of biological chemistry vol. no. 10, 11651-11659, 1982 ... · the journal of biological chemistry...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U. S. A. Vol. 257, No. 19, Issue of October 10, pp 11651-11659, 1982

A Description of the Holes in Human Erythrocyte Membrane Ghosts* (Received for publication, February 8, 1982)

Michael R. Liebert and Theodore L. Steckg From the Department of Biochemistry, University of Chicago, Chicago, Illinois 60637

The holes in hemoglobin-free ghosts generated by osmotic lysis of human erythrocytes have been char- acterized. The efflux from ghosts of water-soluble probes with a Stokes radius of 5,.2 to 61 hi was measured, The kinetics was usually first order, suggesting a homogeneous ghost population, but became complex because of sieving when hole and probe were approximately the same size. The area of exit per ghost, calculated from Fick's law of diffusion, varied widely but the number of holes per ghost, calculated from the sieving of pairs of probes, was always unity. The hole had a circular rather than elliptical shape and a path length of 60 A, approximately the thickness of the membrane.

Ghosts were induced to partially seal so as to trap various probe molecules at diffusional equilibrium. The sustained retention of probes ruled out the possibility that the holes are impermanent, intermittent breaches in membrane continuity. The dispersion of hole size under a given set of conditions was fairly narrow.

Centrifugation on density barriers composed of ap- propriate solutes separated ghosts populations into fractions with holes larger than the solute (which pelleted) and smaller (which floated). The fractional floatation of ghost populations on barriers of dextran, sucrose, mannitol, and CsCl was calibrated with estimates of their hole radii obtained from efflux kinetics to es- tablish a rapid, simple, and precise technique for estimation of mean ghost hole size. The average difference between the hole radius measured by density barrier and equilibrium trapping methods was 4 & 8 S.D. A.

Holes could be reduced at high ionic strength to 7 hi in radius and dilated at very low ionic strength to >lo4 b (1 pm), at which point they became visible in the dark-field microscope as a single round 1esion.The hemolytic hole is thus a continuously-tunable molecular filter, the area of which can be modulated over more than a million-fold in a defined fashion.

The viability of cells depends on the continuity of their plasma membranes. That large and indefinitely stable holes can be opened in erythrocyte membranes by osmotic lysis and closed by warming in saline (1-11) contradicts our conception of biological membranes as being built upon a self-sealing, fluid bilayer,

It was the aim of this research to explore the nature of the

* This research was supported by Grant BC-95 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Grant GM-07281. #Supported by United States Public Health Service Training

Chicago, IL 60637. 8 To whom correspondence should be addressed at 920 E. 58th St.,

hemolytic holes in human erythrocyte ghosts. In this report, we describe the number, size, shape, and other features of these lesions and present a simple centrifugation method for estimating their radius. In a companion paper (12), we explore the dynamics of hole size. Portions of this work have been presented previously in brief (13) and in extenso (14).

EXPERIMENTAL PROCEDURES

Experimental Strategy-Experiments to determine the apparent hole area per ghost were of four types. I) We followed the efflux kinetics of probes of various Stokes radii from ghosts at different stages of sealing. 2) We determined the trapping of probes of various Stokes radii equilibrated into ghosts which were then sealed to different degrees. 3) We measured the degree of floatation of ghosts centrifuged on density barriers composed of various solutes with different Stokes radii. 4) We visualized extremely dilated holes by dark-field microscopy.

Materials-Water was double deionized and carbon-fitered. Chemicals were reagent grade or better from Merck, Fisher, Mallinck- rodt, and Baker. Biochemicals were purchased from Sigma unless specified. Dextrans TI0 and T70 were from Pharmacia. Ferritin was EM grade from Polysciences. ["HISucrose was obtained from New England Nuclear. Blood was obtained either fresh from normal human donors or from outdated units generously provided by the University of Chicago Blood Bank. None of our results varied with the duration of storage in vitro or blood type.

Membrane Preparation-All procedures were at 0-5 "C (typically on ice) and all centrifugations were performed in a Sorvall SS-34 rotor at 15,000 rpm unless specified. Human red cells and hemoglobin- free ghosts were prepared as described (15), except that the hemolysis buffer was 5 m~ NaPi (pH 8), 0.01 mM MgS04 and the membranes were suspended for 10 min in this buffer before each centrifugation to allow hemoglobin to exit fully.

Determination of Hole Size by Efflux Kinetics-Ghost pellets (5- 8 X 10' ghosts/ml) were equilibrated with 50-fold concentrates of various probe solutes for a t least. 10 exit half-times, sealed to varying degrees where indicated, and then injected into a beaker containing a 30-60-fold excess of briskly stirred buffer via a syringe equilibrated to the temperature of the beaker. The buffer in the ghost suspension was the same as in the beaker. Aliquots of the medium were aspirated at intervals through Millipore prefilter/flter assemblies to obtain filtrates (16). The results of some long time courses were corroborated by centrifugation; no systematic differences arose between the two methods of separation.

In the case of glyceraldehyde-3-P dehydrogenase, an endogenous probe, ghost pellets were used directly, and the prefdters were coated with gelatin as described (16).

According to Fick's Fist law, dnldt = -DA'(dC/dx); dnldt is the rate of diffusion of solute molecules across an apparent area, A', driven by a concentration gradient dC/dx, and D is the diffusion coefficient for the solute. For our purposes this equation was written:

dCldt = (DA'/AX)(l/Ve + l/Vm)(Ceq - Ct) (1)

Integration (see Ref. 14) gives Equation 2:

C - Ct -DA'(l/Vm + 1/Vg)t c,, - co Ax

I n 2 = (2)

C is the concentration of the probe in the filtrate (in moles/cm') at zero time (O), at equilibrium (eq), and at intermediate intervals (t, seconds). Co was estimated by extrapolation to t = 0. Vis the volume of solvent (in cubic centimeters/ghost) in the efflux medium (m) and

11651

11652 Holes in Erythrocyte Membrane Ghosts

in the ghosts (8). We estimated V, by multiplying the volume per ghost in the injected sample (i.e. sample volume/ghost number) by the factor (C, - Cd/Ceq. V, was the total medium volume per ghost minus va. Equation 2 was solved for A’, the apparent area of the path of escape from the ghosts (in square centimeters). A’ differs from the true hole area, A , because of sieving; i.e. the reduced permeability of pores to solutes of non-negligible size. Whereas A = nnr’, A’ = nn(r - a)’; the number of holes is n, and the radii of the holes and probes are r and a , respectively (see below). The diffusion path length is designated Ax.

Literature values for D, the diffusion coefficient (in Ficks, square centimeters per s X corrected to 2 “C in water), and the Stokes radius (in angstroms) for the various probes were as follows: ferritin, 2.03 and 61.0 (17, 18); FITCI-Dextran T70, 2.15 and 57.7 (19-21); glyceraldehyde-3-P dehydrogenase, 2.82 and 43.0 (17.22); FITC-Dex- tran T20, 4.21 and 29.5 (19-21); myoglobin, 5.80 and 20.9 (17); and sucrose, 29.3 and 5.2 (17, 23). For NADH, we calculated D = 18.6 X 10” cm2 s-’ and a Stokes radius of 6.5 A (14). Except in Table I, Ax was taken to be 5 X 10“ cm; i.e. 50 A (24).

We found it justified to assume that the diffusion path was a right cylinder and that the properties of the solvent in the hole (pore) were the same as the bulk solvent; we also ignored viscous drag between solute probes and the walls of the hole, since the diffusion path was not long compared to the molecules used to probe it. These issues are discussed in detail by Solomon (25).

In order to avoid the high background inherent in the use of unbound probes, we also analyzed the efflux of an endogenous probe, glyceraldehyde-3-P dehydrogenase. This enzyme is a natural membrane constituent, bound to an excess of homogeneous, cytoplasmic surface sites with on and off rate constants much greater than the rate constant for its egress (16). KD, the dissociation constant for the binding reaction, was determined in each experiment from ghost concentration, enzyme activity in the filtrate at equilibrium, and total enzyme activity present (16), assuming that there are 1.06 X lo6 binding sites/ghost (26, 27). Since there is a 5- to 10-fold excess of binding sites over enzyme molecules, we assumed that binding site concentration remained constant during efflux. Efflux of the enzyme should then follow first order kinetics analogous to Equation 1 except that C,, now represents the concentration of enzyme in the external space at equilibrium and the volume term (l/V,,, + l/V& becomes (l/Vm + KO/(&. V, + N s ) ) , where N, represents unliganded binding sites in moles/ghost (see Ref. 14 for derivation). The increase in N, during efflux caused no more than a 4% systematic error in the estimation of A’ for which it was unnecessary to correct. Because of the highly avid binding of enzyme and the excess of binding sites, efflux rates (hence estimates of hole area) were remarkably insensitive to V,. We calculated that, in the most unfavorable circumstances, a 2-fold change in V, changed efflux rates by less than 6% (14). We therefore either measured V, by a parallel efflux kinetics experiment, with an unbound probe (e.g. we used the data in Fig. 1 to provide V, in Fig. 2) or assumed V, = 50 pm3 (see “Results”). While the results using this bound enzyme were satisfactory, the complexity of the analysis prompted us to use data from the unbound probe studies for routine kinetic estimation of hole area.

Determination of Hole Size by Probe Trapping at Equilibrium- In one series of experiments, ghosts with large holes were equilibrated on ice for at least 2 h with solutions of prohes and then induced to seal in warm salt solutions. The ghosts were washed free of the excess probe and then incubated for 22 and 28 h on ice in copious buffer to allow complete release of probes from those ghosts which were not sealed. (Prolonged incubation allows the complete escape of probes from ghosts with holes just slightly larger than the solute. For example, we calculate that it takes 28 h for 98% of ferritin (Stokes radius = 61 A) to escape from ghosts with a single hole of r = 62.4 A; during which time only -1% would have escaped from ghosts with r = 61.1 A,) The ghosts were pelleted and assayed for their retention of the probes, which was taken as a measure of the fraction of ghosts with holes which had become smaller than the probe during the sealing step. Values for 100% trapping were obtained by vigorous sealing at 37 “C in isotonic saline for prolonged periods (see “Results”).

The trapping of glyceraldehyde-3-P dehydrogenase after partial sealing was assessed by determining the fraction of the enzyme released by vigorous eluting conditions known to completely disso- ciate the enzyme from the membrane (16). Values for 100% release were determined by dissolving the ghosts in 0.1% Triton X-100.

’ The abbreviation used is: FITC, fluorescein isothiocyanate.

DeterminatLon of Hole Size by Floatation on Density Barriers- Density barrier solutions were prepared from IO-fold concentrates in the same buffer which bathed the ghosts. The final barrier solutions contained Dextran T70 and T10 of density 1.010 g/cm3 (3.07 g of dextran plus 100 g of solvent or 2.67 g of dextran/100 ml of solution), Sucrose of 100 m~ (1.014 g/cm3), mannitol of 150 mM (1.010 g/cmJ), or CsCl of 50 mM (1.005 g/cm’). These barrier concentrations were not critical, falling on a plateau of fractional floatation uersus density, but should be kept constant. It was also important in all cases to match the buffer in the barrier solution to that in the ghost suspension, lest the disparity in density and/or osmotic activity alter the floatation.

Routinely, 0.15-ml aliquots of ghost suspensions (0.8-4 X IO8 ghosts/ml) were layered on 1.35 ml of t.he barrier solution and spun at 15,000 ’pm for 12 min (for dextrans) or 18,000 rpm for 10 min (for sucrose, mannitol, and CsC1) in 3-ml polycarbonate tubes. I t was important to treat all samples uniformly with respect to the time spent on top of the gradient, since the ghosts slowly shrink under the osmotic force, increase in density, and may then sediment through the barrier (see “Results”). The entire supernatant fraction (-1.45 ml) was carefully removed with a Pasteur pipette by aspiration along the side of the tube opposite the pellet until only 50 pl of supernatant remained. The pellet was resuspended to 1.5 ml in the sample buffer. The ghosts in each fraction were diluted in filtered saline and counted in a Coulter Counter. The fraction of ghosts floating was calculated after correction for the buffer blank and coincidence.

The ability of dextran barriers to accurately resolve sealed from unsealed ghosts was tested with mixtures of two ghost preparations, one well sealed and the other unsealed. The fractional floatation of mixtures of varied proportions differed from that predicted from the input stocks by an average of only 2%. The recovery of ghosts from the gradient generally exceeded 90%. Of the ghosts that floated, 75% were in the upper third of the fluid and the remainder in the middle third, so that the error from contamination of the pellet was small.

Assays-The concentrations of ferritin (28), myoglobin (29), and NADH (17) were assayed spectrophotometrically. [‘H]Sucrose was determined by liquid scintillation spectrometry in Aquasol. Glycer- aldehyde-3-P dehydrogenase was determined kinetically (16). FITC- Dextran T20 and T70 were analyzed fluorometrically using excitation and emission wavelengths of 485 and 515 nm. The standard deviations in these assays were within 4% of the means. The numerical density of ghosts was determined with a Coulter Counter model ZB.

RESULTS AND DISCUSSION

Kinetics of Efflux of Unbound Probes from Ghosts-We used an integrated form of Fick‘s law to estimate the cross- sectional area of the holes in ghosts. Probe solutes of defined Stokes radius were allowed to equilibrate into the included space of packed unsealed ghosts. Their exit was then followed after injection into an excess of buffer. Fig. 1 illustrates this method for ferritin. Note these features. (a) The zero time intercept is relatively high, reflecting the magnitude of the extracorpuscular volume in the packed ghost suspension. The internal volume per ghost was estimated from the intercept and plateau values and the ghost concentration in the packed suspension to be 56 pm:’ in this experiment. (6) The time course is hyperbolic with no initial lag and a horizontal plateau. (c) A semilog plot of the data according to Equation 2 was linear (Fig. 1, inset). The kinetics was thus compatible with a homogeneous, first order, two-compartment diffusion process. The linearity also signifies that the average hole size was constant during the experiment. The standard deviation of the slope of the semilog plot was less than 4%. ( 4 The slope of the semilog plot corresponds to an average apparent diffusion window of 1.76 X lo4 A2/ghost, as calculated from Equa- tion 2. This value is approximately one-millionth of the surface area of the ghost (1.4 X 10’” A‘; see Ref. 31) and o&’ 50% greater than the cross-sectional area of the probe itself (1.17

In these experiments, care was taken to allow complete pre- equilibration of the probe with the included volume to avoid biasing the measurement against the least fenestrated ghosts. In addition, we did not wash the membranes after loading

X 104 A?,.

Holes in Erythrocyte Membrane Ghosts 11653

TlYl ,YO”,

0; ; 4 6 Q Ib I2 :: io TIME WIN)

FIG. 1. Ferritin efflux from ghosts. Ghosts were prepared in 5 mM NaPi (pH 8), 0.010 m~ MgS04 and then washed into 5 mM Nap, (pH 7.2). The ghost pellet was made to 25 m~ NaCl, 5 mM Nap, (pH 7.2), 0.1 mM EDTA, 0.0028 m~ ferritin by the addition of concentrates and the suspension allowed to equilibrate for 4 h on ice. Three ml of the suspension (6.3 X lo9 ghosts/ml) were injected into 47 ml of the same buffer (lacking ferritin) stirring briskly in an ice bath. Filtrates were collected at intervals and assayed for ferritin by its absorbance at 320 nm. The inset is a plot of the data according to Equation 2.

(although this imposed a significant background of unincluded probe), since washing would deplete the most porous ghosts and bias the outcome in favor of the best sealed species.

The linearity of the semilog plot of probe escape kinetics is a measure of the homogeneity of the ghost preparation. It might be objected that such experiments would not sense subpopulations of ghosts with holes so small that the probe is not taken up during the loading step. If this were the case, however, the apparent average internal volume per ghost would increase as the probe size decreased. This was not the case. The internal volumes obtained with given pairs of probes of different radii typically agreed to within 5 pm3 and no systematic deviation of internal volume with probe size was observed. The average included volume for probes of all sizes (sucrose to ferritin) was approximately 50 pm3. This value is similar to that found previously (8-10,30) and is one-third of the maximal volume for ghosts with a surface area of 140 pm2

The potential contribution of an unstirred layer of solvent (31).

was evaluated using the equation of Dainty (32):

l / P o b h 1 / P t m e +- P / D (3)

where P is the permeability (observed or true) and p is the thickness of the unstirred layer (taken to be 5 X cm (33)). Since p /D was never more than 2.5 X lo3 s/cm and l/P,,b, was never less than 5 X lo5 s/cm, the observed permeability represented the true permeability to within 0.5%; hence, the unstirred layer was negligible. Confirmation of this conclusion was provided by comparing the kinetics of efflux of FITC- Dextran T70 into buffer stirred vigorously or not at all. The results were indistinguishable.

We also considered the possibility that efflux rates could be reduced by the inhomogeneity in distribution of a probe within a ghost that would arise when the rate of diffusion out of the ghost exceeded that within the included space. Accord- ing to Macey (34), the solute concentration within a cell of

radius R remains uniform if D / R . P >> 0.13. In our case, D / R . P was never less than 612. Hence, uniformity of the probe within ghosts is assured.

Interpretation of the data according to a simple, two-compartment model required that marker molecules did not bind to or otherwise partition unequally between the ghost and medium compartments. This was shown to be the case by measuring the equilibrium distribution between ghosts and medium of the following probes: FITC-Dextran T20, FITC- Dextran T70, ferritin, myoglobin, [3H]sucrose, and NADH. In no case did the distribution vary by more than 4% from that predicted. The mean distribution was 1.00 f 0.01 S.D. for all such experiments. Similarly, we demonstrated that none of these probes bound significantly to the filtration apparatus; that is, filtrates of such solutions in the absence of ghosts contained 100 f 1.5% S.D. of the unfiltered stock.

Another test of the ideality of the system was the lack of dependence of efflux kinetics on probe concentration. This was shown to be the case for FITC-Dextran T70 when varied over a 50-fold range of concentration (0.3 to 15 p ~ ) .

Probe charge and chemical composition could conceivably affect efflux kinetics, hence the apparent size of the diffusion window they reported. To test this premise, we measured the simultaneous efflux of probes of very similar radius but markedly different conformation and composition. The escape of 1.4 ~ L M FITC-Dextran T70 and 2.8 p~ ferritin from the same ghosts into 20 volumes of 5 mM Napi (pH 7.2) gave apparent diffusion areas of 1.40 X lo5 and 1.35 X lo5 A2/ghost, respectively. In a related experiment, ghosts were double loaded with 1 mM NADH and 4.2 KIM [3H]sucrose and sealed by warming at 37 “C in 50 mM NaC1,5 m~ Napi (pH 7.2). Efflux kinetics gave an apparent escape window of 424 A’/ghost for NADH and 481 A2/ghost for [3H]sucrose. While the difference in the areas reported by the two probes in each pair is within the error of the method, the small differences may actually reflect greater sieving of the larger probe of each pair (see below). We conclude that the composition of the probes did not affect the area reported.

Kinetics of Efflux of a Bound Probe-Glyceraldehyde-3-P dehydrogenase is specifically associated with band 3 at the cytoplasmic surface of red cell membranes but can be rapidly eluted by NADH and electrolytes (16). This enzyme therefore provides an endogenous, low background probe, as illustrated in Fig. 2. In this experiment, ghosts were pre-equilibrated with a solution of NADH plus NaCl chosen so that the endogenous enzyme present would be almost entirely bound when packed in a pellet but would be almost entirely released by mass action when diluted 16.7-fold. The efflux proceeded hyperbol- ically without an appreciable blank. The absence of an initial lag suggests that the enzyme equilibrates between the membrane and the internal aqueous space much more rapidly than it exits. This inference is warranted; enzyme release from inside out vesicles or from ghosts treated with saponin to increase their leakiness proceeds to completion in 1 s or less (16), two orders of magnitude faster than the rates seen here.

The data in Fig. 2 conform to a linear semilog plot (inset) with a slope which reflects the dissociation equilibrium as well as the exit step (see “Experimental Procedures” and Ref. 14). The apparent diffusion window in this experiment was estimated as described above to be 1.17 X lo5 L’/ghost.

The Nature of the Ghost Sealing Phenomenon-It has long been observed that warming ghosts in salt solutions partially restores the impermeability of the membrane (1-11). The literature is unclear, however, as to whether some ghosts seal fully while others remain highly porous or all seal partially. These alternatives can be distinguished by a comparison of the exit window sensed by the kinetics of probe efflux with


I f , 1 I I

0.8 -

0 2 .E 0.6 ii

- - c .- c u e

0.4 -

i

\ 0.2 1 1 OB O ‘ t

0.0 0 4 8 12 27

Time (min) FIG. 2. Glyceraldehyde-3-P dehydrogenase efflux from

ghosts. Packed ghosts bearing endogenous glyceraldehyde-3-P dehydrogenase, the same as in Fig. l, were equilibrated with 25 mM NaC1, 5 m~ Nap, (pH 7.2), 1 mM dithiothreitol, 0.1 mM EDTA, 0.01 mM NADH. Three ml of the suspension (6.3 X 10’ ghosts/&) were injected into 47 ml of the same buffer and the fraction of the enzyme in the filtrate determined at the indicated intervals. The inset is a plot of the data according to the modification of Equation 2 described under “Experimental Procedures.”

that reported by the fractional trapping of the same probe equilibrated between ghosts and medium prior to sealing.We therefore set about to ascertain whether a partial reduction in the porosity measured by the efflux kinetics of glyceraldehyde- 3-P dehydrogenase signified that (a) some of the ghosts had become entirely impermeable to the enzyme (hence trapping it) while the remainder of the ghosts did not change porosity; (b) all of the ghosts became less porous but none trapped it; or (c) all of the ghosts became less porous and some of them trapped the enzyme.

Packed ghosts were incubated in a dilute salt solution at 25 “C for various intervals to reduce their permeability to the glyceraldehyde-3-P dehydrogenase bound inside without eluting it. The efflux kinetics of this enzyme were then measured. As seen in Fig. 3 (lower), the apparent escape area per ghost, estimated as described above, decreased markedly over 15 min at 25 “C, from approximately 16 times the cross-section of the probe to a value roughly equal to it.

At each time point, the ghosts were also resuspended in a large volume of 10 ~ L M NADH for 2 h to elute the enzyme and allow its complete escape from those ghosts with sufficiently large holes (Fig. 3, upper). The extent of enzyme release remained high during the rapid drop in efflux rates. That the ghosts remained generally permeable to the enzyme at a time when the efflux rate had become very slow, indicates that the sealing process did not reflect the complete closure of an increasing fraction of the ghosts but a partial reduction in the porosity of all of the ghosts. Furthermore, that the ghosts ultimately trapped most of the enzyme, indicates that they seal permanently and do not alternate between permeable and impermeable configurations. Thus, the reduction in the apparent escape area sensed by efflux kinetics does not reflect a reduction of the time fraction in whch flickering holes remain open but rather stable hole closure.

The Trapping of Different Probes a t Equilibrium by Ghost Sealing-Further evidence for graded hole closure during

0 2 I \ i

0 0 IO 20 30

Time (min)

FIG. 3. Ghost hole areas determined by enzyme efflux kinetics and enzyme trapping. Packed ghosts bearing endogenous glyceraldehyde-3-P dehydrogenase were prepared on ice in 25 mM NaC1, 5 mM NaP, (pH 7.2), 0.1 mM EDTA as in Fig. 1. Aliquots were warmed at 25 “C for various times to partially seal the ghosts, then chilled. Lower, kinetic assay. One ml of each aliquot was injected into 49 ml of the same buffer containing 5 p~ NADH and efflux kinetics analyzed as in Fig. 2. The apparent area of the efflux path was calculated from the slope of linear semilog plots according to the modification of Equation 2 described under “Experimental Procedures.” Upper, trapping assay. Portions of each aliquot were extracted with 25 mM NaCl, 5 mM Nap,, 1 mM dithiothreitol, 0.1 mM EDTA, 0.01 mM NADH (pH 7.2) to release all the enzyme not trapped in sealed ghosts, the ghosts pelleted, and the soluble enzyme measured. The values were the same after 1 and 2 h, signifying that the release of enzyme was complete.

ghost sealing was obtained by equilibrating unsealed ghosts with probes of varied Stokes radius and then partially sealing them in warm saline for 0 to 90 min. The fractional retention of probes after extensive washing reflected those ghosts with holes which had become smaller than the probe (assuming a constant ghost volume). The results of such an experiment are shown in Fig. 4. As sealing progressed, each of the probes became increasingly trapped, with larger probes always more retained than the smaller. The retention of smaller probes exhibited an initial lag, reflecting the interval during which the holes were of an intermediate size.

As in Fig. 3, the sealing time course in Fig. 4 can best be interpreted as the graded reduction of the radius of the holes in the ghosts. These data rule out abrupt transitions from fully open to fully closed configurations and exclude the possibility, not addressed by the kinetic method, that the hemolytic hole is statistical, opening and closing or oscillating widely in size about some average.

Hole radii were estimated from the equilibrium trapping data in Fig. 4, as follows. The fractional trapping of each probe at various times of incubation was converted into a probit. Plots of the probits a t a given time point as a function of area (rather than hole radius) had a Gaussian distribution in the ghost population. The mean cross-sectional area of the holes at various points along the time course was estimated from the probit plots. The values obtained showed a close parallel to a similar time course measured by an entirely different technique, the efflux kinetics of a bound probe (Fig. 3). This


Time (min) FIG. 4. Marker trapping by resealed ghosts. Ghosts were

equilibrated with 25 m~ NaC1, 5 mM Nap, (pH 7.2), 0.1 mM EDTA containing either 0.24 m~ myoglobin (X), 1.4 pM ferritin (M), or 1.4 PM FITC-Dextran T70 (0) on ice. Glyceraldehyde-3-P dehydrogenase (A) was endogenous. Aliquots of these mixtures were induced to seal at 25 "C for the indicated intervals, then chilled. Ghosts were freed of untrapped probes by fmt washing at 0-5 "C with the same buffer containing 1 mM dithiothreitol, 0.01 mM NADH (which released the untrapped endogenous enzyme) and then incubated in 24 volumes of this buffer on ice. After 22 and 28 h, samples were centrifuged and the probes retained in the pellets determined. That egress was complete was assured by the fact that probe release after 22 and 28 h incubation were indistinguishable in all cases. The retention of probes in the ghosts was taken as a measure of the fraction of ghosts with holes which became smaller than the probe during the sealing step. Values for 100% trapping were obtained with an aliquot of the ghosts which was vigorously sealed at 37 "C for 2 h in the aforementioned buffer. We believe that these ghosts were well sealed to these probes for two reasons. First, their mean hole radius, as determined by fractional floatation (see below), was 17 A; hence, it was less than the smallest probe, myoglobin (Stokes radius = 21 A). Second, the trapped (intracorpuscular) myoglobin space was estimated to be 72 pm3, a larger value than that routinely measured for ghost volume in kinetic and Coulter Channelyzer experiments (namely, 50-60 pm3). The ghosts incubated with FITC-Dextran T70 (M) were also analyzed for fractional floatation (see below) on density barriers of Dextran T70 (M).

finding suggests that each hole sensed by probes a t equilibrium has the same cross-section as the total escape area per ghost sensed by probe efflux kinetics. This result suggests that there is but one hole per ghost. This inference is substantiated in the next section.

Hole size was also monitored in the experiment by ghost floatation on density barriers, a method described below. These data are also plotted in Fig. 4 (M). The equilibrium trapping and density barrier techniques showed an average difference of 4 f 8 S.D. 8 between matched values for the hole radius at different time points.

A qualitative appreciation of the dispersion of hole size at any point can also be obtained from Fig. 4. After 22 min, for example, when 50% of the ghosts trapped FITC-Dextran T70 (Stokes radius = 58 A), more than 85% trapped ferritin (Stokes radius = 61 8) and only 3% trapped myoglobin (Stokes radius

The Size, Shape, Length, and Multiplicity of Hemolytic Holes-Let us assume that the true cross-sectional area of the

= 21 A).

diffusion path per ghost is distributed among n equivalent holes of radius r ; i.e. A = nm?. We can estimate neither n nor r from the total area per ghost. Furthermore, because sieving restricts diffusion, the values measured for the apparent area of such holes, A', underestimate the true area. The apparent radius of a hole of radius r sensed by a probe of radius a is, in fact, ( r - a) (35). Fortunately, the true area and number of the holes can be estimated by comparing the apparent areas reported by two probe solutes of unequal Stokes radius, a and b, exiting through the same holes, as follows. The total apparent areas reflected by the two probes are Ah = nm(r - a)z and A6 = nn(r - b)'. These two equations can be solved for the two unknowns, r and n:

Ah Ab n(r - a)' - m(r - b)2

n = - (5)

We equilibrated various packed ghost preparations with pairs of probes of different sue so as to measure their efflux kinetics simultaneously. The two apparent areas sensed for the common diffusion pathway were calculated for the probes and used to estimate n and r, hence the true area of diffusion. The radius per hole varied widely with conditions. For example, for standard ghosts in 5 m~ Napi, 0.01 mM MgS04 (pH 8.0), r = 140 2 21 8; in contrast, freshly prepared ghosts immediately warmed at 37 "C in isotonic saline had hole radii as low as 7 A (see Ref. 12 for details). Unlike the variation in hole size, n was invariably unity (Table I). We conclude that osmotically lysed membranes have but a single hole larger than sucrose, which is 5.2 A in radius.

These results can be illustrated graphically. Knowing probe Stokes radii a and b and assuming a series of values for r, we constructed families of theoretical curves for Ah uersus Ab for assigned hole numbers, n (Figs. 5 and 6). Our measured values for the apparent areas of probe pairs always feil close to the theoretical curve for n = 1, independent of the conditions of hemolysis or diffusion area.

We sought to verify that this method was capable of de-

TABLE I Hemolytic hole number and radius calculated from the

simultaneous efflux kinetics of probe pairs Several ghost preparations were sealed to a varying degree and

equilibrated with pairs of probe solutes whose efflux was then followed simultaneously. Several apparent areas of the diffusion pathway were calculated from equation 2 by assuming different path lengths. The corresponding hole number and the true radius per hole were then calculated from equations 4 and 5. The values shown here were selected so that the mean hole number was unity. The corresponding path length is 60 A. The data in Experiments 1 and 2 are those used in Figs. 5 and 6, except that Ax was taken to be 50 A in the figures.

Experiment Probe pair Hole number Hole radius

1 Ferritin Dextran T20

Dextran T70 Myoglobin

NADH Sucrose

Mean S.D.

1.4 1.2 1.2 0.5

0.8 1 .o 1.0

0.8

1.0 0.3

A

154 121 94 89

155 112 105

22


APPARENT AREA (8f x 16' FOR DEXTRAN T20

FIG. 5. Determination of the multiplicity of ghost holes using ferritin and FITC-Dextran T20 escape kinetics. Ghosts were prepared either in 5 nm Nap,, 0.01 mM MgSO, as described under "Experimental Procedures" (0, ., 0) or by gradual hemolysis in a dialysis bag equilibrated with this buffer (0). The ghosts were washed in ice-cold 5 rn Nap, (pH 7.2), lacking (0, 0) or containing (., 0) 100 mM NaCl. Concentrates of ferritin and FITC-Dextran T20 were added to each sample to make 2.8 PM and 5.0 PM solutions, respectively, and the suspensions allowed to equilibrate for 3 h on ice. One preparation (0) was then incubated at 30 "C for 2.5 min to further seal the ghosts. A 3-ml aliquot of each preparation was injected into 57 ml of the corresponding buffer and the efflux of both probes determined. Apparent areas -t S.D. (error bars) were calculated for each probe from the slope and standard deviation of the linear semilog plots and are plotted against each other. The solid curues are theoretical plots for ghosts with n holes (indicated at the right) of varied radius, assuming a path length, Ax = 50 A.

I I I

e 4 I- C e - Q 0 / ' - r z

2

0 2! 4

5

IO,

2 4 6

Apparent Area x IO-*(&) for Myoglobin

FIG. 6. Determination of the multiplicity of ghost holes using FITC-Dextran T70 and myoglobin escape kinetics. Ghosts were washed in 5 m~ NaPi (pH 7.2) containing 25 mM NaCl (0,U) or 100 mM NaCl(0). FITC-Dextran T70 and myoglobin were added to make 1.4 and 300 p~ solutions, respectively. Duplicate suspensions were incubated at 25 "C for 1 min (0) or 2 min (0). Efflux into 20 volumes of the same buffer was determined at 0 "C for both probes. Apparent areas 2 S.D. were calculated for each probe and plotted against each other. The solid curues are theoretical plots for ghosts with n holes (indicated at the right) of varied radius, assuming a path length, Ax = 50 A.

tecting a hole multiplicity greater than one. We equilibrated unsealed ghosts with ferritin and FITC-Dextran T20 and sealed them in saline at 37 "C for 1 h, so that r t 1 6 A. The ghosts were then exposed to 0.08% saponin for 20 min at 37 "C. Saponin produces multiple holes in cholesterol-rich membranes (36) and has been shown to increase the permea-

bility of ghosts markedly without dissolving them (16). From the efflux kinetics of the two trapped probes, we estimated that the number of holes per ghost in saponin exceeded 100. Clearly, the technique can distinguish one hole from many.

The theoretical curves in Figs. 5 and 6 were calculated for circles but the shape of the hole could, in general be asym- metric. In that case, the theoretical curves would be displaced significantly to the right of those shown. For example, it can readily be calculated that data for a single elliptical hole per ghost with an axial ratio of 1.5 would lie to the right of the curve for n = 2 circular holes. It is safe to conclude from the data that the hemolytic hole is best described as circular.

The analysis of hole multiplicity by application of Fick's Law depends on an assumption of path length, Ax. While any value for Ax between 30 and 90 A would be consistent with a single hole per ghost, our routine assumption of Ax = 50 A, approximately the thickness of the hydrated bilayer (24), gave an excellent fit of the data to n = 1 (Figs. 5 and 6). On the other hand, if we assume that n = 1.0, Ax can be estimated from the same data to be 60 A (Table I). Our data thus provide a novel hydrodynamic measurement of the thickness of the red cell membrane, at least at the perimeter of the hemolytic hole.

While the outcome of this analysis is highly satisfactory it should be appreciated that if the hole number were greater than one and the hole radii were heterogeneous, this approach would become increasingly unreliable.

Henceforth, we assume that n = 1 and calculate the true hole radius from the apparent area determined according to Equation 2 as r = a + WT.

The Dispersion in Hole Size-When the mean radius of the holes in a ghost population fell within 1.5 times that of a probe, the retardation of efflux by sieving became marked. The dispersion in ghost hole size then became evident in curvilinear semilog plots, since the apparent hole area is proportional to (r - a)'. The average apparent hole area per ghost was calculated in these cases by breaking the curve into chords and weighting the areas determined from each of their slopes according to the fraction of the population they represented. True hole radii determined in this fashion differed by less than 7% from values obtained with the same ghosts using smaller probes which yielded linear semilog plots. (Cases where this treatment was used are the smallest value for A' for ferritin in Fig. 5 and the ["H]sucrose kinetics in Fig. 9.) We conclude that the marked heterogeneity seen previously in the escape kinetics of a ghost population (1, 2) is not incon- sistent with a unimodal distribution of hole sizes when a - r.

Visualization of the Hemolytic Hole-As detailed in a companion paper (12), the diffusion area can be made larger by incubation of ghosts in very low ionic strength, alkaline buffer. The largest hole size demonstrated by ferritin efflux kinetics was r = 322 A. We reasoned that a sufficiently long incubation might make the putative hemolytic holes visible in the light microscope. That this is the case is demonstrated in Fig. 7 . Many ghosts showed a single, circular (often pursed) breach in the membrane, approximately 1-3 pm in diameter. .No ghost had more than one such hole. These lesions fist became visible after approximately 4 h of incubation at low ionic strength and increased in size thereafter. Phosphate buffers more concentrated than 3 mM at pH 8 blocked the development of visible holes. After prolonged incubation with glutaraldehyde-fwed Escherichia coli, some of the ghosts with visible holes appeared to have these bacterial probes inside.

In retrospect, large singular holes were previously observed by dark-field (37) and scanning electron microscopy (38) as the site of membrane breakdown and the release of the


FIG. 7. Visualization of the hemolytic hole. Ghosts were incubated on ice in 24 \.olumes of 0.5 mM NaP,, 0.010 m~ EDTA (pH 7.2) for 8 h. The sample was fixed in 1% glutaraldehyde for 30 min at 0 "C and then selected ghosts photographed by dark-field microscopy. Calibration bar = 10 pm.

submembrane reticulum, known to occur under low ionic strength conditions.

A Density Barrier Assay for Ghost Hole Size-While rig- orous, kinetic analysis is a laborious approach to the determination of hole size. Trapping of probes a t equilibrium is less precise, rather insensitive, and has a strong dependence on internal ghost volume; furthermore, it requires that ghosts be sealed in the presence of several probes. We therefore explored the separation of sealed from unsealed ghosts by centrifugation on selected density barriers (5).

Permeable ghosts take up density barrier solutes and will sediment until they reach the buoyant density of the membrane itself, -1.15 g/cm:' (39). Impermeable ghosts retain an internal aqueous compartment of -1.00 g/cm" and, if fully inflated, would have an overall density as low as 1.001 g/cm". Since water equilibrates very rapidly across the membrane, the impermeant barrier solutes should cause the ghosts to shrink until the osmotic activity within the internal space equals that outside; in our system, they should shrink flat and sediment.

We have found, however, that membrane stiffness retards osmotic shrinkage of impermeable ghosts and renders them buoyant for a period of at least 30 min, during which they can be separated from their unsealed counterparts. One manifes- tation of this nonideality is that ghosts mixed with an appro- priate impermeant barrier medium do not rise upon centrifugation, but ghosts freshly layered upon the same barrier medium do not sink. In other experiments, well sealed ghosts were incubated with solutions of Dextran T70 (1.010 g/cm.') or sucrose (1.014 g/cm:') on ice. At various times, aliquots were fixed with 1% glutaraldehyde and the modal ghost volume determined with a Coulter Counter Channelyzer. (The ChanEelyzer was calibrated according to manufacturer's in- structions, using maximally expanded, spherical red cells and ghosts for which a volume of 150 pm" was assumed; see Ref. 31.) We found that the volume per ghost slowly dropped from 50-60 pm" to plateau values of 30-35 pm," over the 30-40-min

to ghost densities of 1.0043 to 1.0052 g/cm", consistent with their floatation on these barriers.

Another nonideality arises from the fact that barrier solutes slightly smaller than the holes in the ghosts may not come to complete diffusional equilibrium within the brief time span of the assay.

These complexities notwithstanding, we found it possible to use density barriers empirically for hole size analysis by cali- brating them. In Fig. 4, for example, there is excellent agreement between the fraction of ghosts which trap fluorescent Dextran T70 (M) and the fraction which float on barriers of this solute ( C F - 0 ) . A more precise means of calibration, however, was provided by efflux kinetic data.

We sealed ghosts to different degrees by incubation in solutions of varied ionic strength and temperature for defined intervals. We then determined both the fractional floatation on several density barriers and the escape kinetics for each sample. True hole radii were calculated from the kinetic data. The results are summarized in Figs. 8 and 9 in terms of a set of calibration curves for the estimation of ghost hole radii between 7 and 120 A. (Barriers of Dextrans T500 and T2000 did not extend the upper limit of this useful range apprecia- bly.)

The method is sensitive, employing only 1-5 pl of packed ghosts/assay. The method is also precise; the average difference in floatation among 32 pairs of duplicate determinations on dextran barriers was 3.6% and the average difference for 21 pairs of duplicate assays on sucrose, mannitol, and CsCl barriers was 3.5%. Note that because of the nonidealities discussed above, the 50% floatation point does not necessarily correspond closely to the Stokes radius for the barrier solutes. This effect, however, does not reduce the accuracy of the

KINETICALLY DETERMINED RADIUS (1) FIG. 8. Ghost floatation on dextran density barriers as a

function of hole radius. Ghosts were incubated in several buffers at various temperatures in order to reduce their permeability to varying extents. Probe escape kinetics were determined on these preparations using either NADH, ['H]sucrose, myoglobin, FITC-Dextran T20, FITC-Dextran T70, or ferritin as in Fig. 1. Hemolytic hole radii were calculated from these data using Equation 2. The same samples of ghosts were also analyzed for percentage floatation on 1.010 g/cm" barriers of Dextran T70 (0) and Dextran T10 (0) as described under "Experimental Procedures," and the values plotted against hole size. Vertical error bars are standard deviations for floatation. Horizontal error bars correspond to the standard deviation of the slope of the

exposure to these solutes. These internal volumes correspond semilog plot for marker efflux kinetics.


100

80

2

I- Q % 60

0

s 2

!&

W (3

5 40 V

W a a

20

0 1 I I I L -0 10 20

KINETICALLY DETERMINED RADIUS (i) FIG. 9. Ghost floatation on sucrose, mannitol, and CsCl den-

sity barriers as a function of hole radius. Ghosts were rapidly prepared in 5 mM NaPi (pH 8) and promptly incubated in 24 volumes of 150 mM NaC1, 5 m~ Nap,, 0.1 mM EDTA, 4.5 mM [3H]sucrose (pH 6) at 37 "C for 0.5, 1.5, and 50 min. [3H]Sucrose efflux kinetics were used to determine hemolytic hole radius according to Equation 2, except that the semilog plots were curvilinear and the mean hole radius was therefore calculated as a weighted averaged, as described in the text. (Irreversible trapping of [3H]sucrose occurred in 2,22, and 39% of the three ghost preparations.) The percentage floatation of the samples on sucrose (O), mannitol (X), and CsCl (0) density bamers was determined as described under "Experimental Proce- dures.'' The data are presented as in Fig. 8.

calibrated barriers. Fig. 10 presents a time course of ghost sealing as an illustra-

tion of the utility of the density barrier technique. The following points are noted. ( a ) The data suggest very rapid hole closure in isotonic saline at 37 "C; the mean radius dropped from 120 to 20 A within 1 min. ( b ) The holes never sealed completely but reached a stable plateau value of r = 11 A. (c) As shown in the inset, the sealed population had no ghosts permeable to Dextran T10 (Stokes radius = 23.3 A) but more than half took up sucrose (Stokes radius = 5.2 A). These data confirm the impression from Fig. 4 that the dispersion in hole size is not great. (d) In a region of overlap (r = 15 - 35 A), the radii estimated with three barrier solutes differed by an average of only 3.7 A. In several other experiments, the average difference was 5 A. This close agreement among the various barrier solutes employed is to be expected, since they are calibrated against the same kinetic profiles. (e) The continuity of the data in this experiment confwm that there is only one hole per ghost larger than sucrose. If there were several small holes permeable to sucrose but only one permeable to dextrans, a major disagreement would be seen between the sucrose and dextran data, since a larger escape area would be sensed by the smaller probe than the larger probe.

CONCLUSIONS

While the literature on ghost permeability and its reversal is extensive (cf. Refs. 1-11), it is unclear about the number and size of the holes and about the nature of the sealing process. For example, light microscopy of red cells fixed in the process of hemolysis revealed a single hole in the membrane (40-43), while thin section electron micrographs have sug-

e

100 -

80 - '4 - Le-

v .-

E 6 0 -

40 - t

I I

o l , , I 1 1 1 I , , 0 10 20 30 60

Time (min)

FIG. 10. Time course of hole closure determined with three density barriers. Ghosts were warmed at 37 "C in 24 volumes of 150 mM NaC1,5 mM NaPi, 0.1 m~ EDTA (pH 7.2). At the times indicated, aliquots were chilled and assayed for fractional floatation on density barriers of Dextran T70 (O), Dextran T10 (X) , and sucrose (0) as described under "Experimental Procedures." The mean radius of the holes was calculated from the calibration curues shown in Figs. 8 and 9 from the fraction of the ghosts floating on each barrier a t each time point (inset).

gested a pattern of extensive slits and tears (44). In both cases, the area of the lesions was orders of magnitude greater than that observed in studies with molecular probes (3-11). Fur- thermore, the process of sealing has generally been considered to reflect the abrupt transition of individual ghosts from a highly permeable to a highly impermeable state rather than the uniform reduction of hole radius in each ghost.

Our results provide the following simple picture of the unsealed ghost. Its permeability is consistent with a single, right circular hole, the area of which can be widely varied. The phenomenon of ghost sealing reflects the graded closure of the hole to a radius less than that of the test probe. The dispersion of hole area within the ghost population is reason- ably narrow; yet, when probe and hole size are closely matched, efflux rates vary markedly from ghost to ghost due to sieving.

In a comparison report (12), we have attempted to charac- terize the dynamics of the hemolytic hole using the density barrier method described herein.

Acknowledgments-We thank Donald P. Madden for his excellent technical assistance, and Yvonne Lange, Ferenc Kezdy, John Westley, Robert Gunn, and Wolf Epstein for their valuable comments on this work.

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