This paper is concerned with the application of two new techniques, both of which depend essentially on improved instrumentation, to the study of the electrophoretic mobility of red cells and their ghosts. The first technique has been developed to overcome difficulties which are encountered in the measurement of the mobility of ghosts. If the ghosts are substantially haemoglobinized, they can be seen, although with some difficulty, with the ordinary microscope and their mobility can be measured in an electrophoresis cell such as that described by Abramson (1934); if they are relatively Hb-free, on the other hand, they are almost invisible under the ordinary microscope, and can be seen satisfactorily only with phase contrast. The great majority of existing measurements of the mobility of ghosts, accordingly, are measurements of the mobility of ghosts which contain relatively large amounts of Hb. The new vertical cell to be described uses phase-contrast optics, and is fitted with removable electrodes which have several advantages over those usually employed. The second technique is the result of the observation that the Antweiler electrophoresis apparatus with a Philpot-Svensson attachment, although designed for the separation and observation of the mobilities of protein fractions, can also be used for measuring the relative mobilities of red cells and their ghosts.

The two principal conclusions reached in the investigations with the ordinary microscope and the Abramson horizontal electrophoresis cell (Abramson, Furch-gott & Ponder, 1939; Furchgott & Ponder, 1941) were that the isoelectric point of the haemolysed red cell is in the neighbourhood of pH 2-0, and that ghosts, regardless of the way in which they are prepared (with the exception of the ghost prepared by lysis in CO2-saturated hypotonic saline), have the same mobility as that of the red cells from which they are prepared. The first conclusion can be confirmed by the improved methods used here, but the second is an oversimplification.

The construction of the new cell (Fig. 1) is based on that of a vertical cell already described (Ponder, 1951). It differs from the latter in that it has removable electrodes and that the objects (red cells and ghosts) are seen by phase contrast.

Fig. 1.

The vertical electrophoresis cell. The very large water jacket is seen at the right. For description, see text.

Fig. 1.

The vertical electrophoresis cell. The very large water jacket is seen at the right. For description, see text.

The flat rectangular cell C and the tubes which lead to it are held rigidly on a square frame F of glass rod. Two 3-way stopcocks and S2 lead to the female parts of the ground joints of the electrodes E1 and E2, into which the removable non-polarizable electrodes fit as male parts. Each electrode has a short horizontal part and a short vertical part. The end of the horizontal part which fits into the ground joint is closed with a plate of fritted glass about 1 mm. thick, into which some plaster of Paris is rubbed. The end of the vertical part is closed with a snugly fitting rubber stopper through which a short rod of copper passes. A piece of fine insulated wire, ending in a lug, attaches the copper rod to one of the two terminals of the electrical circuit; by detaching the lug from the terminal, the electrode can be detached from the electrical circuit, and it can also be detached from the electrophoresis cell at the ground joint. The cell is filled by suction through a rubber tube attached at A and empties at B. A 2-way stopcock S3 has been introduced above ; when S3 is closed, the column of fluid below it behaves as if it were a closed column, almost regardless of the positions of S1 and S2.

The frame F is mounted on a metal plate about 1 mm. thick, with a large central aperture through which a long focus phase condenser can project. The plate is clamped, in such a way that it can be removed and returned to exactly the same position, to the square stage of a microscope which is arranged with its stage vertical and its body tube horizontal. The stage and the substage of the standard microscope have to be altered in order that the long-focus phase condenser can be brought close enough to the back of the electrophoresis cell.* The optical system consists of an 8 mm. phase objective (× 21), a long-focus phase condenser (Bausch and Lomb), and a 25 × eyepiece which carries an eyepiece micrometer disk ruled in squares, each covering about 100 μ.2. The light source is a 100 W. projection lamp situated several feet from the electrophoresis cell. A large water jacket and a green filter are inserted in the light path. The electrical circuit consists of a few 45 V. dry batteries, a reversing switch, a milliammeter, and a resistance in series with the cell. The resistance of the cell, when filled with 1 % NaCl, is about 15 ×103 Ω.

The cell, which should be cleaned with soapy water followed by many rinses of distilled water, is filled in the following way. The electrodes are first prepared by filling them with saturated CuSO4, inserting the rubber stoppers with their copper rods, rinsing their outsides with water, and applying a thin layer of stopcock grease to their ground surfaces. The cell, on its frame which is permanently attached to the plate, is removed by unclamping the plate from the microscope stage. The rubber tube leading from A is attached to a Mariotte bottle containing saline or buffer. With the stopcocks S1 and S2 in position 1 (see inset of Fig. 2), the stopcock S3 is opened. This fills the female part of the upper electrode; S3 is closed, and the male part of the upper electrode is inserted into the upper ground joint. Next 51 and S2 are turned into position 2 ; S3 is opened ; the cell and the female part of the lower electrode then fill with saline or buffer. S3 is again closed, and the male part of the lower electrode is inserted into the lower ground joint. S8 is now turned into position 3; S3 is opened, and the tube below S2 is filled. 53 is closed again, and 5X and 52 are turned into position 4. This procedure fills the cell and connects it with the electrodes. There must be no air bubbles.

Fig. 2.

pH-mobility dependences for human red cell ghosts at constant ionic strength. Ordinate: mobility v in μ/sec./V./cm.; abscissa: pH. I.E.P., isoelectric point. Curve A (dotted) is the relation obtained by Furchgott & Ponder in 1941 ; curve B is the relation found with the new electrophoresis cell for systems of red cells and ghosts in Michaelis’s universal buffer. Inset shows the positions of the stopcocks S1 and S2, during the filling of the cell described in the text.

Fig. 2.

pH-mobility dependences for human red cell ghosts at constant ionic strength. Ordinate: mobility v in μ/sec./V./cm.; abscissa: pH. I.E.P., isoelectric point. Curve A (dotted) is the relation obtained by Furchgott & Ponder in 1941 ; curve B is the relation found with the new electrophoresis cell for systems of red cells and ghosts in Michaelis’s universal buffer. Inset shows the positions of the stopcocks S1 and S2, during the filling of the cell described in the text.

The cell is returned to the microscope stage, to which it is clamped. To fill the cell with a suspension of red cells or of ghosts, it is first emptied by turning S1 and S2 into position 3 and opening S3. The suspension can then be drawn up, from a vessel placed below B, by applying suction to the rubber tube attached to A. The suspension should be drawn up above S3, which is then closed. S1 and S2 are turned into position 4. After a few minutes, during which currents in the fluid become negligible, the cell is ready for the making of mobility measurements at the stationary levels. The distance over which a cell or ghost moves during a short period of time (usually 10 sec.) is observed, first with the current flowing in one direction and then in the other, and the small movement due to gravity or to small systematic drifts (liable to occur in vertical cells) is added or subtracted. The time is much more conveniently measured by the beats of a metronome than with a stopwatch. If large systematic drifts develop, the cell should be emptied and refilled; this takes less than a minute to do. After some hours of work, drifts attributable to diffusion, etc., in the neighbourhood of the electrodes develop; the electrodes should therefore be freshly prepared before the cell is filled at the beginning of each day’s work.

Attention should be called to several points. (1) The electrophoresis cell, including the side-tubes which lead to the electrodes, must be filled with the medium in which the cells are suspended. This may be saline or a variety of isotonic buffers, but when the medium is changed the entire filling operation must be carried out anew. If the side-tubes leading to the electrodes are filled with saline, for example, while the cell and the vertical tubes leading to it are filled with a suspension of red cells or ghosts in buffer, the system will soon become unstable and the suspension will begin to stream. (2) Care must be taken not to introduce small air bubbles into suspensions of cells or ghosts, as is easily done when centrifuged material is resuspended by shaking. The upward movement of these small bubbles or of ghosts to which they have stuck is a serious source of instability in the vertical cell. (3) When making measurements of the mobility of ghosts, which are inconspicuous objects even when seen with phase contrast, special care should be taken to be sure that the ghost is in one of the stationary levels. Ghosts tend to move out of these levels more than red cells do, and the best measurements of the mobility of ghosts are those in which the ghost is seen edgewise; when seen edgewise, it almost certainly lies at the stationary level upon which the objective is focused.

The Antweiler microelectrophoresis apparatus (Antweiler, 1952) is essentially a Tiselius apparatus with a Philpot-Svensson attachment, but capable of giving electrophoretic patterns of the proteins in serum, etc., with very small quantities (0·1 ml.) of material and very rapidly (within 10 min.). The light beam which passes through an observation channel is deflected in proportion to the refractive index and therefore to the concentration of the protein in the fluid in the channel, and as the components move under an applied e.m.f., a pattern develops which can be seen with the Philpot-Svensson attachment. If the channel is filled with a suspension of red cells or their ghosts, these may also move when an e.m.f. is applied, and the position of the boundary between the moving objects, e.g. red cells, and the surrounding medium can be observed with the Philpot-Svensson attachment used without its cylindrical lens. Instead, the channel is observed directly, and the rate of movement, under an applied e.m.f., of the advancing front of the columns of red cells, etc., can either be photographed or measured with an eyepiece micrometer.

Success with this moving boundary method depends on a number of factors. (1) The glass electrophoresis cell which contains the observation channel and the red cells in it must be cooled to between 10 and 15°C. (2) The inclusion of even the smallest air bubble must be avoided. (3) It is essential that the entire system of channels leading to the electrodes is filled with the same fluid as that in which the cells are suspended. This may be isotonic saline or an isotonic buffer. The resistance of the electrophoresis cell obviously depends on the ionic strength of this fluid, but the apparatus is fitted with a voltmeter and a milliammeter, so the field strength can always be determined. (4) When an eyepiece micrometer (conveniently divided into squares with 2 mm. sides) is used to measure the movement of the boundary, the time taken for the boundary to move from right to left over a distance of two eyepiece micrometer divisions determines the mobility, but the initial position of the boundary, before the e.m.f. is applied, ought to be a little to the right of the beginning of the first micrometer division. The reason for this is that the slipping of the upper part of the glass electrophoresis cell over the middle part does not always result in a sufficiently sharp boundary, and it is preferable to allow the boundary between the cells and the surrounding medium to move for half a minute or so before the measurement of the rate of movement is begun. (5) All other conditions being the same, the rate of movement of the boundary is a function of the volume concentration p occupied by the red cells or ghosts. The relation is
formula
where t is the time required for the boundary to move through a fixed distance (conveniently two micrometer scale divisions), t0 the time, obtained by extrapolation, for it to move through the same distance if p were zero, and a a constant. The constant a varies from one electrophoresis cell to another and must be determined; its numerical value is usually about 4·0. (6) The most reliable observations are made in systems containing red cells or ghosts in a volume concentration of about 0·2. At smaller volume concentrations, the boundary tends to become diffuse.

The necessity for knowing the volume concentration of each suspension of ghosts, so that the rate of movement of the boundary can be compared with that of a red cell suspension of the same volume concentration, is a real objection to the method. In the case of suspensions of some kinds of ghost, e.g. those produced by saponin, the volume concentration is very difficult to determine by centrifuging, although determinations by conductivity methods are still possible ; a less specific, although still important objection is, however, that the electrophoresis cell has to be cleaned and dried before each determination is made. This is very time consuming.

These are shown in Table 1 and Fig. 2. Apart from showing a general agreement between the relative mobilities obtained with the new vertical cell and with the moving boundary method, the values in Table 1, which are the ratios of the mobility of the object under consideration to the mobility of fresh human red cells under comparable conditions of voltage drop, pH, volume concentration, etc., show that:

Table 1.
graphic
graphic

  • (a) The mobility of red cells which have been stored at 4°C. for long periods of time is less than that of fresh red cells.

  • (b) Fresh watery ghosts have a considerably higher mobility than the red cells from which they are prepared, but the difference decreases with the length of time during which the ghosts are stored at 4°C.

  • (c) The fragmentation of washed human red cells by heat (53°C. for 5 min.) gives fragments which have a higher mobility than that of unheated red cells.

  • (d) Ghosts made by freezing and thawing or by haemolysis by saponin have substantially the same mobility as the red cells from which they are prepared ; this is the conclusion reached by Abramson et al. (1939). On the other hand, ghosts prepared by the CO2 method have a mobility much less than that of the red cells from which they are prepared; this is the conclusion reached by Furchgott & Ponder (1941), except that the reduction in mobility observed here is somewhat larger (a reduction to 0·71 in the red cell mobility instead of the reduction formerly observed, which was 0·82 of the red cell mobility).

These results show that the mobility of ghosts is often different from that of the intact cell. It may sometimes be the same, especially when the ghost is relatively well haemoglobinized, but the mobility is a function of the method by means of which the ghost is prepared and of the time which has elapsed after lysis of the cell. Leaching-out of material from certain types of ghost (Waugh & Schmitt, 1940) may be responsible for the time effects.

There is little doubt that the isoelectric point of the watery ghost is as low as pH 2·0 or even lower. Fig. 2 shows the pH-mobility curve found by Furchgott & Ponder in 1941 and also the curve obtained with the new vertical cell. In the case of the former (dotted in Fig. 2) the pH was controlled by a variety of buffers with ionic strength 0·172, whereas in the case of the latter, Michaelis’s universal buffer (acetate and veronal), which has the same ionic strength at all pH’s between 2·6 and 9·6, was used. The two pH-mobility curves are not identical but are very similar, and the isoelectric point of the ghost, regardless of whether it is the ghost of a red cell haemolysed at a low pH or a watery ghost suspended in a medium of low pH (as in the case of the observations with the vertical electrophoresis cell) is certainly situated at a pH of 2 or even less.

Two new techniques for measuring the electrophoretic mobility of red cells and ghosts are described. In the first, the mobilities are measured in a vertical electrophoresis cell in which they are seen by phase contrast. This enables mobilities to be measured even when ghosts contain so little Hb as to be invisible with the ordinary microscope. The second method uses an Antweiler electrophoresis apparatus to give measurements of the mobility of moving boundaries between columns of red cells or ghosts and the suspension medium.

It can be shown by these methods that the usual statement that the mobilities of ghosts are the same as those of the red cells is an over-simplification. The mobilities, under comparable conditions, are more generally dependent on the method used to prepare the ghosts and on the time which has elapsed after lysis.

The statement that the isoelectric point of watery ghosts is as low as pH 2.0 or even less is confirmed, as is also the general shape of the pH-mobility dependence.

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*

The glass electrophoresis cell was made by E. Machlett and Co. of New York City. The metal plate to which it is attached with its positioning pins and screws, together with the other changes necessary to convert the standard microscope for use with phase contrast are the work of Mr Paul Cutajar of the New York University Machine Shop. The Antweiler apparatus is supplied by Ivan Sorvall of New York City, and was purchased with funds provided by the Eli Lilly Co., Special Grants Committee.