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Osmotic hemolysis contrasted with freeze-thaw hemolysis
CRYOBIOLOGY,
8, 14-24
OSMOTIC
HEMOLYSIS
CONTRASTED
WITH
FREEZE-THAW
HEMOLYSIS’
D. B. PRIBOR Biology
Department,
The University
of Toledo and Research Associate,
Medical Research of The Toledo Hospital, Since hemolysis involves a change in permeability of the plasma membrane to macromolecules, this process must be due to some structuralfunctional change in the membrane. For osmotic hemolysis this change is not a mere bursting of the red cell, but rather it results from a sudden increase in permeability of the entire membrane to all molecules (19). Also, if the membrane develops holes during hemolysis, it repairs itself soon after (31) regaining its impermeability to macromolecules (3) and under appropriate conditions, its relative impermeability to cations (8, 36). Thus, osmotic hemolysis, at least, is due to a membrane alteration which to some extent is reversible. A complete understanding of the mechanism of hemolysis, therefore, requires an understanding of the structural-functional integrity of the red cell membrane. Freezing blood, below -3°C and thawing it without protective agents always brings about some red cell lysis (26). Such red cell lysis usually is interpreted as freeze-thaw damage to the cells. Therefore, to understand the mechanism of freeze-thaw damage to erythrocytes we need to understand the mechanism of hemolysis which in turn requires a better understanding of membrane structure. In recent years the classical theory for membrane structure originally proposed by Danielli and Davson (2) and later modified by Robertson (27) has been challanged by several investigators (6, 7, 14, 37). Korn (11, 12) suggests that no membrane model to date has been adequately proved or disproved. Thus, the field of membrane physiology is in a state of flux. It is for this reason, probably, that there has been a revival of interest in studying erythrocytes. One can isolate from them relatively intact membrane fragments or a complete membrane system-the
Toledo,
Znstitute of
Ohio @SO6
reconstituted ghost. Research which attempts to clarify the mechanism of freeze-thaw damage to erythrocytes may at the same time give new insight to the structural-functional integrity of the red cell membrane and to membranes in general. The red cell semipermeability properties and its biconcave disc shape (5, 15, 20, 24) probably are characteristic of the plasma membrane as a whole. The total membrane ATPase system, consisting of Na-K-Mg-activated ATPase (hereafter referred to as transport ATPase) and the Mg-activated ATPase (hereafter referred to as Mg-ATPase), does not require the complete membrane to be intact (1, 21). However, it does require intact membrane fragments as opposed to solubilized enzymes or even severely disrupted membrane fragments (1, 28,29). Studies of discsphere transformations in erythrocytes strongly imply that the membrane may exist in a number of metastable equilibrium states. Also, the lipoprotein subunit theory for membrane structure implies that all membranes may continuously transform from one metastable equilibrium state to another (6, 10, 12). Using this as a working hypothesis, studies on different types of red cell ghosts of semipermeability changes, capacity for shape changes and changes in ATPase activity, could indicate to what extent the equilibrium state of the membrane had been altered by the process that brought about the hemolysis. In a general way this report may provide additional evidence for the red cell membrane existing in a number of metastable equilibrium states. In particular an attempt is made to answer two questions: (1) Is freeze-thaw hemolysis due to a change in the red cell permeability which subsequently leads to osmotic hemolysis upon thawing. In other words, is freeze-thaw hemolysis simply a special case of colloidal osmotic hemolysis; (2) Does the freeze-thaw process irreversibly disorganize the red cell membrane or does it transform the membrane to a metastable equilibrium state which may be reconverted to a more normal state? The approach used will be to compare ghosts
Received July 13, 1970. 1 Supported by Grant HE12114 (NIH) and a joint, grant from the Institute of Medical Research of The Toledo Hospital and the Medical and Research Foundation of Perth Amboy General Hospital. 14
FREEZE-THAW
VS. OSMOTIC
produced by osmotic hemolysis in the presence of Mg ions (hereafter referred to as Mg ghosts) with ghosts produced by slow free5ing and thawing (hereafter referred to as freezr+thaw ghosts). The properties selected for comparison are: (1) gross structure as seen through the phase microscope, (2) capability of regaining or transforming to the disc shape, and (3) ATPase activity. MATERULB
AND METHODS
Red cells. In all but one set of experiments, cells were obtained from blood freshly drawn by veniptmcture from normal male and female subjects into tubes containing dry heparin (0.3 mg/ml of blood). In the experiment where osmotic ghosts were frozen and thawed and then glycerolated, red cells were obtained from blood drawn from patients at The Toledo Hospital, washed with buffered salt solution, and then pooled.* &&red mlt solution. Throughout the results reference is made to buffered salt solution. This solution contained 150 rnM NaCl, 1 mM MgCIZ. 6H&, 1 rnM Tris-HCl, and enough 0.1 N HCl to make the pH equal 7.4. When ghosts were stored overnight in this solution, a trace of cysteine was added. Special Procedures In the results some procedures either are selfevident or are included along with the report of the results. Special procedures are given below. Preparti of bloodfor hemo1ysi.s. The blood was aentrifuged at SO@for 10 min. The plasma and bt@y layer containing white cells was discarded and the paoked red cell resuspended in buffered salt solution. The cells were then washed three times with the buffered salt solution; after the final washing the supernate was discarded. Pr~bifm of oSn30tkgkmts (Mg gho8t8). One volume of packed cells was rapidly plunged into 5 vol of rapidly stirred uold distilled water containing 1 mu MgClz .SHnOand enough Tris bu&r to adjust the pH to 7.4. This mixture was left to stand stirring in the 4°C cold room for 45 min. z The chemicals, sodium heparinate, d&odium adenosine triphosphate (ATP) , disodium ethylene diamino tartrraeetste (EDTA), ouabain, ~-cyst+ ine, and the reagenti for the ATPase determinations, were obtained from Sigma Chemical Co., St. Louis, MO. The ATP was made up into a 0.1 M stock solution. The ATP solution was adjusted to pH 7.0 with NaOH and stored in the frozen state. Cysteine solutions were discarded after 2 days.
HEMOLYSIS
15
P~~~ti~byfreeairag~tkasrving @wea4mw ghosts). A 39ml Pyrex oe&ifuge tube containing 1 vol of paeked cells and 9 vol of buffered salt solution (the final volume of ghost suspension was about 20 ml) was placed in an alcohol bath previously cooled to -20°C. The red cell suspension froze in a few minutes, It was kept in the frozen state for 30 min, thawed in a water bath at 37”C, centrifuged at 16,990gfor 10 min, and resuspended in buffered salt solution. Preparationof ghost SuSpensionS Of known wnc&rations. Ghost suspensions were counted by means of a phase hemacytometer. Counts were made in each of the two chambers of the hemacytometers and were repeated if the counts did not come within 5 y0 of one another. G~ycerokation of ghQ8t8. In various experiments ghosts were suspended in a 45 $J glycerol solution (v/v) using a 0.01 M Tris-HCl buBer instead of distilled water. The pH was adjusted to 7.4. The ghosts were stored in this solution for 20-24 hr at 4”C, centrifuged at 1O,OOOg, and resuspended in buffered salt solution. Rehemolysis experiments. To 1 vol of ghost suspension containing log ghosts/ml was added 9 vol of a rehemolyzing buffer solution. Three types rehemolyzing solutions were used: (1) control which consisted of 0.01 M Tris-HCl, 0.605 M MgClzm6Hz0, 0.0175 M NaCI; (2) ATP solution which consisted of 0.01 M Tris-HCl, 0.005 M MgClz.6Ha0, 0.01 M NaCI, 0.005 M ATP; (3) EDTA solution which consisted of 0.01 M TrisHCI, 0.0175 M NaCl, 0.005 M EDTA. All solutions were adjusted to pH 7.4. The rehemolysis was done in test tubes at room temperature. The ghost suspensions were thoroughly mixed and either incubated to 37°C for 0.5 hr, centrifuged at 16,06Og,and resuspended in buffered salt solution or allowed to stand for 10 min and then reconstituted. Recanztitution of ghosts. An attempt was made to reconstitute the Mg ghosts and freeze-thaw ghosts by resuspending them in buffered salt solution immediately after hemolysis. This departure from the usual procedure of reconstituting ghosts was necessary so the Mg ghosts and freeze-thaw ghosts would receive the same treatment. However, Mg ghosts and freeze-thaw ghosts which had been glycerolated, resuspended in buffered salt solution, and then rehemolyzed were reconstituted (“reversal of hemolysis”) in the usual manner following the procedure of
16
PRIBOR
Schatzmann (30). Enough solid I~(‘1 was added to the suspensions of rehemolyzed cells to bring t’he concentration to 0.15 M. ATPase activity. ATPase activity was assayed by mea.suring liberated inorganic phosphate (Pi) by a modified Fiske-SubbaRow procedure. The final 1 ml of ghost suspension to be incubated at 37°C for 1 hr contained 118 m&f NaCl, 14 mM buffer, 1 InM L-cysteine, KCl, 20 mM Tris-HCl 0.05 IBM EDTA, 3mM XIg<%~6Hz0, 3 m&I ATP, and 1 X log ghosts. Tubes to be assayed for MgATPase activity also coutained 1 x low2 mM ouabain. The transport ATPase activity was determined by subtracting Rig-ATPase activity from total ATPase activity. d zero-time tube served as an ATP blank and correction for residual phosphate in the ghosts. In this run ATL’ was added to the incubation tube containing ghosbs just prior to killing the reaction with cold TCA. An insignificant percentage of ATP is hydrolyzed during 1-hr incubation at 37°C. All assays were carried out in duplicate. For the .\TPase activities of Rig ghosts which were subsequently frozen and thawed, an ATP blank was used rather than a zero time. The reported ATI’ase activities in this experiment, therefore, probably are somewhat higher than they should be. Photomicroscopy. The ghosts were viewed and photographed through a Nikon phase microscope using a 40 x DLL phase objective.
RF;SULTS
Effect of Freeze-Thaw Process on drg Ghosts and on Vnhemolyzed Cells Mg ghosts tended to be incompletely hemolyzed and to retain the biconcave disc shape (Fig IA). If these ghosts were slowly frozen and thawed, many of them were incompletely hemolyzed and retained the disc shape (Fig. 1B). This was in marked c*ontrast to unhemolyzcd cells from the same blood which were frozen and thawed under the same conditions as the 14g ghosts. The resultant freeze-thaw ghosts were more transparent indicating a more complete hemolysis. They were spherical in shape and showed signs of fragmentation (Fig. l(y). Thus freeze-thawed, unhemolyzed red cells appeared more altered than freeze-thawed Mg ghosts. The freeze-thaw process, however, produced some alteration in the Mg ghosts as indicated by two other experiments. First of all, freeze-thawed Mg ghosts had a higher ATPax activity than nonfrozen Mg ghosts (Table 1). .41x), the freezethawed Mg ghosts fragmented when incubated at 37°C without ATP. This fragmentation was inhibited when 3 mM liTI’ was present, t’hough some fragmentation was still evident. In addition, both types of ghosts were suspended in Trisbuffered 45”/ glycerol solution, stored at 4°C for 12-24 hr, and then resuspended in buffered salt
FIG. 1. Effect of the frer :xe-thaw pr~ess OII Mg ghosts vers, IS its cfrect on Imhemolyzed red cells. A. Mg ghosts; I3 Mg ghosts which were frozen and thawed; C. Vrozewthawed \lnhemolyzed red cells.
FREEZE-THAW
VS. OSMOTIC TABLE
ATPASE
ACTIVITY
Mg ghosts Freeze-thawed ghosts
Mg
Calculated t value for di&renoe of the means
1
OF Mg GHOSTS AND FREEZE-THAWED
Total ATPase
17
HEMOLYSIS
Mg-ATPase
Mg Gaosrsa Transport ATPsse
Mean
SD
Mean
SD
Meful
SD
0.1160 0.2323
0.0557 0.0529
0.0940 0.1481
0.0121 0.0261
0.0220 0.0842
0.0094 0.0387
3.6777
4.6053
3.8281
QThe ATPase activity is expressed in terms of micromoles of inorganic. phosphate per 100 ghosts per hour at 37’C. Three experiments were performed in duplicate; so the means are estimated from an average of six determinations. Note: the t value from the tables for Ni + Nz - 2 = 10 degrees of freedom above which the two means are significantly different at the 1% confidence level is 3.169; for the 6% confidence level the t value is 2.228.
solution. In aliquots taken from packed cells after centrifugation and removal of glycerol supernate, but before resuspension in salt buffer, the Mg ghosts were in&t and many were biconcave discs. In the freeae-thawed Mg ghosts aliquot, however, there were many cell fragments. Upon rehemolysis and resuspension in the b&e& salt solution, many Mg ghosts retained their biconcave shape and showed no evidence of fragmentation. The remaining nonfragmented, freezethawed Mg ghosts were very transparent and spherical in shape.
ghost.9/ml), mmy of t&e Mg ghosts were disc abaped(li”ig,w. salt b&r to give nated spheres. Fur resulted in more and more of them becoming
FIQ. 2. Mg ghosts and freeze-thaw ghosts immediateIy after ysis and resuspension in buffered isotonic salt solution with concentration adjusted to 101 ghosts/ml. A. Mg ghosts; B. freeze-thaw ghosts.
18
PRIBOP,
spherical in shape. Furthermore, if these ghosts were stored at 4°C for 1-3 days, almost all of them became spherical and several apparently swelled and lost more hemoglobin. The freezethaw ghosts immediately after hemolysis and suspension in buffered salt solution (lo9 ghosts/ ml) appeared as smooth spheres of relatively uniform size (Fig. 2B). There was an occasional unhemolyzed cell and some disc-shaped ghosts. Dilution of this suspension resulted in no obvious change in the ghosts escept’ that the unhemolyzed ghosts became spherical. Furt,her washing and cold storage of these ghosts result’ed in some of them breaking up into microspheres. After cold storage and three washes with salt buffer, both Mg ghosts and freeze-thaw ghosts were spherical but differed in that: (1) at least a few Ng ghosts were disc shaped \vhei cas 11011e of the freeze-thaw ghosts were, (2) the i\lg gh:)sts apl)ea*ed daikcr ad easier to see uutlc: l)h:Lse than the freezethaw ghosts, and (3) there was 11’) evidence of
fragmentation of Mg ghosts whereas cell fragments appeared among the freeze-thaw ghosts, Morphological response of Mg ghosts and jreezefhaw ghosts to ATP. Whereas cold-stored, washed
Mg ghosts and freeze-thalv ghosts appeared t,o be similar, they differed considerably in their stability. When Xg ghosts were incubated at 37°C for 0.5 hr, there were no signs of fragmentntion, but when freeze-thaw ghosts were similarly treat,ed a ilumber of microspheres of various sizes appeared among the ghosts (Fig. 3A and C). Furthermore, when Mg ghosts were incubated with 30 pmoles of XTP/lO~ ghosts, they either apl)arently “contracted” to a smaller volume or became disc shaljed. Freeze-thaw ghosts exposed to ll’l?l’ under the same conditions, however, fragmented into microsphere;j of various sizes OI formed what apl)earetl to be membrane filaments (Fig. 31s am1 I>). In five experiments each done in tlul~lieate, 30 pmoles c,f ATI’ always caused the frrczc-thaw ghosts to fragment. The effect
FIG. 3. Effect of 30 @moles of ATP/lOS ghosts on washed, cold-stored Mg ghosts and freeze-thaw ghosts. A. Mg ghosts (control) after 0.5 hr 37°C; B. Mg ghosts plus 30 pmoles ATP after 0.5 hr 37°C; C. freeze-thaw ghosts (control) after 0.5 hr 37°C; I). freezethaw ghosts p111s 30 Mmoles ATP after 0.5 hr 37°C.
FREEZE-THAW
VS. OSMOTIC
of ATP on Mg ghosts was variable. In one experiment the ghosts were greatly contracted but there was no shape transformations. In another experiment the predominant effect was a change in shape from spheres to discs or cups. In the other three experiments the effect consisted of both apparent contractions and shape transformations. In an attempt to obtain more consistent results with the effect of ATP on Mg ghosts, Mg ghosts and freeze-thaw ghosts were rehemolyzed in a dilute Tris-HCl buffer containing MgClz and ATP, or just MgC&, or EDTA. In this regard the results were somewhat disappointing but they did show a dramatic difference between Mg ghosts and freeze-thaw ghosts. Four experiments using blood from three different donors were run in duplicate for a total of eight determinations. In all eight determinations Mg ghosts rehemolyzed with ATP or EDTA appeared as discs or crenated spheres. In five of the eight runs the Mg ghosts rehemolyzed with MgClt were crenated spheres. In the other three runs, there were several discs. In all cases,when the rehemolyzed Mg ghosts were resuspended in buffered salt solution, they remained disc shaped or became discs, crenated discs or crenated and apparently shrunken spheres. Freeze-thaw ghosts remained spherical when diluted with buffered salt solution and when rehemolyzed in the presence of ATP, MgCl,, or EDTA. The rehemolyzed ghosts appeared more transparent. Some of the ghosts rehemolyzed with ATP or EDTA still remained relatively smooth spheres when resuspended in buffered salt solution in contrast to some of the ghosts rehemolyzed with MgClz which appeared shrunken and crenated. In general, when the rehemolyzed freeze-thaw ghosts were resuspended in buffered salt solution, in all casesthey tended to break up into microspheres and the nonfragmented ghosts remained spherical. Response of Mg ghosts and freezt&aw ghosts to bu$ered 46% glycerol solution. This experiment was done to seewhether under any circumstances the freeze-thaw ghosts still are able to take on the disc shape. As usual the freeze-thaw ghosts were spherical and more hemolyzed than the Mg ghosts. Immediately after suspension in 45~~ glycerol the Mg ghosts and the freeze-thaw ghosts were thin discs (Fig. 4A and B). After 20-24 hr in glycerol, the ghosts were resuspended in buffered salt solution. Here both types of ghosts
HEMOLYSIS
19
were smooth spheres. They were then rehemolyzed with ATP, MgClz, or EDTA, allowed to stand for 5-10 mm, and finally reconstituted by adding KC1 to bring the concentration up to 0.15 M. As with the previous rehemolysis experiments, the populations of Mg ghosts rehemolyzed in the three different ways contained crenated spheres and discs and smooth discs (Fig. 4C). The freezethaw ghosts were very transparent and diflicult to see. Of those exposed to ATP and then reconstituted, many were thick discs (Fig. 4D). Of those exposed to MgClz, many were spheres but there were some discs, while of those exposed to EDTA, all were spheres. This experiment was repeated four times with blood from four different donors. ATPase activity of Mg ghosts and freeze-thaw ghosts. As is seen in Table 2, the total ATPase activity and the Mg ATPase activity of freea+ thaw ghosts were significantly higher at the 1% confidence level than that of the Mg ghosts. In contrast, the transport ATPase of freeze-thaw ghosts was significantly higher than that of Mg ghosts only at the 5% confidence level. Stated in a different way, the ratios of ATPase activity of Mg ghosts to freeze-thaw ghosts are as follows: total ATPase, l/2.1; Mg-ATPase, l/2.8; transport ATPase, l/1.5. Thus, the freeze-thaw processappeared to increase the Mg-ATPase activity to a greater extent than it increased the transport ATPase activity. DISCUSSION
Site of Freeze-Thaw Damage: The Whole Cell vs. the Plasma Membrane
In comparing freeze-thawed, unhemolyzed cells to freeze-thawed Mg ghosts morphologically it appears that unhemolyzed cells are damaged to a greater extent than Mg ghosts (Fig. 1). However, freezing and thawing does alter the Mg ghost membrane, for some of these ghosts break up into microspheres (Fig. 1B) ; the ATPase activity is increased (Table l), and they are more unstable, i.e., they tend to break up into microspheres when suspended in buffered 45% glycerol solution and subsequently resuspended in buffered salt solution. Thus the freeze-thaw process does alter the red cell membrane. This is consistent with evidence dealing with other types of cells that the plasma membrane is the site of freeze-thaw damage (22, 23,32).
20
PRIBOR
FIG. 4. Effect of glycerolation, resuspension, rehemolysis and thell reronstitlltion with KC1 011 Mg ghosts and freeze-thaw ghosts. A. Mg ghosts immediately after sllspension in buffered 15% glycerol solution; B. freeze-thaw ghosts immcdi:rtely after sllspension in buffered 45yc glycerol sollltion; C. Mg ghosts resuspended in 0.17 hf salt bllffer, rehemolyzed in the presence of ATP and MgCl? then reconstitrlted with KCl; I). freeze-thaw ghosts resuspended in 0.17 M salt buffer, rehemolyzed in the presence of AT]’ and MgCla then reconstituted with KCl. Extent
oj” Freeze-Thaw Damage to the Red Cell Membrane
Gross structural integrity and stability of the plasma nzembrane oj freeze-thaw ghosts. Immedi-
ately after hemolysis freeze-thaw ghosts are more hemolyzed than 14g ghosts and are spherical a3 opposed to the disc shape of LIg ghosts (Fig. 2). After three washings wit,h buffered salt solution and cold storage, many freeze-t,haw ghosts appear to be intact. However, some change has occurred in the membrane of these ghosts, for when subjected to various kinds of stress the) tend to break up into microspheres whereas >Ig ghosts are relatively unaffected. Freeze-thaw ghosts tend to fragment when washed with buffered salt solution and when rehemolyzed and resuspended in salt buffer, whereas hlg ghosts become crenated discs. Freeze-thaw ghosts COW
pletely fragment when cxpo~ed to 30 ~.lmoles ATP/lOg ghosts at 37’Y: for 0.5 hr, whereas hlg ghosts contract or become discs. Thus freezethaw ghosts arc structurally intact but relatively unstable in contrast to 11g ghosts which are quite stable.
Increased ATI’ase activit,y of freeze-thaw ghosts. It is often assumed that the freeze-thaw process increases LITl’ase activity of red cell ghosts by increasing the membrane permeability to hT1’ and thus making hT1’ more available to this enzyme (4). JIowever, freezing and thawing brings about some fragmrntation. Some microsphcrcs resulting from fragmentation contain hemoglobin and apparently have regained at least some of their semipermeability (25). If there is a net increase in membrane permeability, it may not be as great as originally proposed.
21
FREEZE-THAW VS. OSMOTIC HEMOLYSIS
ATPASE
ACTIVITY
OF
TABLE 2 Mg GHOSTS AND FREEZE-THAW
Total ATPase
l&g ghosts PPraeze-thawghosts C&uleted t value for &fference of the means
GHOSTS” Transport ATPme
Mg-ATPase
Meall
SD
Mobil
SD
MeaIl
SD
0.0790 0.1735
0.0239 0.0616
0.0385 0.1098
0.0192 0.0566
0.0405 0.0636
0.0132 0.0258
4.9355
4.1191
2.7737
6 The ATPase activity is expressed in terms of micromoles of inorganic phosphate per lo@ghosts per hour at 37°C. Six experiments were performed in duplicate; so the means are estimated from an average of 12 determinations. Note: the t value from the tables for N1 + Nn - 2 = 22 degreesof freedom above which the two means are significantly different at the 1% confidence level is 2.819;for the 5% confidence level the t value is 2.074. In the experiments reported in this paper freezing and thawing increases Mg-ATPase activity to a significantly greater extent than transport ATPase activity. According to Marchesi (16) both of these ATPase systems are located on the inner surface of the membrane. If this is the case, with increased permeability the total ATPase activity would increase but the ratio of Mg-ATPase to transport ATPase should remain the same. On the other hand, according to Hoffman (9) and Novikoff (18) the Mg-ATPase is located on the outer membrane surface. If this is correct, then the increased permeability would not account for the marked increase of Mg-ATPase. An alternate hypothesis consistent with this data is that freezing and thawing increase transport ATPase by increasing cell membrane permeability, and it increases Mg-ATPase activity by some other means such as altering the three-dimensional configuration of the membrane proteins or changing the membrane fixed charge distribution. Volume changes of ghosts. Rehemolyzed Mg ghosts appear to shrink when resuspended in buffered salt solution whereas freeze-thaw ghosts treated in the same way tend to fragment or remain spherical. Furthermore, in some preliminary experiments where volume changes of ghosts were determined by a hematocrit method, Mg ghosts had a smaller volume than freeze-thaw ghosts in a buffered 0.17 M NaCl solution. The Mg ghosts increased in volume when placed in a 0.05 M NaCl solution and decreased in volume in a 0.5 M NaCl solution. This is in line with the work of Hoffman (8) and Teorell (36) who reported that osmotic ghosts regain their semipermeability
and low permeability to ions. In contrast, the freeze-thaw ghosts do not increase in volume in the hypotonic solution but do decreasein volume in the hypertonic solution. The experiments should be repeated to determine whether the decrease in hematocrit of the freeze-thaw ghosts is due to fragmentation, increased cell packing, or to a real cell shrinkage. Nevertheless, it appears that Mg ghosts are osmotically active whereas the freeze-thaw ghosts are not. Thus, the freezethaw process has altered the red cell membrane to the extent that under the reconstituting procedure employed, freeze-thaw ghasts do not completely regain their semipermeability. Shape chxcnges of Mg ghosts and freeze-thaw ghosts. It has already been reported that immedi-
ately a.fter hemolysis osmotic ghosts spontaneously take on the biconcave disc shape (3, 8, 38). Likewise, Mg ghosts spontaneously take on the biconcave disc shape immediately after hemolysis and resuspension in buffered salt solution and after rehemolysis as well. Freeze-thaw ghosts, on the other hand, remain spherical and tend to fragment, particularly after rehemolysis and resuspension in buffered salt solution. Also, washed and cold-stored Mg ghosts contract or take on a cup or biconcave disc shape when incubated in the presence of 30 pmoles ATP/lOO ghosts, whereas similarly treated freeze-thaw ghosts from the same blood break up into microspheres or membrane filaments. Thus, the membranes of Mg ghosts exist in a functional state where they revert to the biconcave disc shape spontaneously or in response to ATP. But since freeze-thaw
22
PRIBOI’,
ghosts do not behave in this manner, their menbranes must exist in a different metastable state. The freeze-thaw ghosts, however, have not lost their capacity to revert to the biconcave disc shape. When these ghosts are suspended in a buffered 45% glycerol solution, they become very t’hin discs. This kind of dehydration-induced, sphere-disc transformation has been reported elsewhere (13, 38). When these glycerolated freeze-thaw ghosts are resuspended in buffered salt solution they become very transparent smooth spheres. If they are rehemolyzed in the presence of ATP and reconstituted by adding KCl, many ghosts become thick discs. This is further evidence that the metastable state of the freeze-thaw ghost’s membrane is altered rather than that its structural integrit.y is totally destroyed. Possible reversibility of freeze-thaw damage. It seems clear that the freeze-thaw process alters the red cell membrane to a greater extent than osmotic hemolysis, particularly if the latter process occurs in the presence of Mg ions. However, freeze-thaw ghosts retain their transport AT&se activity. It would be interesting to see whether under appropriate conditions they regain their semipermeability properties. Conditions have been found in which these ghosts become biconcave discs in the presence 450/, glycerol. These disc-shaped, freeze-thawed ghosts may very well have regained their seimpermeability though no evidence for this is presented in this paper. Thus, it may be possible to bring back the freeze-thawed ghost membrane to a more normal structural-functional state. If this is the case, then by preventing loss of hemoglobin immediately following thawing by addition of agents such as PVP (17) or dextran, it may be possible to find conditions where the freeze-thawed, mlhemolyzed red cells can repair themselves. Experimental
Study of Freeze- Thaw Injury
The mechanism of freeze-thaw damage to cells is still unresolved. However, generalizing from the results presented in this paper one may characterize this type of damage as follows: (1) It results in structural-functional disorganization of membrane systems; (2) The damage may not express itself until some time after thawing, i.e., it may be latent injury as studied by Sherman (33, 34) ; (3) Alterations at the molecular level may not be directly apparent at the supramolecu-
lar level, i.e., they may not be tl&($ed by light or electron microscopy; (4) Some of the altcrations may be reversible so that in the l,roper environment the cell may be able to rq)air itself. If the above generalizations are even approximately correct, then it seems evident, t,hat no single approach or experimental technique will be sufficient to illucidnte the mechanism of frcezethaw injury. Several parameters should be mrasured or observed simultaneously in studying freeze-thaw damage to a particular type of cell or tissue. The damage may express itself as altered function of the cell and/or altered enzymatic activity and/or altered structure. If all three types of parameters are studied simultaneously, one has a better opportunity to pinpoint where’ the damage occurred a.nd how it occurred in terms of the particular known physiological and biochemical processes with which it has interfered. Sherman has given a review of the rationale and possible experimental proposals useful for this type of approach (35). Finally, if freeze-thaw damage occurs primarily to membrane systems, then advances in cryobiology will depend upon advances in membrane physiology. We need to know normal membrane structure and function before we can appreciate in detail its alteration by freezing and thawing. However, the dependence may work both ways in that by studying the effect of the freeze-thaw process on membrane systems we may further illucidate the normal structure and function of these systems. SUMMARY Red blood cell ghosts resulting from osmotic hemolysis in the presence of Mg ions (Mg ghosts) and ghosts resulting from slow freeze-thaw process (freeze-thaw ghosts) differ in many respects: (1) Mg ghosts spontaneously take on the disc shape immediately after hemolysis and resuspension in buffered salt solution; whereas freeze-thaw ghosts are spherical; (2) Mg ghosts appear to be less hemolyzed than freeze-thaw ghosts; (3) washed and cold-stored Mg ghosts contract or become biconcave discs when exposed to 30 pmoles of ATP/lOg ghosts at 37°C; whereas freeze-thaw ghosts under similar conditions break up into microspheres and membrane filaments; (4) Mg ghosts become crenated discs and spheres when rehemolyzed and resuspended in buffered salt solution; whereas freeze-thaw ghosts tend to
FREEZE-THAW
VS. OSMOTIC
fretgment; (5) the ATPase activity
of Mg ghosts, particularly the nontransport ATPase activity, is considerably less than that of freeze-thaw ghosts. Sinoe all of these properties characterize the structural-functional integrity of the red cell membrane, the primary site of freeze-thaw damage to erythrocytes is the plasma membrane. Furthermore, freeze-thaw damage to erythrocytes results in loss of hemoglobin by some other mechanism than colloidal osmotic hemolysis. Freeze-thaw ghosts become thin discs in 45% glycerol solution, spherical when resuspended in 0.17 M buffered salt solution, and some become
thick discs when rehemolyzed in the presence of ATP and reconstituted with KCl. Based on this finding, it is suggested that freezing and thawing
alters the metastable functional state of the red cell membrane rather than irreversibly damaging it. ACKNOWLEDGMENTS The author thanks Dr. S. L. Beck for his critical reading of the manuscript and his suggestions and Mrs. Kay Jyung and Mr. Alan Solinger for their technical assistance. REFERENCES 1. Ghan, P. C. Reversible effect of sodium dodecyl sulfate on human erythrocyte membrane adenosine triphosphatase. Biochim. Biophys. Acts, 1366:53-60, 1967. 2. Danielli, J. F., and Davson, H. A contribution to the theory of permeability of thin fiims. J. Cell Physiol., 6: 495-593, 1935. 3. Davson, H., and Ponder, E. Studies on the permeability of erythrocytes. IV. The permeability of “ghosts” to cations. Biochem. J., 8??:756-762, 1938. 4. Dunham, E. T., and Glynh, I. M. Adenosine triphosphatase activity and the active movements of alkab metal ions. J. Physiol. London, 166: 274-293, 1961. 5. Fung, Y. C. B., and Tong, P. Theory of the sphering of red blood cells. Biophys. J., 8: 175-198, 1968. 6. Green, D. E., Allmann, D. W., Bachmann, E., Baum, H., Kopaczyk, K., Korman, E. F., Lipton, S., MacLennan, D. FL, McConnell, D. G., Perdue, J. F., Rieske, J. S., and Tzagoloff, A. Formation of membranes by repeating units. Arch. Biochem. Biophys., 119: 3X2-335,1967. 7. Gross, L. Active membranes for active transport. J. Theor. Biol., 16: 293-396,1967.
HEMOLYSIS
23
8. Hoffman, J. F. Physiological charscteristies of human red blood cell ghosts. J. Gen. Physiol., Q!: 9-23, 1953. 9. Hoffman, J. F. Cation transport and structure of the red-cell plasma membrane. Circulation, 86: 1291-1213, 1962. 10. Kavanau, J. L. In Structure and function in biologicJ membranes, Vois. 1 and 2. HoldenDay, San Francisco, 1965. 11. Korn, E. D. Structure of biological membranes. Science, f6.8: 1491-14931966. 12. Korn, E. D. Structure and function of the plasma membrane-A biochemicJ perapective. J. Gen. Physiol. suppl., 68: 3257-8278, 1963. 13. Lacko, J. Transport of sugars into human red blood cell ghosts. J. Cell Physiol., 67: 591506,1966. 14. Lenard, J., and Singer, S. J. Protein conformation in cell membrane preparations se studied by optical rotatory dispersion and circular dichroism, Proc. Nat. Aaad. Sci. U.S.A., 66: 1323-1335, 1966. 15. Lopez, L., Duck, I. M., and Hunt, W. A. On the shape of the erythroeyte. Biophys. J., 8: 12231235, 1963. 16. Marchesi, V. T., and Palade, G. E. The looelization of Mg-Na-K-activated adenosine triphosphatase on red cell ghost membranes. J. Cell Biol., 86: 335-494, 1967. 17. Meryman, H. T. Review of biological freezing. In Cryobiology, H. T. Meryman, ed., pp. 63-64. Academic Press, New York, 1966. 18. Novikoff, A. B. Ultrastructure of the plasma membrane. Discussion. In symposium of the plasma membrane. Circulation, 86: 11261132, 1962. 19. Parpart, A. K., and Ballentine, R. Molecular anatomy of the red cell plasma msmbrane. 1% Modern trends in physiology and biochemistry, E. S. G. Barron, ed., pp. 135-143. Academic Press, New York, 1952. 29. Ponder, E. Shape changes accompanied by volume changes. In Hemolysis and related phenomena, E. Ponder, ed., pp. 59-114. Grune and Stratton, New York, 1943. 21. Post, R. L., Merritt, C. R., Kinsolving, C. R., and Albright, C. D. Membrane adenosine triphosphatase as a participant in the aotive transport of sodium and potassium in the human erythrocyte. J. Biol. Chem., 886: 1796-1392,1969. 22. Pribor, D. B., and Nara, A. Toxicity and eryoprotection by dimethyl sulfoxide and by glycerol in isolated frog sciatic nerves. Cryobiology 6: 355-365, 1969.
24
PRIBOR
23. Pribor, I). B., and Novak, 1~. The relation of the cryoprotective action of dimethyl sulfoxide to salt damage to frog sciatic nerves. Cryobiology, 6: 126129, 19G9. 24. Rand, R.. P., and Burton, A. C. Mechanical properties of the red cell membrane I. Membrane stiffness and intracellular pressure. Biophys. J., 4: 115-135, 1964. 25. Rand, 1~. P. Some biophysical considerations of the red cell membrane. Fed. Proc. Fed. Amer. Sot. Exp. Biol., 26: 1780-1784, 1967. 26. Rapatz, G., and Luyet, B. Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. Cryobiology, 4: 215-222, 1968. 27. Robertson, J. D. The ultrastruct.ure of cell membranes and their derivatives. Biochem. Sot. Symp., 16: 3-43, 1959. 28. Roelofsen, B., Baadenhuysen, H., and van Deenen, L. L. M. Effects of organic solvents of the adenosine triphosphatase activity of erythrocyte ghost.s. Nature London, 212: 1379-1380, 1966. 29. Schatzmann, H. J. Lipoprotein nature of red cell adenosine triphosphatase. Nature london, 196: 677, 1962. 30. Schatzmann, H. J., and Vincenzi, F. F. Calcium movements across the membrane of human red cells. J. Physiol. London, 201: 369-395, 1969.
31. Seeman, P. Transient holes in the erythrocyte membrane during hypot,onic hcniolysis and stable holes in the membrane aft,er lysis by saponin and lysolecithin. J. Cell Biol., 32: 55-70, 1967. 32. Sherman, J. K. Unusual action of heparin on ascites tumour cells during freezing and and thawing. Nature London, 204: 100-101, 1964. 33. Sherman, J. K. ljimethyl sulfoxide as a protective agent during freezing and thawing of human spermatozoa. Proc. Sot. Exp. Biol. Med., 117: 2Gl-264, 19G4. 34. Sherman, J. K. Freeze-thaw-induced latent injury as a phenomenon in Cryobiology. Cryobiology, 3: 407-413, 1967. 35. Sherman, J. K. Freeze-thaw induced structural changes in cells. III. Experimental approach to analysis of ultrastructural cryoinjury. J. Cryosurg., 2: 189-205, 1969. 36. Teorell, T. Permeability properties of erythrocyte ghosts. J. Gen. Physiol., 355: 0%i01, 1952. 37. Wallach, D. F. H., and Zahler, I’. H. Protein conformations in cellular membranes. Proc. Nat. Acad. Sci. U.S.A., 56: 1552-1559, 196F. 38. Weed, K. I., Reed, C. F., and Berg, G. Is hemoglobin an essential structural component of the human erythrocyte membranes? J. Clin. Invest., 42: 581-588, 1963.
8, 14-24
OSMOTIC
HEMOLYSIS
CONTRASTED
WITH
FREEZE-THAW
HEMOLYSIS’
D. B. PRIBOR Biology
Department,
The University
of Toledo and Research Associate,
Medical Research of The Toledo Hospital, Since hemolysis involves a change in permeability of the plasma membrane to macromolecules, this process must be due to some structuralfunctional change in the membrane. For osmotic hemolysis this change is not a mere bursting of the red cell, but rather it results from a sudden increase in permeability of the entire membrane to all molecules (19). Also, if the membrane develops holes during hemolysis, it repairs itself soon after (31) regaining its impermeability to macromolecules (3) and under appropriate conditions, its relative impermeability to cations (8, 36). Thus, osmotic hemolysis, at least, is due to a membrane alteration which to some extent is reversible. A complete understanding of the mechanism of hemolysis, therefore, requires an understanding of the structural-functional integrity of the red cell membrane. Freezing blood, below -3°C and thawing it without protective agents always brings about some red cell lysis (26). Such red cell lysis usually is interpreted as freeze-thaw damage to the cells. Therefore, to understand the mechanism of freeze-thaw damage to erythrocytes we need to understand the mechanism of hemolysis which in turn requires a better understanding of membrane structure. In recent years the classical theory for membrane structure originally proposed by Danielli and Davson (2) and later modified by Robertson (27) has been challanged by several investigators (6, 7, 14, 37). Korn (11, 12) suggests that no membrane model to date has been adequately proved or disproved. Thus, the field of membrane physiology is in a state of flux. It is for this reason, probably, that there has been a revival of interest in studying erythrocytes. One can isolate from them relatively intact membrane fragments or a complete membrane system-the
Toledo,
Znstitute of
Ohio @SO6
reconstituted ghost. Research which attempts to clarify the mechanism of freeze-thaw damage to erythrocytes may at the same time give new insight to the structural-functional integrity of the red cell membrane and to membranes in general. The red cell semipermeability properties and its biconcave disc shape (5, 15, 20, 24) probably are characteristic of the plasma membrane as a whole. The total membrane ATPase system, consisting of Na-K-Mg-activated ATPase (hereafter referred to as transport ATPase) and the Mg-activated ATPase (hereafter referred to as Mg-ATPase), does not require the complete membrane to be intact (1, 21). However, it does require intact membrane fragments as opposed to solubilized enzymes or even severely disrupted membrane fragments (1, 28,29). Studies of discsphere transformations in erythrocytes strongly imply that the membrane may exist in a number of metastable equilibrium states. Also, the lipoprotein subunit theory for membrane structure implies that all membranes may continuously transform from one metastable equilibrium state to another (6, 10, 12). Using this as a working hypothesis, studies on different types of red cell ghosts of semipermeability changes, capacity for shape changes and changes in ATPase activity, could indicate to what extent the equilibrium state of the membrane had been altered by the process that brought about the hemolysis. In a general way this report may provide additional evidence for the red cell membrane existing in a number of metastable equilibrium states. In particular an attempt is made to answer two questions: (1) Is freeze-thaw hemolysis due to a change in the red cell permeability which subsequently leads to osmotic hemolysis upon thawing. In other words, is freeze-thaw hemolysis simply a special case of colloidal osmotic hemolysis; (2) Does the freeze-thaw process irreversibly disorganize the red cell membrane or does it transform the membrane to a metastable equilibrium state which may be reconverted to a more normal state? The approach used will be to compare ghosts
Received July 13, 1970. 1 Supported by Grant HE12114 (NIH) and a joint, grant from the Institute of Medical Research of The Toledo Hospital and the Medical and Research Foundation of Perth Amboy General Hospital. 14
FREEZE-THAW
VS. OSMOTIC
produced by osmotic hemolysis in the presence of Mg ions (hereafter referred to as Mg ghosts) with ghosts produced by slow free5ing and thawing (hereafter referred to as freezr+thaw ghosts). The properties selected for comparison are: (1) gross structure as seen through the phase microscope, (2) capability of regaining or transforming to the disc shape, and (3) ATPase activity. MATERULB
AND METHODS
Red cells. In all but one set of experiments, cells were obtained from blood freshly drawn by veniptmcture from normal male and female subjects into tubes containing dry heparin (0.3 mg/ml of blood). In the experiment where osmotic ghosts were frozen and thawed and then glycerolated, red cells were obtained from blood drawn from patients at The Toledo Hospital, washed with buffered salt solution, and then pooled.* &&red mlt solution. Throughout the results reference is made to buffered salt solution. This solution contained 150 rnM NaCl, 1 mM MgCIZ. 6H&, 1 rnM Tris-HCl, and enough 0.1 N HCl to make the pH equal 7.4. When ghosts were stored overnight in this solution, a trace of cysteine was added. Special Procedures In the results some procedures either are selfevident or are included along with the report of the results. Special procedures are given below. Preparti of bloodfor hemo1ysi.s. The blood was aentrifuged at SO@for 10 min. The plasma and bt@y layer containing white cells was discarded and the paoked red cell resuspended in buffered salt solution. The cells were then washed three times with the buffered salt solution; after the final washing the supernate was discarded. Pr~bifm of oSn30tkgkmts (Mg gho8t8). One volume of packed cells was rapidly plunged into 5 vol of rapidly stirred uold distilled water containing 1 mu MgClz .SHnOand enough Tris bu&r to adjust the pH to 7.4. This mixture was left to stand stirring in the 4°C cold room for 45 min. z The chemicals, sodium heparinate, d&odium adenosine triphosphate (ATP) , disodium ethylene diamino tartrraeetste (EDTA), ouabain, ~-cyst+ ine, and the reagenti for the ATPase determinations, were obtained from Sigma Chemical Co., St. Louis, MO. The ATP was made up into a 0.1 M stock solution. The ATP solution was adjusted to pH 7.0 with NaOH and stored in the frozen state. Cysteine solutions were discarded after 2 days.
HEMOLYSIS
15
P~~~ti~byfreeairag~tkasrving @wea4mw ghosts). A 39ml Pyrex oe&ifuge tube containing 1 vol of paeked cells and 9 vol of buffered salt solution (the final volume of ghost suspension was about 20 ml) was placed in an alcohol bath previously cooled to -20°C. The red cell suspension froze in a few minutes, It was kept in the frozen state for 30 min, thawed in a water bath at 37”C, centrifuged at 16,990gfor 10 min, and resuspended in buffered salt solution. Preparationof ghost SuSpensionS Of known wnc&rations. Ghost suspensions were counted by means of a phase hemacytometer. Counts were made in each of the two chambers of the hemacytometers and were repeated if the counts did not come within 5 y0 of one another. G~ycerokation of ghQ8t8. In various experiments ghosts were suspended in a 45 $J glycerol solution (v/v) using a 0.01 M Tris-HCl buBer instead of distilled water. The pH was adjusted to 7.4. The ghosts were stored in this solution for 20-24 hr at 4”C, centrifuged at 1O,OOOg, and resuspended in buffered salt solution. Rehemolysis experiments. To 1 vol of ghost suspension containing log ghosts/ml was added 9 vol of a rehemolyzing buffer solution. Three types rehemolyzing solutions were used: (1) control which consisted of 0.01 M Tris-HCl, 0.605 M MgClzm6Hz0, 0.0175 M NaCI; (2) ATP solution which consisted of 0.01 M Tris-HCl, 0.005 M MgClz.6Ha0, 0.01 M NaCI, 0.005 M ATP; (3) EDTA solution which consisted of 0.01 M TrisHCI, 0.0175 M NaCl, 0.005 M EDTA. All solutions were adjusted to pH 7.4. The rehemolysis was done in test tubes at room temperature. The ghost suspensions were thoroughly mixed and either incubated to 37°C for 0.5 hr, centrifuged at 16,06Og,and resuspended in buffered salt solution or allowed to stand for 10 min and then reconstituted. Recanztitution of ghosts. An attempt was made to reconstitute the Mg ghosts and freeze-thaw ghosts by resuspending them in buffered salt solution immediately after hemolysis. This departure from the usual procedure of reconstituting ghosts was necessary so the Mg ghosts and freeze-thaw ghosts would receive the same treatment. However, Mg ghosts and freeze-thaw ghosts which had been glycerolated, resuspended in buffered salt solution, and then rehemolyzed were reconstituted (“reversal of hemolysis”) in the usual manner following the procedure of
16
PRIBOR
Schatzmann (30). Enough solid I~(‘1 was added to the suspensions of rehemolyzed cells to bring t’he concentration to 0.15 M. ATPase activity. ATPase activity was assayed by mea.suring liberated inorganic phosphate (Pi) by a modified Fiske-SubbaRow procedure. The final 1 ml of ghost suspension to be incubated at 37°C for 1 hr contained 118 m&f NaCl, 14 mM buffer, 1 InM L-cysteine, KCl, 20 mM Tris-HCl 0.05 IBM EDTA, 3mM XIg<%~6Hz0, 3 m&I ATP, and 1 X log ghosts. Tubes to be assayed for MgATPase activity also coutained 1 x low2 mM ouabain. The transport ATPase activity was determined by subtracting Rig-ATPase activity from total ATPase activity. d zero-time tube served as an ATP blank and correction for residual phosphate in the ghosts. In this run ATL’ was added to the incubation tube containing ghosbs just prior to killing the reaction with cold TCA. An insignificant percentage of ATP is hydrolyzed during 1-hr incubation at 37°C. All assays were carried out in duplicate. For the .\TPase activities of Rig ghosts which were subsequently frozen and thawed, an ATP blank was used rather than a zero time. The reported ATI’ase activities in this experiment, therefore, probably are somewhat higher than they should be. Photomicroscopy. The ghosts were viewed and photographed through a Nikon phase microscope using a 40 x DLL phase objective.
RF;SULTS
Effect of Freeze-Thaw Process on drg Ghosts and on Vnhemolyzed Cells Mg ghosts tended to be incompletely hemolyzed and to retain the biconcave disc shape (Fig IA). If these ghosts were slowly frozen and thawed, many of them were incompletely hemolyzed and retained the disc shape (Fig. 1B). This was in marked c*ontrast to unhemolyzcd cells from the same blood which were frozen and thawed under the same conditions as the 14g ghosts. The resultant freeze-thaw ghosts were more transparent indicating a more complete hemolysis. They were spherical in shape and showed signs of fragmentation (Fig. l(y). Thus freeze-thawed, unhemolyzed red cells appeared more altered than freeze-thawed Mg ghosts. The freeze-thaw process, however, produced some alteration in the Mg ghosts as indicated by two other experiments. First of all, freeze-thawed Mg ghosts had a higher ATPax activity than nonfrozen Mg ghosts (Table 1). .41x), the freezethawed Mg ghosts fragmented when incubated at 37°C without ATP. This fragmentation was inhibited when 3 mM liTI’ was present, t’hough some fragmentation was still evident. In addition, both types of ghosts were suspended in Trisbuffered 45”/ glycerol solution, stored at 4°C for 12-24 hr, and then resuspended in buffered salt
FIG. 1. Effect of the frer :xe-thaw pr~ess OII Mg ghosts vers, IS its cfrect on Imhemolyzed red cells. A. Mg ghosts; I3 Mg ghosts which were frozen and thawed; C. Vrozewthawed \lnhemolyzed red cells.
FREEZE-THAW
VS. OSMOTIC TABLE
ATPASE
ACTIVITY
Mg ghosts Freeze-thawed ghosts
Mg
Calculated t value for di&renoe of the means
1
OF Mg GHOSTS AND FREEZE-THAWED
Total ATPase
17
HEMOLYSIS
Mg-ATPase
Mg Gaosrsa Transport ATPsse
Mean
SD
Mean
SD
Meful
SD
0.1160 0.2323
0.0557 0.0529
0.0940 0.1481
0.0121 0.0261
0.0220 0.0842
0.0094 0.0387
3.6777
4.6053
3.8281
QThe ATPase activity is expressed in terms of micromoles of inorganic. phosphate per 100 ghosts per hour at 37’C. Three experiments were performed in duplicate; so the means are estimated from an average of six determinations. Note: the t value from the tables for Ni + Nz - 2 = 10 degrees of freedom above which the two means are significantly different at the 1% confidence level is 3.169; for the 6% confidence level the t value is 2.228.
solution. In aliquots taken from packed cells after centrifugation and removal of glycerol supernate, but before resuspension in salt buffer, the Mg ghosts were in&t and many were biconcave discs. In the freeae-thawed Mg ghosts aliquot, however, there were many cell fragments. Upon rehemolysis and resuspension in the b&e& salt solution, many Mg ghosts retained their biconcave shape and showed no evidence of fragmentation. The remaining nonfragmented, freezethawed Mg ghosts were very transparent and spherical in shape.
ghost.9/ml), mmy of t&e Mg ghosts were disc abaped(li”ig,w. salt b&r to give nated spheres. Fur resulted in more and more of them becoming
FIQ. 2. Mg ghosts and freeze-thaw ghosts immediateIy after ysis and resuspension in buffered isotonic salt solution with concentration adjusted to 101 ghosts/ml. A. Mg ghosts; B. freeze-thaw ghosts.
18
PRIBOP,
spherical in shape. Furthermore, if these ghosts were stored at 4°C for 1-3 days, almost all of them became spherical and several apparently swelled and lost more hemoglobin. The freezethaw ghosts immediately after hemolysis and suspension in buffered salt solution (lo9 ghosts/ ml) appeared as smooth spheres of relatively uniform size (Fig. 2B). There was an occasional unhemolyzed cell and some disc-shaped ghosts. Dilution of this suspension resulted in no obvious change in the ghosts escept’ that the unhemolyzed ghosts became spherical. Furt,her washing and cold storage of these ghosts result’ed in some of them breaking up into microspheres. After cold storage and three washes with salt buffer, both Mg ghosts and freeze-thaw ghosts were spherical but differed in that: (1) at least a few Ng ghosts were disc shaped \vhei cas 11011e of the freeze-thaw ghosts were, (2) the i\lg gh:)sts apl)ea*ed daikcr ad easier to see uutlc: l)h:Lse than the freezethaw ghosts, and (3) there was 11’) evidence of
fragmentation of Mg ghosts whereas cell fragments appeared among the freeze-thaw ghosts, Morphological response of Mg ghosts and jreezefhaw ghosts to ATP. Whereas cold-stored, washed
Mg ghosts and freeze-thalv ghosts appeared t,o be similar, they differed considerably in their stability. When Xg ghosts were incubated at 37°C for 0.5 hr, there were no signs of fragmentntion, but when freeze-thaw ghosts were similarly treat,ed a ilumber of microspheres of various sizes appeared among the ghosts (Fig. 3A and C). Furthermore, when Mg ghosts were incubated with 30 pmoles of XTP/lO~ ghosts, they either apl)arently “contracted” to a smaller volume or became disc shaljed. Freeze-thaw ghosts exposed to ll’l?l’ under the same conditions, however, fragmented into microsphere;j of various sizes OI formed what apl)earetl to be membrane filaments (Fig. 31s am1 I>). In five experiments each done in tlul~lieate, 30 pmoles c,f ATI’ always caused the frrczc-thaw ghosts to fragment. The effect
FIG. 3. Effect of 30 @moles of ATP/lOS ghosts on washed, cold-stored Mg ghosts and freeze-thaw ghosts. A. Mg ghosts (control) after 0.5 hr 37°C; B. Mg ghosts plus 30 pmoles ATP after 0.5 hr 37°C; C. freeze-thaw ghosts (control) after 0.5 hr 37°C; I). freezethaw ghosts p111s 30 Mmoles ATP after 0.5 hr 37°C.
FREEZE-THAW
VS. OSMOTIC
of ATP on Mg ghosts was variable. In one experiment the ghosts were greatly contracted but there was no shape transformations. In another experiment the predominant effect was a change in shape from spheres to discs or cups. In the other three experiments the effect consisted of both apparent contractions and shape transformations. In an attempt to obtain more consistent results with the effect of ATP on Mg ghosts, Mg ghosts and freeze-thaw ghosts were rehemolyzed in a dilute Tris-HCl buffer containing MgClz and ATP, or just MgC&, or EDTA. In this regard the results were somewhat disappointing but they did show a dramatic difference between Mg ghosts and freeze-thaw ghosts. Four experiments using blood from three different donors were run in duplicate for a total of eight determinations. In all eight determinations Mg ghosts rehemolyzed with ATP or EDTA appeared as discs or crenated spheres. In five of the eight runs the Mg ghosts rehemolyzed with MgClt were crenated spheres. In the other three runs, there were several discs. In all cases,when the rehemolyzed Mg ghosts were resuspended in buffered salt solution, they remained disc shaped or became discs, crenated discs or crenated and apparently shrunken spheres. Freeze-thaw ghosts remained spherical when diluted with buffered salt solution and when rehemolyzed in the presence of ATP, MgCl,, or EDTA. The rehemolyzed ghosts appeared more transparent. Some of the ghosts rehemolyzed with ATP or EDTA still remained relatively smooth spheres when resuspended in buffered salt solution in contrast to some of the ghosts rehemolyzed with MgClz which appeared shrunken and crenated. In general, when the rehemolyzed freeze-thaw ghosts were resuspended in buffered salt solution, in all casesthey tended to break up into microspheres and the nonfragmented ghosts remained spherical. Response of Mg ghosts and freezt&aw ghosts to bu$ered 46% glycerol solution. This experiment was done to seewhether under any circumstances the freeze-thaw ghosts still are able to take on the disc shape. As usual the freeze-thaw ghosts were spherical and more hemolyzed than the Mg ghosts. Immediately after suspension in 45~~ glycerol the Mg ghosts and the freeze-thaw ghosts were thin discs (Fig. 4A and B). After 20-24 hr in glycerol, the ghosts were resuspended in buffered salt solution. Here both types of ghosts
HEMOLYSIS
19
were smooth spheres. They were then rehemolyzed with ATP, MgClz, or EDTA, allowed to stand for 5-10 mm, and finally reconstituted by adding KC1 to bring the concentration up to 0.15 M. As with the previous rehemolysis experiments, the populations of Mg ghosts rehemolyzed in the three different ways contained crenated spheres and discs and smooth discs (Fig. 4C). The freezethaw ghosts were very transparent and diflicult to see. Of those exposed to ATP and then reconstituted, many were thick discs (Fig. 4D). Of those exposed to MgClz, many were spheres but there were some discs, while of those exposed to EDTA, all were spheres. This experiment was repeated four times with blood from four different donors. ATPase activity of Mg ghosts and freeze-thaw ghosts. As is seen in Table 2, the total ATPase activity and the Mg ATPase activity of freea+ thaw ghosts were significantly higher at the 1% confidence level than that of the Mg ghosts. In contrast, the transport ATPase of freeze-thaw ghosts was significantly higher than that of Mg ghosts only at the 5% confidence level. Stated in a different way, the ratios of ATPase activity of Mg ghosts to freeze-thaw ghosts are as follows: total ATPase, l/2.1; Mg-ATPase, l/2.8; transport ATPase, l/1.5. Thus, the freeze-thaw processappeared to increase the Mg-ATPase activity to a greater extent than it increased the transport ATPase activity. DISCUSSION
Site of Freeze-Thaw Damage: The Whole Cell vs. the Plasma Membrane
In comparing freeze-thawed, unhemolyzed cells to freeze-thawed Mg ghosts morphologically it appears that unhemolyzed cells are damaged to a greater extent than Mg ghosts (Fig. 1). However, freezing and thawing does alter the Mg ghost membrane, for some of these ghosts break up into microspheres (Fig. 1B) ; the ATPase activity is increased (Table l), and they are more unstable, i.e., they tend to break up into microspheres when suspended in buffered 45% glycerol solution and subsequently resuspended in buffered salt solution. Thus the freeze-thaw process does alter the red cell membrane. This is consistent with evidence dealing with other types of cells that the plasma membrane is the site of freeze-thaw damage (22, 23,32).
20
PRIBOR
FIG. 4. Effect of glycerolation, resuspension, rehemolysis and thell reronstitlltion with KC1 011 Mg ghosts and freeze-thaw ghosts. A. Mg ghosts immediately after sllspension in buffered 15% glycerol solution; B. freeze-thaw ghosts immcdi:rtely after sllspension in buffered 45yc glycerol sollltion; C. Mg ghosts resuspended in 0.17 hf salt bllffer, rehemolyzed in the presence of ATP and MgCl? then reconstitrlted with KCl; I). freeze-thaw ghosts resuspended in 0.17 M salt buffer, rehemolyzed in the presence of AT]’ and MgCla then reconstituted with KCl. Extent
oj” Freeze-Thaw Damage to the Red Cell Membrane
Gross structural integrity and stability of the plasma nzembrane oj freeze-thaw ghosts. Immedi-
ately after hemolysis freeze-thaw ghosts are more hemolyzed than 14g ghosts and are spherical a3 opposed to the disc shape of LIg ghosts (Fig. 2). After three washings wit,h buffered salt solution and cold storage, many freeze-t,haw ghosts appear to be intact. However, some change has occurred in the membrane of these ghosts, for when subjected to various kinds of stress the) tend to break up into microspheres whereas >Ig ghosts are relatively unaffected. Freeze-thaw ghosts tend to fragment when washed with buffered salt solution and when rehemolyzed and resuspended in salt buffer, whereas hlg ghosts become crenated discs. Freeze-thaw ghosts COW
pletely fragment when cxpo~ed to 30 ~.lmoles ATP/lOg ghosts at 37’Y: for 0.5 hr, whereas hlg ghosts contract or become discs. Thus freezethaw ghosts arc structurally intact but relatively unstable in contrast to 11g ghosts which are quite stable.
Increased ATI’ase activit,y of freeze-thaw ghosts. It is often assumed that the freeze-thaw process increases LITl’ase activity of red cell ghosts by increasing the membrane permeability to hT1’ and thus making hT1’ more available to this enzyme (4). JIowever, freezing and thawing brings about some fragmrntation. Some microsphcrcs resulting from fragmentation contain hemoglobin and apparently have regained at least some of their semipermeability (25). If there is a net increase in membrane permeability, it may not be as great as originally proposed.
21
FREEZE-THAW VS. OSMOTIC HEMOLYSIS
ATPASE
ACTIVITY
OF
TABLE 2 Mg GHOSTS AND FREEZE-THAW
Total ATPase
l&g ghosts PPraeze-thawghosts C&uleted t value for &fference of the means
GHOSTS” Transport ATPme
Mg-ATPase
Meall
SD
Mobil
SD
MeaIl
SD
0.0790 0.1735
0.0239 0.0616
0.0385 0.1098
0.0192 0.0566
0.0405 0.0636
0.0132 0.0258
4.9355
4.1191
2.7737
6 The ATPase activity is expressed in terms of micromoles of inorganic phosphate per lo@ghosts per hour at 37°C. Six experiments were performed in duplicate; so the means are estimated from an average of 12 determinations. Note: the t value from the tables for N1 + Nn - 2 = 22 degreesof freedom above which the two means are significantly different at the 1% confidence level is 2.819;for the 5% confidence level the t value is 2.074. In the experiments reported in this paper freezing and thawing increases Mg-ATPase activity to a significantly greater extent than transport ATPase activity. According to Marchesi (16) both of these ATPase systems are located on the inner surface of the membrane. If this is the case, with increased permeability the total ATPase activity would increase but the ratio of Mg-ATPase to transport ATPase should remain the same. On the other hand, according to Hoffman (9) and Novikoff (18) the Mg-ATPase is located on the outer membrane surface. If this is correct, then the increased permeability would not account for the marked increase of Mg-ATPase. An alternate hypothesis consistent with this data is that freezing and thawing increase transport ATPase by increasing cell membrane permeability, and it increases Mg-ATPase activity by some other means such as altering the three-dimensional configuration of the membrane proteins or changing the membrane fixed charge distribution. Volume changes of ghosts. Rehemolyzed Mg ghosts appear to shrink when resuspended in buffered salt solution whereas freeze-thaw ghosts treated in the same way tend to fragment or remain spherical. Furthermore, in some preliminary experiments where volume changes of ghosts were determined by a hematocrit method, Mg ghosts had a smaller volume than freeze-thaw ghosts in a buffered 0.17 M NaCl solution. The Mg ghosts increased in volume when placed in a 0.05 M NaCl solution and decreased in volume in a 0.5 M NaCl solution. This is in line with the work of Hoffman (8) and Teorell (36) who reported that osmotic ghosts regain their semipermeability
and low permeability to ions. In contrast, the freeze-thaw ghosts do not increase in volume in the hypotonic solution but do decreasein volume in the hypertonic solution. The experiments should be repeated to determine whether the decrease in hematocrit of the freeze-thaw ghosts is due to fragmentation, increased cell packing, or to a real cell shrinkage. Nevertheless, it appears that Mg ghosts are osmotically active whereas the freeze-thaw ghosts are not. Thus, the freezethaw process has altered the red cell membrane to the extent that under the reconstituting procedure employed, freeze-thaw ghasts do not completely regain their semipermeability. Shape chxcnges of Mg ghosts and freeze-thaw ghosts. It has already been reported that immedi-
ately a.fter hemolysis osmotic ghosts spontaneously take on the biconcave disc shape (3, 8, 38). Likewise, Mg ghosts spontaneously take on the biconcave disc shape immediately after hemolysis and resuspension in buffered salt solution and after rehemolysis as well. Freeze-thaw ghosts, on the other hand, remain spherical and tend to fragment, particularly after rehemolysis and resuspension in buffered salt solution. Also, washed and cold-stored Mg ghosts contract or take on a cup or biconcave disc shape when incubated in the presence of 30 pmoles ATP/lOO ghosts, whereas similarly treated freeze-thaw ghosts from the same blood break up into microspheres or membrane filaments. Thus, the membranes of Mg ghosts exist in a functional state where they revert to the biconcave disc shape spontaneously or in response to ATP. But since freeze-thaw
22
PRIBOI’,
ghosts do not behave in this manner, their menbranes must exist in a different metastable state. The freeze-thaw ghosts, however, have not lost their capacity to revert to the biconcave disc shape. When these ghosts are suspended in a buffered 45% glycerol solution, they become very t’hin discs. This kind of dehydration-induced, sphere-disc transformation has been reported elsewhere (13, 38). When these glycerolated freeze-thaw ghosts are resuspended in buffered salt solution they become very transparent smooth spheres. If they are rehemolyzed in the presence of ATP and reconstituted by adding KCl, many ghosts become thick discs. This is further evidence that the metastable state of the freeze-thaw ghost’s membrane is altered rather than that its structural integrit.y is totally destroyed. Possible reversibility of freeze-thaw damage. It seems clear that the freeze-thaw process alters the red cell membrane to a greater extent than osmotic hemolysis, particularly if the latter process occurs in the presence of Mg ions. However, freeze-thaw ghosts retain their transport AT&se activity. It would be interesting to see whether under appropriate conditions they regain their semipermeability properties. Conditions have been found in which these ghosts become biconcave discs in the presence 450/, glycerol. These disc-shaped, freeze-thawed ghosts may very well have regained their seimpermeability though no evidence for this is presented in this paper. Thus, it may be possible to bring back the freeze-thawed ghost membrane to a more normal structural-functional state. If this is the case, then by preventing loss of hemoglobin immediately following thawing by addition of agents such as PVP (17) or dextran, it may be possible to find conditions where the freeze-thawed, mlhemolyzed red cells can repair themselves. Experimental
Study of Freeze- Thaw Injury
The mechanism of freeze-thaw damage to cells is still unresolved. However, generalizing from the results presented in this paper one may characterize this type of damage as follows: (1) It results in structural-functional disorganization of membrane systems; (2) The damage may not express itself until some time after thawing, i.e., it may be latent injury as studied by Sherman (33, 34) ; (3) Alterations at the molecular level may not be directly apparent at the supramolecu-
lar level, i.e., they may not be tl&($ed by light or electron microscopy; (4) Some of the altcrations may be reversible so that in the l,roper environment the cell may be able to rq)air itself. If the above generalizations are even approximately correct, then it seems evident, t,hat no single approach or experimental technique will be sufficient to illucidnte the mechanism of frcezethaw injury. Several parameters should be mrasured or observed simultaneously in studying freeze-thaw damage to a particular type of cell or tissue. The damage may express itself as altered function of the cell and/or altered enzymatic activity and/or altered structure. If all three types of parameters are studied simultaneously, one has a better opportunity to pinpoint where’ the damage occurred a.nd how it occurred in terms of the particular known physiological and biochemical processes with which it has interfered. Sherman has given a review of the rationale and possible experimental proposals useful for this type of approach (35). Finally, if freeze-thaw damage occurs primarily to membrane systems, then advances in cryobiology will depend upon advances in membrane physiology. We need to know normal membrane structure and function before we can appreciate in detail its alteration by freezing and thawing. However, the dependence may work both ways in that by studying the effect of the freeze-thaw process on membrane systems we may further illucidate the normal structure and function of these systems. SUMMARY Red blood cell ghosts resulting from osmotic hemolysis in the presence of Mg ions (Mg ghosts) and ghosts resulting from slow freeze-thaw process (freeze-thaw ghosts) differ in many respects: (1) Mg ghosts spontaneously take on the disc shape immediately after hemolysis and resuspension in buffered salt solution; whereas freeze-thaw ghosts are spherical; (2) Mg ghosts appear to be less hemolyzed than freeze-thaw ghosts; (3) washed and cold-stored Mg ghosts contract or become biconcave discs when exposed to 30 pmoles of ATP/lOg ghosts at 37°C; whereas freeze-thaw ghosts under similar conditions break up into microspheres and membrane filaments; (4) Mg ghosts become crenated discs and spheres when rehemolyzed and resuspended in buffered salt solution; whereas freeze-thaw ghosts tend to
FREEZE-THAW
VS. OSMOTIC
fretgment; (5) the ATPase activity
of Mg ghosts, particularly the nontransport ATPase activity, is considerably less than that of freeze-thaw ghosts. Sinoe all of these properties characterize the structural-functional integrity of the red cell membrane, the primary site of freeze-thaw damage to erythrocytes is the plasma membrane. Furthermore, freeze-thaw damage to erythrocytes results in loss of hemoglobin by some other mechanism than colloidal osmotic hemolysis. Freeze-thaw ghosts become thin discs in 45% glycerol solution, spherical when resuspended in 0.17 M buffered salt solution, and some become
thick discs when rehemolyzed in the presence of ATP and reconstituted with KCl. Based on this finding, it is suggested that freezing and thawing
alters the metastable functional state of the red cell membrane rather than irreversibly damaging it. ACKNOWLEDGMENTS The author thanks Dr. S. L. Beck for his critical reading of the manuscript and his suggestions and Mrs. Kay Jyung and Mr. Alan Solinger for their technical assistance. REFERENCES 1. Ghan, P. C. Reversible effect of sodium dodecyl sulfate on human erythrocyte membrane adenosine triphosphatase. Biochim. Biophys. Acts, 1366:53-60, 1967. 2. Danielli, J. F., and Davson, H. A contribution to the theory of permeability of thin fiims. J. Cell Physiol., 6: 495-593, 1935. 3. Davson, H., and Ponder, E. Studies on the permeability of erythrocytes. IV. The permeability of “ghosts” to cations. Biochem. J., 8??:756-762, 1938. 4. Dunham, E. T., and Glynh, I. M. Adenosine triphosphatase activity and the active movements of alkab metal ions. J. Physiol. London, 166: 274-293, 1961. 5. Fung, Y. C. B., and Tong, P. Theory of the sphering of red blood cells. Biophys. J., 8: 175-198, 1968. 6. Green, D. E., Allmann, D. W., Bachmann, E., Baum, H., Kopaczyk, K., Korman, E. F., Lipton, S., MacLennan, D. FL, McConnell, D. G., Perdue, J. F., Rieske, J. S., and Tzagoloff, A. Formation of membranes by repeating units. Arch. Biochem. Biophys., 119: 3X2-335,1967. 7. Gross, L. Active membranes for active transport. J. Theor. Biol., 16: 293-396,1967.
HEMOLYSIS
23
8. Hoffman, J. F. Physiological charscteristies of human red blood cell ghosts. J. Gen. Physiol., Q!: 9-23, 1953. 9. Hoffman, J. F. Cation transport and structure of the red-cell plasma membrane. Circulation, 86: 1291-1213, 1962. 10. Kavanau, J. L. In Structure and function in biologicJ membranes, Vois. 1 and 2. HoldenDay, San Francisco, 1965. 11. Korn, E. D. Structure of biological membranes. Science, f6.8: 1491-14931966. 12. Korn, E. D. Structure and function of the plasma membrane-A biochemicJ perapective. J. Gen. Physiol. suppl., 68: 3257-8278, 1963. 13. Lacko, J. Transport of sugars into human red blood cell ghosts. J. Cell Physiol., 67: 591506,1966. 14. Lenard, J., and Singer, S. J. Protein conformation in cell membrane preparations se studied by optical rotatory dispersion and circular dichroism, Proc. Nat. Aaad. Sci. U.S.A., 66: 1323-1335, 1966. 15. Lopez, L., Duck, I. M., and Hunt, W. A. On the shape of the erythroeyte. Biophys. J., 8: 12231235, 1963. 16. Marchesi, V. T., and Palade, G. E. The looelization of Mg-Na-K-activated adenosine triphosphatase on red cell ghost membranes. J. Cell Biol., 86: 335-494, 1967. 17. Meryman, H. T. Review of biological freezing. In Cryobiology, H. T. Meryman, ed., pp. 63-64. Academic Press, New York, 1966. 18. Novikoff, A. B. Ultrastructure of the plasma membrane. Discussion. In symposium of the plasma membrane. Circulation, 86: 11261132, 1962. 19. Parpart, A. K., and Ballentine, R. Molecular anatomy of the red cell plasma msmbrane. 1% Modern trends in physiology and biochemistry, E. S. G. Barron, ed., pp. 135-143. Academic Press, New York, 1952. 29. Ponder, E. Shape changes accompanied by volume changes. In Hemolysis and related phenomena, E. Ponder, ed., pp. 59-114. Grune and Stratton, New York, 1943. 21. Post, R. L., Merritt, C. R., Kinsolving, C. R., and Albright, C. D. Membrane adenosine triphosphatase as a participant in the aotive transport of sodium and potassium in the human erythrocyte. J. Biol. Chem., 886: 1796-1392,1969. 22. Pribor, D. B., and Nara, A. Toxicity and eryoprotection by dimethyl sulfoxide and by glycerol in isolated frog sciatic nerves. Cryobiology 6: 355-365, 1969.
24
PRIBOR
23. Pribor, I). B., and Novak, 1~. The relation of the cryoprotective action of dimethyl sulfoxide to salt damage to frog sciatic nerves. Cryobiology, 6: 126129, 19G9. 24. Rand, R.. P., and Burton, A. C. Mechanical properties of the red cell membrane I. Membrane stiffness and intracellular pressure. Biophys. J., 4: 115-135, 1964. 25. Rand, 1~. P. Some biophysical considerations of the red cell membrane. Fed. Proc. Fed. Amer. Sot. Exp. Biol., 26: 1780-1784, 1967. 26. Rapatz, G., and Luyet, B. Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. Cryobiology, 4: 215-222, 1968. 27. Robertson, J. D. The ultrastruct.ure of cell membranes and their derivatives. Biochem. Sot. Symp., 16: 3-43, 1959. 28. Roelofsen, B., Baadenhuysen, H., and van Deenen, L. L. M. Effects of organic solvents of the adenosine triphosphatase activity of erythrocyte ghost.s. Nature London, 212: 1379-1380, 1966. 29. Schatzmann, H. J. Lipoprotein nature of red cell adenosine triphosphatase. Nature london, 196: 677, 1962. 30. Schatzmann, H. J., and Vincenzi, F. F. Calcium movements across the membrane of human red cells. J. Physiol. London, 201: 369-395, 1969.
31. Seeman, P. Transient holes in the erythrocyte membrane during hypot,onic hcniolysis and stable holes in the membrane aft,er lysis by saponin and lysolecithin. J. Cell Biol., 32: 55-70, 1967. 32. Sherman, J. K. Unusual action of heparin on ascites tumour cells during freezing and and thawing. Nature London, 204: 100-101, 1964. 33. Sherman, J. K. ljimethyl sulfoxide as a protective agent during freezing and thawing of human spermatozoa. Proc. Sot. Exp. Biol. Med., 117: 2Gl-264, 19G4. 34. Sherman, J. K. Freeze-thaw-induced latent injury as a phenomenon in Cryobiology. Cryobiology, 3: 407-413, 1967. 35. Sherman, J. K. Freeze-thaw induced structural changes in cells. III. Experimental approach to analysis of ultrastructural cryoinjury. J. Cryosurg., 2: 189-205, 1969. 36. Teorell, T. Permeability properties of erythrocyte ghosts. J. Gen. Physiol., 355: 0%i01, 1952. 37. Wallach, D. F. H., and Zahler, I’. H. Protein conformations in cellular membranes. Proc. Nat. Acad. Sci. U.S.A., 56: 1552-1559, 196F. 38. Weed, K. I., Reed, C. F., and Berg, G. Is hemoglobin an essential structural component of the human erythrocyte membranes? J. Clin. Invest., 42: 581-588, 1963.