Activity in Red Cell Membranes of Freezing Regimen1
I. TAKEHSRA” The New
The damage which occurs when intact red blood cells are frozen has been associatedwith alterations in the integrity of the cell membrane by a number of cryobiological studies (5, 10, 17). The adenosinetriphosphatase(ATPase) of red blood cellsis thought to be a major protein componentof the membranes(14,15). Nakao, et al. (13) have reported that the shapeof red cell ghosts changeson the addition of BTP, which suggeststhe presenceof a membrane ATPase associatedwith the control of cell shape. A membraneATPase is alsoinvolved in the active transport of sodium and potassiumin erythrocytes (21). Changesin ATPase activity could be expected, therefore, to be indicative of the extent of changesin membraneintegrity induced by the freezing of isolatedred cell membranes. Doebbler, et al. (4, 6) noted that a marked increasein ATPase act#ivity resulted from freezing and thawing of intact red cells, and this activity appearedto be associatedwith the cell membranes.Other investigators (7, 9) in their studiesof red cell membraneATPase, have used freezing and thawing as a meansof preparation of the enzyme. In the present work, changesin ATPase act)ivity of red cell membranesinduced 1j.v different freezing conditions were studied in order to elucidatemechanismsof freezing injury. EXPERIMENT,4L
membrane. Fresh human blood drawn in ACD-A solution was used in all experiment,s.The cells were washed four times wit#hisot,onicsaline.White cells were Preparation
Rccrix.ed 1 Supported tional Heart ‘Visiting Temperature pore. Japan. 31nvestigator ology, The St.rerlt. Sew
June 11. 1971. by Research Grants from Institute, NIH (HE-09011). scientist from the Institute Scirncc. Hokkaido University. and Head, Laboratory Xew York Blood Center, York. N.Y. 10021.
of CryobiEast 67th
of Cryobiology, York
as a Function
removed by suction, along with the supernate, after each washing. Red cell membraneswere prepared by one-stephemolysisin 20 milliosmolar phosphatebuffer of pH 5.8, accordingto the method of Dodge, et al. (2). After four washings with the same buffer the membranes were washed three times with 0.01 M histidine-imidazole buffer of pH 7.2, and finally reconstituted to the original blood volume with the latter buffer solution. This membrane suspension was called “pH 5.%preparation,” and was used in all the experimentsexcept for “pH 7.2-preparation” in Table 9. The pH 7.2-preparation was prepared with a phosphate buffer of pH i.2 insteadof pH 5.8. Freezing procedures. 0.5 ml of the original membranesuspension and 0.5 ml of 0.01 M histidine-imidazolc buffer, or additive solutionswere pipetted into test tubes (18 x 150 mm). The test tubes containing membranesuspensions of 1 ml were immersedin baths at various temperatures, or into liquid nitrogen (-196°C) for various periods.When spontaneousfreezing did not occur, freezing was initiated by touching the outsideof the test t’ubeswith a spatula cooledin liquid nitrogen. Frozen membrane suspensions were t,hawed by immersionin a water bath at, 37-40°C. and t’hen stored in ice for no more than 2 hr unt,il STPnse activity was measured. Duplicate specimenswere frozen eat+ time’ under the samecondit,ions. Measurement of ATPase activity. ATPasc :ICtivitv wasmeasuredby the method of Post, rt al. (16) wit,h somemodificat8ions. The reaction mixture contained 2 m&r A4TP (Na-salt. Sigma Chemical Co.), 2 mM M&l,, 80 m&r histidineimidazolo buffer (pH 7.2), 80 rnlx SaCI. 33 m&r KCI. Five-tenths of a milliliter of the original membranesuspension. and 0.5 ml of the 0.01 M histidine-imidazolebuffer or nddit,ivc solutioii were added to 1.5 ml of this reaction mixture 5,) that the final volume was 2.5 ml. Thp .<:amples wcrc incubat’cd at 37°C for 60 min. :~url the
reaction was stopped by the addition of 1.5 ml of cold 8% perchloric acid. iZfter filtration, inorganic phosphate in an aliquot of the filtrate was determined by a modification of the FiskeSubbaRow method (8). ATPase activity was expressed as micrograms of orthophosphatic phosphorus released per hour from ATP by the original membrane suspension of 0.5 ml. A sample without membrane suspension was run as a control for correction of a small amount of nonenzymatic breakdown of ATP. Although some inorganic phosphate was contained in all the membrane suspension, there was no additional TABLE T~;FFECT
release of inorganic phosphate from membranes during incubation without ATP. ATPase activity measured in the present experiment was the total activit#y of both (Na+ + K+)-dependent and nondependent ATPase, and no attempt was made to distinguish bet’ween them. All ATPase activity results are mean values from duplicate samples. Absolute values of STPase activity varied between each preparation of red cell membranes, and therefore the same preparation was used under any one experimental condition. RESULTS Efect of freezing temperature. As shown in Table 1, ATPnse activity in the membrane suspension increased after freezing by immersion of the tubes in baths of various temperatures. The results showed that the extent of increase in activity seemed to depend on the freezing bath temperatures, although t’he absolute values of ATPase activit,y after freezing at the same temperature differed between preparations. This increase in membrane *4TPase activity by freezing was not due to the release of ATPase from membranes or the fragment,ation of membranes. If the release of ATPase, or the fragment#ation of membranes had occurred by freezing, some ATPase activity would have been demonstrated in the supernatant. In fact, ATPase acbivity was not demonstrated in the supernat,ant of frozen membrane suspensions after centrifugation at 20,000 g for 15 min. Table 2 shows that the greatest increase in ATPase activity occurs in a short period of time following freezing; longer storage does not cause much further increase.
1 ON ATPASIZ
MEMBRANES” pg Pi/O.5 ml suspension/hr
12.3 23.4 35.6
-5 -10 -20 -83 -98 -196
5.3 17.2 30.7 46.4
(16) (36) (57)
(0) (18) (45) (63)
89.2 92.4 83.0
(108) (111) (100)
3 All specimens were frozen in freezing baths at each temperature for 15 hr (*), or 1 hr (**), and thawed
at, a bath
were used. Preparations
5.8.membrane preparation” prepared different samples of red blood cells. parentheses show percentage of increase relative to that obtained at, - 196°C.
Duplicate are “pH from three Figures in in activity
OF INCREASE FREEZING AT
2 OF ATPME ACTIVITY -5°C AND -196”Ca ATPase
1 2 3
-5 -196 - 196
2.0 5.8 4.0
16.2 49.2 42.8
a Preparations l-3 are “pH 5.8-preparation” IIuplirate specimens were frozen at -5”C, a water bath at 37p40°C.
from three for various
20.8 57.4 -
red blood cell samples. indicated, and thawed in
ATPASE ACTIVITY TABLE ATP~SEACTIVITYSFTER FOLLOWED HY RAPID
IN RED CELL
FREEZING AT-~‘C FREEZING TO -196°C” SLOW
ATPnse Expt. no.
activity, IS Pi/O.5 ml/hr
1 2 3 4 5 6 7 8
No freezing -5”C, 5 min -5”C, 60 min -5”C, 60 min + -196”C, -5”C, 5 min + - 196”C,
30 min 30 min -5”C, 60 min thaw O”C, 10 min + - 196”C, 30 min -5”C, 5 min thaw O”C, 10 min + -196”C, 30 min -19G”C, 30 min
1.2 5.8 12.6 13.0 25.2 33.0 55.2
initiat,ion of freezing at -5”C, temperature was about -1°C for about 6 min. Samples in Expts. 4 and 5, after freezing at -5”C, and without thawing, were immersed into liquid nitrogen. Samples in Expts. 6 and 7 were thawed once at a bath temperature of 37-1O”C after freezing at -5”C, and then kept in ice for 10 min before refreezing at - 196°C. Duplicate samples were treated under the freezing conditions mentioned above.
Table 3 shows the effect of slow freezing to -5°C followed by transfer of the preparation to -196°C as compared to fast freezing it directly to -196°C. The membranes frozen to -5°C for 60 min exhibited no further increase of ATPase activity in spite of transfer to -196°C (Expts. 3 and 4). On the other hand, ATPase activity inrre:lsed markedly in the membranes frozen at --5°C for 5 min. and then transferred t.o -196°C (Expt. 5), in comparison with those not transferred t,o -196°C (Expt. 2). More increase of ATPase act,ivity was found in the membranes t,harved once before transfer to -196°C (Expts. 6 and i). Such an increase was greater in the membranes frozen at -5°C for 5 min (Expt. ‘i), than for 60 min (Expt. 6). These result,s suggest t,hnt. the increase of ATPase activity by freezing
by the condition of prefreezing at -5°C. Data in Table -1-showed no marked difference in ATPasc activity in membranes frozen at -lWC,
lowed by transfer to -5” slllts indicate that ATPase
brane, once frozen at -196”C, is practically unaffected by the changes in the state of ice crystals or in other factors. Table 5 shows the effect of repeated freezing and thawing on ATPase activity at -so and -196°C. Repeated freezing and thnwing at -5°C increased ATPase activity slightly. but. there was virtually no additional increase on repeated freezing and thawing at -196°C. From these results, it would seem that maximum increase of ATPase activity occurs in membranes once frozen at -196”C, and that no further changes of activity occur from subsequent freezing. The results from repeated freezing and thawing at -5°C suggest that t’he slight increase
.z Samples in Expts. 2 and 3 were frozen at -5°C only for 5 and 60 min, no transfer to - 196°C. After
or -10°C. These reactivity of the mem-
OF TRANSFER FROM -196’C IV -so .1x11 ON .4TPas~: ACTIVITY OF 1: I,.I) C'~:r,r. kfEMIiKr\NES
No freezing -196”C, 10 min -196”C, 10 min
2.2 X6.0 ix.8 Xi.8 I-1.8 28.4
10 min 4 -lo%,
-5”C, 60 min - lO”C, 60 min
OF REPLATED FREEZING ON ATPas*; ACTIVITY ~%M~IRANES' Freezing
No freezing -5’C, 30 min -5”C, 180 min -5”C, 30 min X 3 -5”C, 30 min X 6
AT -5°C OF
ATPase activity, pg Pi~‘0.s mlthr
3 2 13.0 IS.2 Ifi.
:’ L’ No freezing - 196°C 60 min !)4.4 -1964: 20 min X 3 !bl h -19(i”C, 10 min X 6 !E.N -a Membrane suspensions were frozen at -5Y: and - 196°C for the indicated periods, and thawed at bath temperature of 37-40°C. After keeping in ice for about 5 min, the,y were frozen again. Fr~zing and thawing was repeated in the same W:I~ :i OI 6 times.
OF COOLING OF MEMBRANES
ROTE ON ATP~sE ACTIVITY FI~OZEN AT -60°C activity, Pi/O.5 ml/hr
Cooling rate, “C/&n
2.0 18.0 20.6 23.4 33.6
2.4 4.0 8.8 22.0
EFFECT OF FINAL TEMPEKATURE ON ACTIVITY ON MEMBRANES FROZEN SIMILAR COOLI~VC RATE” Final temperature,
ATPase activity, Pi/O.5 ml/hr
-25” -35” -60” -115O
4.6 64.0 68.8 77.8 78.2
a Freezing carried out by immersion of test tubes containing membrane suspension in liquid nitrogen followed by withdrawing just before reaching final temperature. The tube was immediately transferred to a water bath at 3740°C for thawing. Cooling rate was 330420”C/min.
e.g., about l”/min at -5”C, and about 5OO”/min at -196°C. Table 6 shows the effect of cooling rate on the increase of ATPase activity induced by freezing. When the membrane suspensions were cooled to -6O”C, with various cooling rates, ATPase activity was the highest in the membrane frozen at the highest rate. On the other hand, the data in Table 7 showed that in cooling to various temperatures using constant cooling rates, the degree of activity increase did not differ markedly among the specimens cooled to temperatures from -25” to -196°C. These results show that cooling rate is a more effective factor than final temperature in t’he increase of ATPase activity by freezing. Ef7ect of cryoprotective additives. Table 8 shows the effect of some cryoprotective subst’ances on ATPase activity by freezing at -196°C for 60 min. Except for methanol, the compounds were relatively effective in inhibiting the increase of ATPase activity induced by freezing. Under phase microscopy, there was almost no difference in the appearance of memTABLE EFFECT
OF SOME ADDITIVES ON INCREASE ATPASE ACTIVITY BY FREEZING&
% of ATPase activity may be due to slight increase of activity in almost all the membranes rather than to maximal increase in some of the membranes; otherwise more increase of activity would be expected to occur in the membranes repeatedly frozen and thawed at -5°C. The microscopic appearance of membranes after freezing at -5°C for 30 min also showed that the great majority of membranes were strikingly altered in shape as compared with nonfrozen membranes. Effect of cooling rate. The results so far obtained indicate that the lower the freezing temperature the greater the increase of activity. However, there still remains the question of whether it is the rate of freezing or the freezing temperature itself which affects the increase of ilTPase activity, because, in the freezing methods used, there are wide differences in cooling rates among the temperatures of freezing;
none 5y0 glycerol 4v0 glycerol 3y0 glycerol
2% 1% 5% 5% 5% 5y0 5% 5% 5’$$
23 50 19 20 23 25 30 45 100
glycerol glycerol sucrose dimethylsulfoxide glucose
lactose ethylene mannitol methanol
a All additives
were present at the final concen-
tration shown in membrane suspension of 1 ml. All specimens were frozen by immersion in liquid nitrogen (-196°C) for 60 min, and thawed in a bath at 37-4O”C. b Percentage of degree of increase in ATPase activity compared to degree of increase of ATPase activity without additives.
IN RED CELL TABLE
pH 5.8-Preparation Freezing temp. “C
Hematacrit x0 S&l
0 -5 - 196
Ratio x 100
9 pH 5% 1~1) 7.2-b~EMISltt\NE
__ ATPase activity, pg Pi/O.5
pH i.2-Preparation --.--.~-. ~--~Hematocrit value, % Ratio x 100 0.3 Y L’o NaCl N&l
6.8 14.8 67.6
ATPase activity /A; P,, o.,? ml ‘hr
:34.‘l 33.8 :
1~Membrane suspensions in 0.01 M histidine-imidazole buffer (pH 7.2) were frozen for 30 min at -5’C, and - 196°C. Histidine-Imidazole buffer (0.01 M, pH 7.2) was added to the one part (no N&l), and NaCl solution was added to the other to give the final concentration of 0.3 M (0.3M NaCl). Hematocrit values wit,h both membrane suspensions were measured by centrifugation at 11,500 rpm for 15 min. branes before and after freezing
with the effec-
tive additives. Menzbrane permeability and ATPase activity. Hematocrit measurementsof membranes in media of different concentrations was used to demonstratewhether membranesare semipermeable or not, in the sensethat water will pass rapidly in either direction to maintain isotonicity with the external medium.Hematocrit values in 0.01 M buffer solution containing no NaCl, and 0.3 M NaCl were measuredwith two diierent membrane preparations, one with low ATPase activity, and one with rather high activit,y, without freezing. Hemxtocrit valuesin Table 9 showedthat t,he pH 5%membranepreparation ,shrank in 0.3 M SaCl solution, but the pH 7.2-prepurat,iondid not. This suggeststhat the pH S.&preparation may be impermeableand the pH 7.2-preparationmay be freely permeable to X:1+ ;~ndalso possibly to ATP, and that for this reason,ATPase activity in the pH 5%preparation is very much lower than in the pH 7.2preparation. It would seemfrom these results that :YTPase activity is closely related to the semipermeability of membranes, and, accordingly, to the accessibility of ATP t,o iZTPase. Aftclr freezing at, --5” and -196”C, ATPase activity increasedmarkedly in pH 5%preparation. On the other hand, ,1TPaseactivity in pH 7.2preparation did not change after freezing at hot,h temperatures. From these results it seems likely that t’he increaseof ATPase activity intlurc~l by freezing may be due to t’he changein scmipermeabilitvof membranes.
The rate of cooling appears to be a main factor in the increaseof membraneATPase activity
branes,the fast’er rates causinggreater increase of activity (Tables 6 and 7). Various factors may contribute t,o membrane damagebrought on by freezing?but differentiation betweenthem is difficult. Our result’salso suggestthat the action of ice crystals on membranesmay be an important factor in the increaseof ATPase activity, especially under the conditions of rapid cooling. Lusena and Rose (11) observed that rapid rates of ice growt8hwere more damagingto red cellsthan slow rates, regardlessof temperature. Under rapid cooling, tberefore. the effect’s Of cryO]JrOtective
;lgc!ntd 011 the COmpounds
Table 8 may be explainedin part by the modifica,tion of ire crystal growth through H-bonding of water, and competition with ice for available unfrozen water. Concentrated electrolytes, however, may not be a main factor for the increase: of activity in the present work at least, because significant increaseof STPase activity WRPnot, observed on membranessuspendedin 1.2 M NaCl solution for 15 hr at 1°C (unpublished data). Marchesi and Palade (12) demonstratedthat both Mg-ATPase and Sa-K-hTPase of guinea pig red cell ghostswere localized on the inner surfacesof the ghos;tmembranes.Our dat,a wit#h unfrozen membranesin Table 9 showed t,hat, membranes with semipermeability properties (pH 5Xpreparwtion) had very low ATPnse a~-
tivit,y, while membranes without marked semipermeability (pH 7%preparation) had rather high act)ivity. These results suggest that ATPase activity of red cell ghosts is closely correlated to the accessibility of ATP to ATPase localized on the inside of membranes. AccordingIy, the increase of ATPase activit’y by freezing probably dependa upon leakiness or semipermeability changes resulting from damage of membranes V,9). Doebbler, et 01. (6) have noted that the level of ATPase activity of red cells depended upon the methods of lysis, including freezing, hypertonic solutions, mechanical agitation, etc. Their results suggested to us that the changes in membrane ATPase activity may likely refer to a measure of disruption of red cell membranes, and changes in semipermeability characteristics. However, ihe possibility that membrane STPase itself might be activated by freezing cannot be excluded. although no evidence has thus far been obtained. The results in Table 3 also may be explained with the abovementioned considerations of the increase in ATPase act.ivity by freezing and of the localization of STPase on membranes. It appears that low act.ivity in the membrane frozen for 5 min, and not otherwise treated, is due to incomplete freezing (Expt. 2). Further increase of activity after freezing at -5°C for 60 min (Espt,. 3) suggests that the shrinkage of ghosts by dehydration, and the mechanical stress of ice attendant on the completion of freezing may affect the structure of membranes resulting in some destruction of membranes and in the activity increase. On the other hand, freezing at -5°C reduced the effects of freezing at -196°C (Expt. 5), or made them ineffective (Expt. 4) when the membrane suspensions frozen at -5°C were directly transferred to liquid nitrogen. These results would be somewhat similar to the effect of dehydration by a prefreezing method which is remarkably effective for survival freezing at superlow t,emperatures in plant tissues and insects (1). It suggests the shrinkage of ghosts by dehydration with freezing at -5°C prevents the membrane destroying effect of freezing at -196”C, which renders ATP accessible to BTPase, resulting in increase of its activity. Such effe& of dehydration were irreversible in part, since ATPase activity was not fully increased by freezing at -196°C even after
thawing from -5”C, especiaily when the membranes kept at -5°C for 60 min, as compared with the activit,y in the membranes directly frozen at -196°C (Expts. 6 and 7). This may be due to the irreversible blockage of active sites by shrinkage of ghosts. ilTPase activity differs considerably in membranes from different preparations (16, 19, 20). In the present study, freezing resulted in an increase in ATPase activity, only with the pH 5%preparation, but., not with the pH 72-prcparation (Table 9). In our preliminary experiment (18) with rabbit red cell membranes prepared by osmotic lysis by phosphate buffer of pH 7.3, inactivation of ATPase occurred a,fter freezing at -5”C, -lO”C, and -20°C (not at -196°C). Red cell ghosts prepared by Whittam (20) did not exhibit ATPasc activity at all when ATP was added to the medium. Our results and those of others suggest that ATPase activity is closely associated with t,he membrane prcparation used and t,hus with the charact)eristic of isolat’ed membranes. *4TPase, therefore, appears useful as a marker for the integrity of membranes. SUMMrlRY The effects of freezing on the membrane of red blood cells were examined using membranes prepared from fresh human blood by one-step hemolysis with phosphate buffer of pH 5.8. adenosinetriphosphatase Membrane-associated (ATPase) was used as an indicator of freezing effect,s. Freezing and thawing resulted in marked inrrease of ATPase activity, as compared to that of unfrozen membranes. The rate of freezing was a main factor in the increase of ATPase activity, the faster rate causing the great’er increase. The increase of STPase activity by freezing appeared attributable to the increase in accessibility of ,4TP to ATPase arising from disrupt’ion in membranes. REFERElCCES 1. Asahina, E. Prefreezing animals to surrire low temperature. (1959). 2. Dodge, J. T., Mitchell,
The preparation of hemoglobin-free
as a method enabling freezing at an extremely Nnture 184, 1003-1004
C., and Hanahan, D. J. and chemical characteristics ghosts
IN RED CELL
Arch. Biochem. Biophys. 100, 119-130 (1963). 3. Dorhblcr. G. F., and Rinfret,. A. P. A biochemi(.a1 basis of hcmolysis of erythrocytes by freezing :and tltxwinp. Fed. Proc. 21, 67 (1962). 4. Doehbler. G. F. Xncleoside triphosphatase wtivity of red cells lysed by freezing. Z?rt. f'ongr. Biochem. Vol. 8, dbst. 649 (1964). .5. I)cwhhkr. G. F.. and Rinfret, A. P. Rapid freezing and tlrxlving of blood. Physical and chemical ronsitlrration of injury and protrction. Cryobiology 1, 205-211 (1965). 6. Docbbler, G. F.. Role. -4. W.. and Rinfret, A. I’. Freezing of mammalian blood and its c,onstituents. ZU “Cryobiology” (H. T. Meryman, rd.). pp. 407-450. Academic* Press, 1,ondon. 1966. 7. Dunham. E. ‘I‘.. and Glynn, I. M. Adenosinetriphosphatasr activity and the active movements of alkali metal ions. J. Physiol. 156, 274-293 (1961). 8. Fiske. C. II., and SubbaRow, Y. The colorimetric determination of phosphorus. J. Biol. cytrs.
iion of Mg-Nx-K-activated adenosiw triphosphntase. .I. Cell Biol 35, 385-401 (1967). 13. Nakao. M.. Xnkao, T.. Tatibnna. M.. and Yoslrikaw-n. H. Shape transformation of erytlrrot~ytf~ ghosts on addition of :tdcno
kl.. Sak:~o. T.. Yamnzoe. s.. and ‘I~o~hiltnn;t, H. .L\denosine triphosph:lt(’ :tnti s11:ipr of rq?hroc~tc. .z. Bioclrem. 49, 487492 (1!)61).
15. Ohnishi. T. Estraction of actin- an11 m!.osinlike proteins from erythrocyte mt~ilihr:tnc~. J.Biocire7n.
16. Poh(, 1~. I... Merritt. C. R.. KinsolT-ing. C. Ii.. and .\lbright. C. D. Membrane :rdruosino tri~,llo~:l)hntn~(~ as a participant in 11113:tcntivc tmnsl)ort of sodium and potnssitun in the 11uman erythrocyte. J. Riol. Chrt/,. 235, 17OtG-1802 17.
9. Heinz, E.. ant1 Hoffman, J. F. Phosphate incorporation and Sa, K-ATPase activity in human red blood cell ghosts. J. Cell. Camp. Z’hysiol. 65, 31-44 (1965). 10. lovelock. J. E. The denaturation of lipid-protein complex as :I cause of damage by freezing, Proc. Roy. SOC. B 147, 427-433 (1957). 11. Lusena. C. V., and Rose, D. Effect of rate of ice-crystal gro\vth on hcmolysis of erythroc.ytcs. ilrch. Biochenz. Biophys. 65, 534-544 (1956). 12. Marchesi. V. T., ancl Palade, G. E. The localiza-
Rowe. .\. W. Biochemical aspeck oi qvoprotec~tive agents in freezing and t,haning. (‘ruebiology 3,12-18 (1966). Tnlwhnr:r. 1. Effwt of freezing on the hTP:lse in wtl rell mcmbranrs of rabbits. Lorr Temp. sci. Sec. B 25, 155-158 (1967) (in Japanese). Weed, R. I.. Reed, C. F.. and Berg, G. Is hemoglobin ;tn essential structural componrnt~ of human erythroq-to membranes? .I. (‘lit/ Inuesf. 42, 581-588 (1963). Whittam. 1~. The asymmetrical stimulation of a nwmbrnne :~tlcnosine triphosphatsse in relation to active cation transport. Biochem. J. 84, 110-118 (1962). Whittam. R, In “Transport and Diffusion in Red Blood Cells” p. 228. The Williams and 1Vilkins (“(0.. Thrltimoro. 1964.