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Electrophoresis of human leukocytes
ARCHLVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Electrophoresis
350-355 (1969)
136,
of Human
Leukocytes’
P. S. VASSAR AND h/I. J. KENDALL Department
of Pathology, Faculty
of Medicine,
University
of British
Columbia
Vancouver 9, British Columbia, Canada AND
G. V. F. SEAMAN Division of Neurology, University oj Oregon ilfedical School, Portland, Oregon 97301 Received July 10, 1969; accepted September 17, 1969 The peripheral regions of human polymorphonuclear leukocytes and erythrocytes contain at least two types of ionogenic group, a neuraminate ion and unidentified groups of respectively pKa. e 4.0 for the polymorphonuclear leukocytes and pK, N 3.4 for erythrocytes. These pK, values suggest p or r-carboxyl groups and ol-carboxyl groups of acidic amino acids in the membrane-protein for the polymorphonuclear leuckocytes and red blood cells respectively. The presence of ribonucleic acids could not be demonstrated in the peripheral regions of these cell types by electrophoretic methods. Both the intact polymorphonuclear leukocyte and erythrocyte lacked significant numbers of cationic groups at their surfaces.
Abramson (1) discussed the earlier literature on the electrophoresis of leukocytes. Bangham, Pethica, and Seaman (2) examined the effect of pH on the electrophoretie mobility of sheep polymorphonuclear leukocytes (PMN) and deduced the presence on the surface of a singly charged acidic group of pK, 3.7, probably a carboxyl group. No evidence for the presence of a significant number of basic groups could be found. Problems relating to the periphery of cells and intercellular phenomena have been recently reviewed (3, 4). MATERIALS
AND METHODS
Microelectrophoresis was performed as previously described (5). The majority of the experiments were performed in triplicate and the average t.aken of between 30 to 160 times of migration per experiment. All reagents were of analytical grade. Standard i This research was supported by Grant MA3040 from the Medical Research Council of Canada and by Grant CA-10422 from the National Cancer Institute.
saline consisted of 0.145 M aqueous sodium chloride solution with the pH adjusted to pH 7.2 f 0.2 by means of the addition of 0.5 M aqueous sodium bicarbonate solution. Isotonic aqueous solutions of hydrochloric acid or sodium hydroxide were added to standard saline for studies of variation of the electrophoretic mobilities of the cells with PH. The neuraminidase (Behringwerke) had an activity of 500 units/ml where 1 unit of activity is that quantity of enzyme which would liberate 1 H of sialic acid from a glyeoprotein substrate in 15 min at 37”. Neuraminidase as a crystalline receptor-destroying enzyme supplied by Dr. Ada was also used (6). Ribonuclease (Worthington, 3~ crystallized, beef pancreas, chromatographitally prepared) was found to have an activity of 1000 units/mg measured by the method of Kalnitsky et al. (7). Blood was obtained fresh from normal healthy donors of various blood groups using one-fortieth the volume of the blood of a 5y0 aqueous solution of disodium EDTA as the anticoagulant. All glassware was routinely siliconized. Leukocytes were separated from whole blood using isotonic aqueous ammonium chloride solution (8, 9). The separated leukocytes (1 vol) were finally washed twice with 50 vol of standard saline 350
ELECTROPHORESIS
OF HUMAN
at room temperature (1009; 7 min). Leukocytes washed in this manner will be termed “standard washed” cells. At this stage of the preparation the suspension of cells from the NH&l separations consisted of 859Ooj, polymorphonuclear leukocytes with no red blood cells or platelets remaining. It should be emphasized that the separated leukocytes have unimpaired viability (8, 9) and show a high phagocytic index for latex particles or st,arch grains in the presence of fresh serum and calcium ions. Red blood cells (RBC) were prepared for electrophoretic examination as described previously (10). Neuraminidase treatment. Standard washed and centrifugally packed human RBC and PMN were treated with neuraminidase as described by Seaman and Uhlenbruck (11). Control cells were incubated in 0.145 M aqueous sodium chloride solution containing 0.002 M aqueous calcium chloride solution or in this system containing the appropriate concentration of neuraminidase which had been inactivated by boiling for 20 min. The supernatant fluids from the treated cells and appropriate controls were analyzed for sialic acid by the methods of Svennerholm (12) and Warren (13). Cell counts were carried out in duplicate with a Coult.er counter. treatmenl. Packed and standard Ribonuclease washed human erythrocytes or polymorphonuclear leukocytes were treated with ribonuclease for 30 min at 37” (0.1 mg ribonuclease per ml of standard saline) using the procedure described by Mayhew and Weiss (14). Red blood cell suspensions were also treatedwith a 1 mg/ml solution of ribonuclease in standard saline. Control cells were incubated with standard saline. The enzyme-treated cells were washed in standard saline and then examined by microelectrophoresis. Acetaldehyde treatment. PMN were fixed for at
351
LEUKOCYTES
least 20 days in a 2% (w/v) solution of acetaldehyde in phosphate-buffered saline at pH 7.4. Preparation of this solution was previously described (15). The reagent grade acetaldehyde used was shown to be pure by spectroanalysis which revealed a single peak with a maximum at 278 pm (16). Fixed PMN were washed four times in 0.145 M aqueous NaCl before further procedures were performed. Electrophoretic mobilities were determined on the following: (a) acetaldehydetreated cells alone; (b) acetaldehyde-treated cells followed by neuraminidase treatment; and (e) neuraminidase-treated cells followed by acet.aldehyde fixation. RESULTS
All the mobility measurements reported in Table I were performed on “standard washed” cells suspended in standard saline, pH 7.2. The standard deviations are given with the number of measurements upon which a particular value is based in parentheses. The effects of neuraminidase, inactivated neuraminidase, and ribonuclease are listed. Only active neuraminidase produced a significant reduction in the mobility of the polymorphonuclear leukocytes (p < 0.001). The effects of the enzymes on the electrophoretic mobility of human erythrocytes are included in the table. Ribonuclease was also tried at a concentration of 1 mg/ml and found not to change the mobility of human erythrocytes. In Fig. 1 is given the pH versus electrophoretic mobility relationship for human PM/IN suspended in standard saline; open circles for the normal and full circles for the
TABLE
I
ELECTROPHORETIC MOBILITIES OF HUMAN RED BLOOD CELLS AND POLYMORPHONUCLEAR LEUKOCYTES PREPARED IN VARIOUS WAYS AND TREATED WITH NEURAMINIDASE AND RIBONUCLEASE CELLS SUSPENDED IN STANDARD SALINE, pH 7.2 f 0.2, 25” Electrophoretic mobilities, p/set/V/cm + SD Control not incubated 1 Inactive neuraminidase -0.90
f
0.037
PMN -1.08
(170) f 0.03
RBC
(160)
-0.82 f 0.028 0.001 < p < 0.01 (40) -1.08 f 0.03 p = 1.0 (30)
Neuraminidase -0.36 f 0.040 p < o.ool (110) -0.34 * 0.04 p < 0.001 (30)
Control for ribonuclease -0.87
+ 0.015
(‘33 -1.07
zk 0.03
Ribonuclease
, -0.88 f 0.021 0.4 > p > 0.3 (40) -1.07 f 0.03 p = 1.0 (30)
352
VASSAR, KENDALL, AND SEAMAN
Bulk pH
-o---o-o-d--A
_____ .., ____ A----p------A--A
FIG. 1. pH versus electrophoretic mobility relationships for human erythrocytes; normal (A----), neuraminidase-treated (A----) and polymorphonuclear leukocytes; normal (O-), and neuraminidase-treated (o-). Cells suspended in standard saline at 25”. TABLE II
absent in the untreated cells. The PMN, like the RBC, does not display a true isoelectric point as there is no detectable migration toward the cathode down to a pH of 1.8. The electrophoretic mobility of the untreated cell is constant between pH 5 and 10. Electrophoretic mobilities, &x/V/cm f standard deviation All measurements were obtained under conditions of electrokinetic stability, that is, AcetalAcetalphyde Neuraminidase the mobility under the given conditions did dehyde+ !2I$ incubated neuraminidase acetaldehyde not change with time and the changes in the control -~ -____ electrophoretic mobility produced by pH were completely reversible over the period -0.89 -0.90 -0.57 -0.56 10.032 +0.035 *to.035 &to.04 of time required to perform the measurep > 0.1 p < 0.001 p < 0.001 ments. PMN (130) (120) (220) (40) Neuraminidase treatment of the PMN and RBC released 2.0 and 0.4 pmoles of sialic acid per lOlo cells respectively, as asneuraminidase-treated PMN. The pH versayed by both the Svennerholm (17) and sus mobility relationship for RBC’s are Warren (13) methods. The theoretical represented by dashed lines with open triquantities of membrane-bound sialic acid angles for the normal and full triangles for released by neuraminidase were calculated the neuraminidase-treated RBC. The mean on the assumption that each electron charge pK, for the PMN surface is approximately lost at the cell periphery corresponded to one 3.4, whereas the pK for the acidic groupings neuraminate ion. The molecular weight of which remain after neuraminidase treatment the sialic acid was taken as 309 (N-acetyl is about 4.0. The mean pK, for the RBC derivative). The mean area of the RBC was surface is 2.9 and of the anionic groupings taken as 1.63 X 1O-6 cm2 (18) and that of the left after neuraminidase treatment the pK, PMN as 2.84 X 10-6cm2 as calculated from is approximately 3.4. Both the neuraminidase-treated PMN and RBC have some an assumed volume of 4.5 X lo-lo cm3 (19). The calculated charge density decreases cationic character as evidenced by positive after neuraminidase treatment implied that branches to the pH versus mobility relationships at low pH; this cationic character is 0.2 pmoles and 0.15 Fmoles of sialic acid ELECTROPHORETIC MOHILITIES OF POLYMORPHONUCLEAR LEUKOCY~FES TREATED WITH ACETALDEHYDE AND NEURAMINIDASE CELLS SUSPENDED IN STANDARD SALINE, pH 7.2 + 0.2, 25”
ELECTROPHORESIS
OF HUMAN
were released from lOlo cells of PMN and RBC respectively. Electrophoretic mobilities of acetaldehyde-treatNed PMN are shown in Table II. On comparing these results with those in Table I it can be seen that: (a) acetaldehyde fixation has no effect on the electrophoretic mobility of the normal PMN; (b) acetaldehyde treatment after neuraminidase reveals an increase in negative mobility of about 22 % compared with neuraminidasetreated P&IN alone; and (c) acetaldehyde treatment shows similar results whether cells are fixed before or after neuraminidase treatment. DISCUSSION
The mean pK, value for the human polymorphonuclear leukocytes obtained from the pH at which the electrophoretic mobility was one half of the plateaux value suggests that the ionogenic groups are probably carboxy1 groups. The constant mobility between pH 5.0 and 10.0 and the lack of a positive branch to the pH versus mobility curve at low pH suggests the absence of a significant number of basic groups such as amino groups. Treatment of the P-MN’s with neuraminidase produces about a 60% decrease in the electrokinetic charge density at neutral pH with the release of a membranebound sialic acid. The changes which occur in red blood cell charge after treatment with neuraminidase are already well documented Gw Comparison of the normal and neuraminidase-treated RBC (Fig. 1) shows that neuraminate ions constitute about 60% of the anionic groups on the untreated erythrocyte. After these groups have been removed by neuraminidase the remaining 40% have a pK, of about 3.4. Svennerholm (12) estimated the pK, of free N-acetylneuraminic acid to be about 2.6, a value close to that estimated for the membrane-bound neuraminate ion (15). A survey of literature values shows that this figure is close to the pK, of a-carboxyl groups in polypeptides or proteins. The pK, of the surface groups which remain on the PMN after neuraminidase treatment is circa 4.0 and close to that for the y-carboxylic acid groups of glutamic or
LEUKOCYTES
353
&carboxylic acid groups of aspartic acid (21). Although the nature of the ionogenic groups in the peripheral regions of the RBC and PMN which are not neuraminate ions is unknown, it can be inferred from the pK, values that these groups are probably carboxylate ions originating from the acidic amino acids in the membrane protein. Any changes which occur in the surface properties of cells after treatment with an enzyme must be evaluated carefully since these alterations could have arisen in ways other than by simple enzyme action (11). Kraemer (22) has questioned the purity of commercially available neuraminidase. He found that neuraminidase from Clostridium perfrinyens contains cytotoxic, hemolytic, and phospholipase contaminants. However, the neuraminidase samples used in our studies on membrane-bound sialic acid were obtained from Vibrio choler& and do not contain the same contaminants (22), nor any detectable proteolytic activity when subjected to the Azocoll test (23). The small net positive electrokinetic charge carried by the neuraminidase-treated PMN and RBC at low pH (Fig. 1) is attributed to conformational changes in the cellular periphery. Both the normal and neuraminidase-treated PMN show electrokinetic reversibility over the range pH 2-10 which renders unlikely the irreversible adsorption of intracellular leakage products at the extremes of pH. The absence of significant proteolytic activity in the neuraminidase makes the origin of the positive character of the neuraminidase-treated PMN by proteolysis of membrane protein improbable. Adsorption of the enzyme or of any impurities in the enzyme although possible is considered unlikely as an explanation for the positive character of the PMN and RBC at low pH (24). Neuraminidase releases about 2.0 clmoles of sialic acid per lOlo PMN. The theoretical yield of sialic acid was calculated to be 0.2 pmoles on the basis of the change in electrokinetic charge produced by neuraminidase treatment, assuming that the sialic acid when bound in the membrane has its carboxylic acid group fully effective at the electrophoretic plane of shear. The IO-fold divergence between the analytical and theo-
354
VASSAR,
KENDALL,
retical yields indicates that sialic acid is being released from sites deep with respect to the electrophoretic plane of shear and even perhaps from intracellular organelles (25). The similar though smaller discrepancy between the experimental and theoretical yields of sialic acid from neuraminidasetreated human RBC has been discussed in previous publications (11, 24). The observations of Hackel and Smolker (26) that ribonuclease affects the Rh and Lu antigenic sites on human erythrocytes and, furthermore, that anti-Rh and antiLutheran sera are inhibited by ribonucleic acid derivatives (27) suggested that ribonucleic acids might be associated with the peripheral regions of cells. More recently Burka et nl. (28) have shown that a membrane-bound RNA is present in mammalian erythroid cells. Vassar et al. (29) found that ribonuclease did not alter the eleetrophoretic properties of a number of cell types but a series of recent studies which indicate the probable presence of surface RNA in cellular systems has prompted us to re-examine the action of ribonuclease on the cellular electrophoretic properties of the human RBC and PMN (14, 30, 31). Ribonuclease appeared to be without significant influence on the electrophoretic properties of both types of cell. The absence of a change in the electrophoretic mobility of normal PMN after their treatment with acetaldehyde implies the absence of an appreciable number of cationogenic groups in the cellular peripheral region of this blood element. For changes in the electrophoretic mobility to be significant the mobility values would have to differ from the control values by at least 5 %. Now the normal human PMN has about 1.8 X 10’ anionogenic sites, so that the presence of up to 4.5 X lo5 (5% of the anionic sites) cationic sites would not be detected by electrophoretic methods. Treatment of the PMN with neuraminidase, in -addition to producing about a 60% decrease in the anodic mobility at pH 7, also leads to the appearance of a significant number of cationogenic groups as may be seen from an examination of the pH versus mobility relationship particularly in the low pH region. Treatment of the neuraminidase-modified
AND
SEA&IAN
PMN with acetaldehyde leads to an increase in the anodic mobility (Table II) up to the value obtained by neuraminidase treatment of acetaldehyde-fixed cells. The reason for the appearance of the cationic groups after treatment of the PMN with neuraminidase is unknown since it is a glycosidase lacking any significant protease activity. The results presented here show that the peripheral region of PMN and RBC contain at least two types of ionogenic group, a neuraminate ion and an unidentified ionogenie group, probably a carboxyl group from the acidic amino acids in the membrane protein. No ribonucleic acids could be demonstrated in the peripheral regions of these cell types by electrophoretic methods. Both the intact RBC and PMN lacked any significant number of cationogenic groups in their cell peripheries. REFERENCES 1. ABRaMSON, H. A., J. Exp. Med. 46, 987 (1927). 2. BANGHAM, A. D., PETHICA, B. A., AND SEAMAN, G. V. F., Biochem. J. 69, 12 (1958). 3. BROOKS, D. E., MILLAR, J. S., SEAMAN, G. V. F., AND VASS.~R, P. S., J. Cell Physiol. 69, 155 (1967). 4. WEISS, L. “ The Cell Periphery, Metastasis and Other Contact Phenomena,” North-Holland Publ., Amsterdam (1967). SEAMAN, G. V. F., AND HE.IRD, D. H., Blood 18, 599 (1961). AD.~, G. L., .\ND FRENCH, L. E., Nature 183, 1740 (1959). KALNITSKY, G., HUMMEL, J. P., AND DIERKS, C., J. Biol. Chem. 234.1512 (1959). DIOGUARDI, N., AGOSTINI, A., FIORBLLI, G., AND LOMANTO, B., J. Lab. Clin. Med. 61, 713 (1963). 9. CARRUTHERS, B. M., Can. J. Physiol. Pharmacol. 44, 475 (1966). 10. SEAMAN, G. V. F., AND HEARD, D. H., J. Gen. Physiol. 44, 251 (1960). 11. SEAMEN, G. V. F., AND UHLENBRUCK, G., Arch. Biochem. Biophys. 100, 493 (1963). L., Acta Sot. Med. Upsalien. 12. SVENNERHOLM, 61, 75 (1956). L., J. Biol. Chem. 234, 1971 (1959). 13. WARREN, E., AND WEISS, L., Exptl. Cell Res. 14. MAYHEW, 60, 441 (1968). 15. HAYDON, D. A., AND SEAMAN, G. V. F., Arch. Biochem. Biophys. 122, 126 (1967). 16. BELL, R. P., AND CLUNIE, J. C., Trans. Faraday Sot. 48, 439 (1952).
ELECTROPHORESIS
OF HUMAN
17. SVENNERHOLM, L., B&him. Biophys. Ada 24, 604 (1957). 18. PONDER, E., “Hemolysis and Related Phenomena,” Grune & Stratton, New York (1948) . 19. GAUTHIER J., AND HAREL, P., Can. Med. Assoc. J. 97, 793 (1967). 20. SEAMAN, G. V. F., AND COOK, G. M. W., in “Cell Electrophoresis” (E. J. Ambrose, ed.), Churchill, London (1965). 21. EDSALL, J. T., AND WYMAN, J., “Biophysical Chemistry”, Vol. 1. Academic Press, New York (1958). 22. KRAEMER, P. M., Biochim. Biophys. Acta 167, 205 (1968). 23. SIZAMAN, G. V. F., JACKSON, L. J., AND UHLENBRUCK, G., Arch. Biochem. Biophys. 122, 605 (1967).
LEUKOCYTES
355
24. COOK, G. M. W., HEARD, D. H., AND SEAMAN, G. V. F., Nature 191, 44 (1961). 25. WALLACH, D. F. H., AND EYLAR, E. H., Biochim. Biophys. Acta 62, 594 (1961). 26. HACKEL, E., AND SMOLKER, R. E., Nature 18’7, 1036 (1960). 27. HACKEL, E., SMOLKER, R. E., AND FENSKE, S. A., Box Sanguinis 3,402 (1958). 28. BURKA, E. R., SCHREML, W., AND KICK, C. J., Biochemistry 6, 2840 (1967). 29. VASSAR, P. S., SEAMAN, G. V. F., AND BROOKS, D. E., Proc. Can. Cancer Res. Conf. 7th Honey Harbour, Ontario. p. 268. Macmillan (Permagon), New York (1967). 30. WEISS, L., AND MAYHEW, E., J. Cell Physiol. 68, 345 (1966). 31. WEISS, L., AND MAYHEW, E., J. Cell Physiol. 69, 281 (1967).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Electrophoresis
350-355 (1969)
136,
of Human
Leukocytes’
P. S. VASSAR AND h/I. J. KENDALL Department
of Pathology, Faculty
of Medicine,
University
of British
Columbia
Vancouver 9, British Columbia, Canada AND
G. V. F. SEAMAN Division of Neurology, University oj Oregon ilfedical School, Portland, Oregon 97301 Received July 10, 1969; accepted September 17, 1969 The peripheral regions of human polymorphonuclear leukocytes and erythrocytes contain at least two types of ionogenic group, a neuraminate ion and unidentified groups of respectively pKa. e 4.0 for the polymorphonuclear leukocytes and pK, N 3.4 for erythrocytes. These pK, values suggest p or r-carboxyl groups and ol-carboxyl groups of acidic amino acids in the membrane-protein for the polymorphonuclear leuckocytes and red blood cells respectively. The presence of ribonucleic acids could not be demonstrated in the peripheral regions of these cell types by electrophoretic methods. Both the intact polymorphonuclear leukocyte and erythrocyte lacked significant numbers of cationic groups at their surfaces.
Abramson (1) discussed the earlier literature on the electrophoresis of leukocytes. Bangham, Pethica, and Seaman (2) examined the effect of pH on the electrophoretie mobility of sheep polymorphonuclear leukocytes (PMN) and deduced the presence on the surface of a singly charged acidic group of pK, 3.7, probably a carboxyl group. No evidence for the presence of a significant number of basic groups could be found. Problems relating to the periphery of cells and intercellular phenomena have been recently reviewed (3, 4). MATERIALS
AND METHODS
Microelectrophoresis was performed as previously described (5). The majority of the experiments were performed in triplicate and the average t.aken of between 30 to 160 times of migration per experiment. All reagents were of analytical grade. Standard i This research was supported by Grant MA3040 from the Medical Research Council of Canada and by Grant CA-10422 from the National Cancer Institute.
saline consisted of 0.145 M aqueous sodium chloride solution with the pH adjusted to pH 7.2 f 0.2 by means of the addition of 0.5 M aqueous sodium bicarbonate solution. Isotonic aqueous solutions of hydrochloric acid or sodium hydroxide were added to standard saline for studies of variation of the electrophoretic mobilities of the cells with PH. The neuraminidase (Behringwerke) had an activity of 500 units/ml where 1 unit of activity is that quantity of enzyme which would liberate 1 H of sialic acid from a glyeoprotein substrate in 15 min at 37”. Neuraminidase as a crystalline receptor-destroying enzyme supplied by Dr. Ada was also used (6). Ribonuclease (Worthington, 3~ crystallized, beef pancreas, chromatographitally prepared) was found to have an activity of 1000 units/mg measured by the method of Kalnitsky et al. (7). Blood was obtained fresh from normal healthy donors of various blood groups using one-fortieth the volume of the blood of a 5y0 aqueous solution of disodium EDTA as the anticoagulant. All glassware was routinely siliconized. Leukocytes were separated from whole blood using isotonic aqueous ammonium chloride solution (8, 9). The separated leukocytes (1 vol) were finally washed twice with 50 vol of standard saline 350
ELECTROPHORESIS
OF HUMAN
at room temperature (1009; 7 min). Leukocytes washed in this manner will be termed “standard washed” cells. At this stage of the preparation the suspension of cells from the NH&l separations consisted of 859Ooj, polymorphonuclear leukocytes with no red blood cells or platelets remaining. It should be emphasized that the separated leukocytes have unimpaired viability (8, 9) and show a high phagocytic index for latex particles or st,arch grains in the presence of fresh serum and calcium ions. Red blood cells (RBC) were prepared for electrophoretic examination as described previously (10). Neuraminidase treatment. Standard washed and centrifugally packed human RBC and PMN were treated with neuraminidase as described by Seaman and Uhlenbruck (11). Control cells were incubated in 0.145 M aqueous sodium chloride solution containing 0.002 M aqueous calcium chloride solution or in this system containing the appropriate concentration of neuraminidase which had been inactivated by boiling for 20 min. The supernatant fluids from the treated cells and appropriate controls were analyzed for sialic acid by the methods of Svennerholm (12) and Warren (13). Cell counts were carried out in duplicate with a Coult.er counter. treatmenl. Packed and standard Ribonuclease washed human erythrocytes or polymorphonuclear leukocytes were treated with ribonuclease for 30 min at 37” (0.1 mg ribonuclease per ml of standard saline) using the procedure described by Mayhew and Weiss (14). Red blood cell suspensions were also treatedwith a 1 mg/ml solution of ribonuclease in standard saline. Control cells were incubated with standard saline. The enzyme-treated cells were washed in standard saline and then examined by microelectrophoresis. Acetaldehyde treatment. PMN were fixed for at
351
LEUKOCYTES
least 20 days in a 2% (w/v) solution of acetaldehyde in phosphate-buffered saline at pH 7.4. Preparation of this solution was previously described (15). The reagent grade acetaldehyde used was shown to be pure by spectroanalysis which revealed a single peak with a maximum at 278 pm (16). Fixed PMN were washed four times in 0.145 M aqueous NaCl before further procedures were performed. Electrophoretic mobilities were determined on the following: (a) acetaldehydetreated cells alone; (b) acetaldehyde-treated cells followed by neuraminidase treatment; and (e) neuraminidase-treated cells followed by acet.aldehyde fixation. RESULTS
All the mobility measurements reported in Table I were performed on “standard washed” cells suspended in standard saline, pH 7.2. The standard deviations are given with the number of measurements upon which a particular value is based in parentheses. The effects of neuraminidase, inactivated neuraminidase, and ribonuclease are listed. Only active neuraminidase produced a significant reduction in the mobility of the polymorphonuclear leukocytes (p < 0.001). The effects of the enzymes on the electrophoretic mobility of human erythrocytes are included in the table. Ribonuclease was also tried at a concentration of 1 mg/ml and found not to change the mobility of human erythrocytes. In Fig. 1 is given the pH versus electrophoretic mobility relationship for human PM/IN suspended in standard saline; open circles for the normal and full circles for the
TABLE
I
ELECTROPHORETIC MOBILITIES OF HUMAN RED BLOOD CELLS AND POLYMORPHONUCLEAR LEUKOCYTES PREPARED IN VARIOUS WAYS AND TREATED WITH NEURAMINIDASE AND RIBONUCLEASE CELLS SUSPENDED IN STANDARD SALINE, pH 7.2 f 0.2, 25” Electrophoretic mobilities, p/set/V/cm + SD Control not incubated 1 Inactive neuraminidase -0.90
f
0.037
PMN -1.08
(170) f 0.03
RBC
(160)
-0.82 f 0.028 0.001 < p < 0.01 (40) -1.08 f 0.03 p = 1.0 (30)
Neuraminidase -0.36 f 0.040 p < o.ool (110) -0.34 * 0.04 p < 0.001 (30)
Control for ribonuclease -0.87
+ 0.015
(‘33 -1.07
zk 0.03
Ribonuclease
, -0.88 f 0.021 0.4 > p > 0.3 (40) -1.07 f 0.03 p = 1.0 (30)
352
VASSAR, KENDALL, AND SEAMAN
Bulk pH
-o---o-o-d--A
_____ .., ____ A----p------A--A
FIG. 1. pH versus electrophoretic mobility relationships for human erythrocytes; normal (A----), neuraminidase-treated (A----) and polymorphonuclear leukocytes; normal (O-), and neuraminidase-treated (o-). Cells suspended in standard saline at 25”. TABLE II
absent in the untreated cells. The PMN, like the RBC, does not display a true isoelectric point as there is no detectable migration toward the cathode down to a pH of 1.8. The electrophoretic mobility of the untreated cell is constant between pH 5 and 10. Electrophoretic mobilities, &x/V/cm f standard deviation All measurements were obtained under conditions of electrokinetic stability, that is, AcetalAcetalphyde Neuraminidase the mobility under the given conditions did dehyde+ !2I$ incubated neuraminidase acetaldehyde not change with time and the changes in the control -~ -____ electrophoretic mobility produced by pH were completely reversible over the period -0.89 -0.90 -0.57 -0.56 10.032 +0.035 *to.035 &to.04 of time required to perform the measurep > 0.1 p < 0.001 p < 0.001 ments. PMN (130) (120) (220) (40) Neuraminidase treatment of the PMN and RBC released 2.0 and 0.4 pmoles of sialic acid per lOlo cells respectively, as asneuraminidase-treated PMN. The pH versayed by both the Svennerholm (17) and sus mobility relationship for RBC’s are Warren (13) methods. The theoretical represented by dashed lines with open triquantities of membrane-bound sialic acid angles for the normal and full triangles for released by neuraminidase were calculated the neuraminidase-treated RBC. The mean on the assumption that each electron charge pK, for the PMN surface is approximately lost at the cell periphery corresponded to one 3.4, whereas the pK for the acidic groupings neuraminate ion. The molecular weight of which remain after neuraminidase treatment the sialic acid was taken as 309 (N-acetyl is about 4.0. The mean pK, for the RBC derivative). The mean area of the RBC was surface is 2.9 and of the anionic groupings taken as 1.63 X 1O-6 cm2 (18) and that of the left after neuraminidase treatment the pK, PMN as 2.84 X 10-6cm2 as calculated from is approximately 3.4. Both the neuraminidase-treated PMN and RBC have some an assumed volume of 4.5 X lo-lo cm3 (19). The calculated charge density decreases cationic character as evidenced by positive after neuraminidase treatment implied that branches to the pH versus mobility relationships at low pH; this cationic character is 0.2 pmoles and 0.15 Fmoles of sialic acid ELECTROPHORETIC MOHILITIES OF POLYMORPHONUCLEAR LEUKOCY~FES TREATED WITH ACETALDEHYDE AND NEURAMINIDASE CELLS SUSPENDED IN STANDARD SALINE, pH 7.2 + 0.2, 25”
ELECTROPHORESIS
OF HUMAN
were released from lOlo cells of PMN and RBC respectively. Electrophoretic mobilities of acetaldehyde-treatNed PMN are shown in Table II. On comparing these results with those in Table I it can be seen that: (a) acetaldehyde fixation has no effect on the electrophoretic mobility of the normal PMN; (b) acetaldehyde treatment after neuraminidase reveals an increase in negative mobility of about 22 % compared with neuraminidasetreated P&IN alone; and (c) acetaldehyde treatment shows similar results whether cells are fixed before or after neuraminidase treatment. DISCUSSION
The mean pK, value for the human polymorphonuclear leukocytes obtained from the pH at which the electrophoretic mobility was one half of the plateaux value suggests that the ionogenic groups are probably carboxy1 groups. The constant mobility between pH 5.0 and 10.0 and the lack of a positive branch to the pH versus mobility curve at low pH suggests the absence of a significant number of basic groups such as amino groups. Treatment of the P-MN’s with neuraminidase produces about a 60% decrease in the electrokinetic charge density at neutral pH with the release of a membranebound sialic acid. The changes which occur in red blood cell charge after treatment with neuraminidase are already well documented Gw Comparison of the normal and neuraminidase-treated RBC (Fig. 1) shows that neuraminate ions constitute about 60% of the anionic groups on the untreated erythrocyte. After these groups have been removed by neuraminidase the remaining 40% have a pK, of about 3.4. Svennerholm (12) estimated the pK, of free N-acetylneuraminic acid to be about 2.6, a value close to that estimated for the membrane-bound neuraminate ion (15). A survey of literature values shows that this figure is close to the pK, of a-carboxyl groups in polypeptides or proteins. The pK, of the surface groups which remain on the PMN after neuraminidase treatment is circa 4.0 and close to that for the y-carboxylic acid groups of glutamic or
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&carboxylic acid groups of aspartic acid (21). Although the nature of the ionogenic groups in the peripheral regions of the RBC and PMN which are not neuraminate ions is unknown, it can be inferred from the pK, values that these groups are probably carboxylate ions originating from the acidic amino acids in the membrane protein. Any changes which occur in the surface properties of cells after treatment with an enzyme must be evaluated carefully since these alterations could have arisen in ways other than by simple enzyme action (11). Kraemer (22) has questioned the purity of commercially available neuraminidase. He found that neuraminidase from Clostridium perfrinyens contains cytotoxic, hemolytic, and phospholipase contaminants. However, the neuraminidase samples used in our studies on membrane-bound sialic acid were obtained from Vibrio choler& and do not contain the same contaminants (22), nor any detectable proteolytic activity when subjected to the Azocoll test (23). The small net positive electrokinetic charge carried by the neuraminidase-treated PMN and RBC at low pH (Fig. 1) is attributed to conformational changes in the cellular periphery. Both the normal and neuraminidase-treated PMN show electrokinetic reversibility over the range pH 2-10 which renders unlikely the irreversible adsorption of intracellular leakage products at the extremes of pH. The absence of significant proteolytic activity in the neuraminidase makes the origin of the positive character of the neuraminidase-treated PMN by proteolysis of membrane protein improbable. Adsorption of the enzyme or of any impurities in the enzyme although possible is considered unlikely as an explanation for the positive character of the PMN and RBC at low pH (24). Neuraminidase releases about 2.0 clmoles of sialic acid per lOlo PMN. The theoretical yield of sialic acid was calculated to be 0.2 pmoles on the basis of the change in electrokinetic charge produced by neuraminidase treatment, assuming that the sialic acid when bound in the membrane has its carboxylic acid group fully effective at the electrophoretic plane of shear. The IO-fold divergence between the analytical and theo-
354
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KENDALL,
retical yields indicates that sialic acid is being released from sites deep with respect to the electrophoretic plane of shear and even perhaps from intracellular organelles (25). The similar though smaller discrepancy between the experimental and theoretical yields of sialic acid from neuraminidasetreated human RBC has been discussed in previous publications (11, 24). The observations of Hackel and Smolker (26) that ribonuclease affects the Rh and Lu antigenic sites on human erythrocytes and, furthermore, that anti-Rh and antiLutheran sera are inhibited by ribonucleic acid derivatives (27) suggested that ribonucleic acids might be associated with the peripheral regions of cells. More recently Burka et nl. (28) have shown that a membrane-bound RNA is present in mammalian erythroid cells. Vassar et al. (29) found that ribonuclease did not alter the eleetrophoretic properties of a number of cell types but a series of recent studies which indicate the probable presence of surface RNA in cellular systems has prompted us to re-examine the action of ribonuclease on the cellular electrophoretic properties of the human RBC and PMN (14, 30, 31). Ribonuclease appeared to be without significant influence on the electrophoretic properties of both types of cell. The absence of a change in the electrophoretic mobility of normal PMN after their treatment with acetaldehyde implies the absence of an appreciable number of cationogenic groups in the cellular peripheral region of this blood element. For changes in the electrophoretic mobility to be significant the mobility values would have to differ from the control values by at least 5 %. Now the normal human PMN has about 1.8 X 10’ anionogenic sites, so that the presence of up to 4.5 X lo5 (5% of the anionic sites) cationic sites would not be detected by electrophoretic methods. Treatment of the PMN with neuraminidase, in -addition to producing about a 60% decrease in the anodic mobility at pH 7, also leads to the appearance of a significant number of cationogenic groups as may be seen from an examination of the pH versus mobility relationship particularly in the low pH region. Treatment of the neuraminidase-modified
AND
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PMN with acetaldehyde leads to an increase in the anodic mobility (Table II) up to the value obtained by neuraminidase treatment of acetaldehyde-fixed cells. The reason for the appearance of the cationic groups after treatment of the PMN with neuraminidase is unknown since it is a glycosidase lacking any significant protease activity. The results presented here show that the peripheral region of PMN and RBC contain at least two types of ionogenic group, a neuraminate ion and an unidentified ionogenie group, probably a carboxyl group from the acidic amino acids in the membrane protein. No ribonucleic acids could be demonstrated in the peripheral regions of these cell types by electrophoretic methods. Both the intact RBC and PMN lacked any significant number of cationogenic groups in their cell peripheries. REFERENCES 1. ABRaMSON, H. A., J. Exp. Med. 46, 987 (1927). 2. BANGHAM, A. D., PETHICA, B. A., AND SEAMAN, G. V. F., Biochem. J. 69, 12 (1958). 3. BROOKS, D. E., MILLAR, J. S., SEAMAN, G. V. F., AND VASS.~R, P. S., J. Cell Physiol. 69, 155 (1967). 4. WEISS, L. “ The Cell Periphery, Metastasis and Other Contact Phenomena,” North-Holland Publ., Amsterdam (1967). SEAMAN, G. V. F., AND HE.IRD, D. H., Blood 18, 599 (1961). AD.~, G. L., .\ND FRENCH, L. E., Nature 183, 1740 (1959). KALNITSKY, G., HUMMEL, J. P., AND DIERKS, C., J. Biol. Chem. 234.1512 (1959). DIOGUARDI, N., AGOSTINI, A., FIORBLLI, G., AND LOMANTO, B., J. Lab. Clin. Med. 61, 713 (1963). 9. CARRUTHERS, B. M., Can. J. Physiol. Pharmacol. 44, 475 (1966). 10. SEAMAN, G. V. F., AND HEARD, D. H., J. Gen. Physiol. 44, 251 (1960). 11. SEAMEN, G. V. F., AND UHLENBRUCK, G., Arch. Biochem. Biophys. 100, 493 (1963). L., Acta Sot. Med. Upsalien. 12. SVENNERHOLM, 61, 75 (1956). L., J. Biol. Chem. 234, 1971 (1959). 13. WARREN, E., AND WEISS, L., Exptl. Cell Res. 14. MAYHEW, 60, 441 (1968). 15. HAYDON, D. A., AND SEAMAN, G. V. F., Arch. Biochem. Biophys. 122, 126 (1967). 16. BELL, R. P., AND CLUNIE, J. C., Trans. Faraday Sot. 48, 439 (1952).
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17. SVENNERHOLM, L., B&him. Biophys. Ada 24, 604 (1957). 18. PONDER, E., “Hemolysis and Related Phenomena,” Grune & Stratton, New York (1948) . 19. GAUTHIER J., AND HAREL, P., Can. Med. Assoc. J. 97, 793 (1967). 20. SEAMAN, G. V. F., AND COOK, G. M. W., in “Cell Electrophoresis” (E. J. Ambrose, ed.), Churchill, London (1965). 21. EDSALL, J. T., AND WYMAN, J., “Biophysical Chemistry”, Vol. 1. Academic Press, New York (1958). 22. KRAEMER, P. M., Biochim. Biophys. Acta 167, 205 (1968). 23. SIZAMAN, G. V. F., JACKSON, L. J., AND UHLENBRUCK, G., Arch. Biochem. Biophys. 122, 605 (1967).
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24. COOK, G. M. W., HEARD, D. H., AND SEAMAN, G. V. F., Nature 191, 44 (1961). 25. WALLACH, D. F. H., AND EYLAR, E. H., Biochim. Biophys. Acta 62, 594 (1961). 26. HACKEL, E., AND SMOLKER, R. E., Nature 18’7, 1036 (1960). 27. HACKEL, E., SMOLKER, R. E., AND FENSKE, S. A., Box Sanguinis 3,402 (1958). 28. BURKA, E. R., SCHREML, W., AND KICK, C. J., Biochemistry 6, 2840 (1967). 29. VASSAR, P. S., SEAMAN, G. V. F., AND BROOKS, D. E., Proc. Can. Cancer Res. Conf. 7th Honey Harbour, Ontario. p. 268. Macmillan (Permagon), New York (1967). 30. WEISS, L., AND MAYHEW, E., J. Cell Physiol. 68, 345 (1966). 31. WEISS, L., AND MAYHEW, E., J. Cell Physiol. 69, 281 (1967).