 103.
FURCX~GOTT,R. F., and PONDER,E., 1941, o7. Gen. Physiol., 24,447.
HARDY, W. G., 1905, o7. Phsyiol., 33,251.
PONDER, E., and PONDER,R. V., 1955, o7. Exp. Biol., 32, 175.
PONDER, E., and PONDER,R. V., 1959, Rev. hhnatol., 13,506.
Downloaded from on June 16, 2017
E. LIPID COMPLEXES The lipid complexes used were provided by the
American Lecithin Company, the name applied being "Alcolec granules."
These granules form a finely divided sol in Michaelis buffer and consist of 95
per cent phosphatides made up of 30 per cent lecithin, 30 per cent cephalin,
and 30 per cent lipositol. T h e sol is prepared by adding 1 gm. of the granules
to 100 ml. of Michaelis buffer at pH's between 9.4 and 4.2, allowing the
mixture to stand for 48 hours with occasional agitation, and then filtering
through glass wool; the final concentration is about 0.5 per cent. The particles
of the sol are spherical and vary from 0.5 to 5 # in diameter; they contain about
85 per cent of water and can be easily observed in the phase-lit vertical electrophoresis cell. The sol can be further diluted with Michaelis buffer at the same
p H as that used for its preparation.
The electrophoretic mobility as a function of pH of these lipid complexes is
shown in Fig. 1. The particles of the sol move more rapidly than do washed
h u m a n red cells, but the general form of the curve is the same and the point
of zero mobility is at about p H 2, although again this has to be found by
extrapolation because the granules of the sol agglutinate between p H 3,0 and
p H 2.5.
The surface of the red cell is not made up of the same lipids in the same
proportion as are contained in the Alcolec granules, but these lipid complexes
are nevertheless interesting. They give an electrophoretic pH-mobility
dependence curve not unlike that for washed red cells, and like the latter,
they can be coated both with trypsin and dextran; they also form a precipitate at all pH's between 2.3 and 9.0 with 0.5 per cent protamine, a n d their
electrophoretic mobility is altered very little by Cohn's fraction V (kindly
given to us by the American National Red Cross) or with globulin (Cohn's
fraction II plus III).