Biochtmicu et Btophy.~t~a A~'ta. 1079 (1991! 152-160 I'~;i [ l~.~*cr S~.i~'ncc Publi,,hc~ t3 V. All right,, rc~,er~cd (1t67-4.~38/91/$(13.50 A D O N I S (! 1674N3~t~ I 1~1266K
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Hemoglobin polymerization in sickle cells studied by circular polarized light scattering C o r n e l i u s T . G r o s s z..~, H u g h S a l a m o n
3 A r i o n J. H u n t
~, R o b e r t I. M a c e y
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F r a n k O r m e ~ a n d A l e x a n d r e T. Q u i n t a n i l h a t.2.3 : ,4pp/ied Science Ditt~ion. Lawren~'e Berkel~ Laborator~ Berkeh% CA ¢U.S.A ~ 2 Center fi~r X-ray Optie.~, l_awrence Berkeley Laboratory, Berkeley C.4 ¢U.S.A. J and ~Department off Cell attd ,~,l~decular Biok~ga'. Dt~ i.~icn~ of Btophy.sies and ('eli Ph~'~tolox'y, £.'m~ er~tt) of ('al(fornta~ Berkeley. ('A ¢U..~.A.
(Rccci~cd 28 February 19911
Ke} ~ords: ltemoglobin S: Polymerization: Light Scattering: Sickle cell
We have studied intracellular polymerization of hemoglobin S in suspensions of small populations of sickle cells using circular polarized light scattering. We argue that the preferential scattering of right circular polarized light (as expressed by measurements of the Sl4 Mueller scattering matrix element) directly reflects the a m o u n t of polymer inside cells. This technique has made it possible to investigate the effect of oxygen tension, cell density and osmotic stress on intraeellular hemoglobin polymerization. Using S l ~ to determine hemoglobin polymer, we show that the polymer increases with deoxyhemoglobin concentration, that cells containing higher hemoglobin concentrations show significantly more polymer than cells containing less hemoglobin, and tha'. polymerization occurs in sickle-trait cells in hypertonic solutions as the oxygen tension in the suspension is reduced. We also present kinetic measurements of polymerization, including that induced by osmotic shock. Finally, we demonstrate that the total light scattered (St~ Mueller scattering matrix element) that is routinely measured simultaneously with St4 can be used to estimate the percent of reduced ~deoxy) Hb in the sample. These experiments demonstrate the potential of this technique to monitor hemoglobin polymerization simultaneously with oxygen dissociation under a wide variety of physiological conditions.
Introduction
The polymerization of hemoglobin S plays a key role in the pathology of sickle cell anemia [1], The presence of intracellular hemoglobin (Hb) polymer reduces the deformability of sickle cells and is a major cause of blood vessel blockage in sickle cell disease. Since Hb polymerization is believed to be the first step in the development of pathologs' it is desirablc to be able to ,letcct and predict this polymerization within cells under various physiological conditions. Aside from a few notable exceptions [2-5], most extensive studies of hemoglobin polymerization have been limited to measurements on cell-free solutions of hemoglobin. In this paper, we present a practical new circular polarized light scattering technique for studying ooth the equilib-
Correspondence: R.I. Macey. Department o1 ('ell and Molecular Biology. DiviMon of Biophysics and Cell Phy~,iolog~,. Uni~cr,,ily of Calilornia. Berkeley, CA 94720. U.S.A.
rium concentration and kinetics of formation of intracellular hemoglobin polymer in very dilute suspensions of sickle cell, ~Vtth this technique it is possible to measure the effect of oxygen tension, cell density, osmotic stress, temperature, acidity, electrolytes and drugs on the polymerization process. Other researchers have u ~ d polarized light ~ a t t e r ing to study biological macromolecules. Crossed polarizers have been used to detect hemoglobin S polymer, and linear dichrc, i~m s:udies of single ,sickle cells have produced images of hemoglobin polymer orientation [5,6]. Several researchers [7,8] have suggested circular polarized light .scattering for detection of helical structures. Maestre e~ al. [9] have reported circular polarized light .scattering signals from the helical octopus sperm head. Our method is based on the tendency of right helical polymers to scatter right circular polarized light when the wavelength of the light approaches the pitch of the helix [7,8]. Our results show that ~ a t t e r e d light signals conform to a number of criteria for polymer measurements, it, addition we present preliminary
153 data on kinetics o f polymerization, on polymer fl~rmation in sickle trait cells, as well as evidence that the total scattering can be used to measure ,~x3/gen saturation simultaneously with polymerization on the same dilute sample of blood. An abstract describing .some of these results has a p p e a r e d [ 10].
equation h3 a gcncr:d~z~..d ~,cattering r~mHrix ~cprc~,cR~ins the blood suspension gi~c~ the final ~,c~ttcred li~hl: s..s.~s~ S~ ~'~-~ iS
Materials and Methods
T h e light ~ a t t e r i n g a p p a r a t u s ( n e p h e l o m e t e r ) used for these m e a s u r e m e n t s was developed by H u n t [I 1] and consists of a laser ( H e C d 441.6 nm, model 405(I. Liconix, M o u n t a i n View, C A ) ~ h o s c light is directed t h r o u g h a polarizer and a photoelastic m o d u l a t o r ( P E M - 8 0 Hinds International. Portland. O R ) into a d a r k e n e d c h a m b e r containing the sample cuvette and the d e t e c t o r (photomultiplier tube, Vincent As~,~3ciates. Rochester, N Y ) m o u n t e d on a rotating arm (Fig. 1). T h e photoelastie modular'or consists o f a fused silica block mechanically driven by an adjacent piezoelectric quartz crystal at 50 kHz. T h e periodic compressions of the silica m o d u l a t e the polarization of the laser beam. As a result, the light which impinges on the sample cuvette has a complex polarization which c h a n g e s periodically at 50 kHz. T o describe this process mathematically, it is convenient to divide ar, y light beam into four c o m p o n e n t s . each normali2cd to unity and g r o u p e d together as a vector. T h e first c l e m e n t is the total intensity of the beam. T h e second element is the intensity of horizontal minus vertical polarized light; the third is the intensity o f + 4 5 ° minus - 4 5 ° polarized light; a n d the fourth is the intensity o f right minus left circular lx~larized light. A n y complex mixture of polarized light can be r e p r e s e n t e d by such a vcctor (for a complete discu,,sion o f this representation see Ref. 12). in ot, r case. the light beam incident on the photoelastic m o d u l a t o r is horizontally polarized: its fl)ur c o m t x m e n t s are I. I. 0,0. Using the Stokes formalism, the action of the pholoelastic m o d u l a t o r can be completely described by the following four-by-four Mueller matrix v, hich o p e r a t e s on the incident light b e a m vector.
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This matrix takes account o f the phase retardation = A In ~ t intn~luced by, the m(rdu[ator, where A = the p h a ~ retardation at m a x i m u m c~'stal compression and oJ = 51) kHz. Multiplying the m o d u l a t e d b e a m from the a b t ~ c
].~-. - S-.~o~- ~'~- S:~ ",in ,~ i
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St4 is the scattering matrLx element used to measure hemoglobin polymer. It relates the circular polarization c o m p o n e n t of the m o d u l a t e d beam to the unlx~larized c o m p o n e n t of the ~'attered light. It can be i n t e ~ r e t e d as the t e n d e n c y of the sample to ~ ' a t t c r right circular over !eft circular polarized light. T h e S~.~ matrix element is determined by measuring the first e l e m e n t of the vector on the right in the abo~e equation, v, hich r c p r c ~ n t s the l/me ~.aDing intensity of the unpolarized ~ a t t e r e d light+ Substituting for the phase r e t a r d a t m n and expanding the trigonometric terms as sums ot- Bessel functions gives [13]: Inlen',,it~
:
S:~Sj:IJ.(-11-2Jq.4)cL~.2.,t-
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t o 134: (J;~(t3~':)= 0. w e Re[ 13). A lock-in analyzer (model 52(M, Princeton Applied Research. Princeton. N J) .,,clectivcly detects the 51} kHz c o m p o n e n t (sin a,t c o m p o n e n t ) of the signal, which is then directly prol'~rtional to S~. The D C c o m p o n e n t of the signal, on the o t h e r hand. is the total ~ a t t e r i n g . or S,~. in ~his paper "S~=" rotors either t o Sj4 or to the ratio Sis di~.ided by S~+. rcsi~'cliveb called +non-normalized" and "normalized S ~,'. T h e r a t i o o f S++ to the total ~+attered light is m a r c con+enicnt because it is independent of fluctuations m beam intensit5 o r n u m b e r of scattering particles. It is obtained with the aid of a feedback kwap which adjusts the high xoltage on the photomuhiplier tube to maintain a constant current from the delector, thus effeetl~'ely normalizing Ihc S~.~ signal to the total ~.'attercd light. This ratio ts al.,,,o referred to as circular inten.,,it~ differential ~ a t t e r i n g (CIDS). T o measure the non-normalized S~.: signal, or to measure the St, signal by itself. the feedback l t ~ p could be switched off. With either oi~tical setup, a positive Ss4 signal reflects a preference for scattering right circular polarized light. "l'hc sample cuvette is an annealed silica eTlinder (7.0 e m × 4.b cm diameter) ~hich stands on an adjustable platform. A flat entry, window" for incident light avoids outicai elfects from the cur~'ed cuvette walls. In addition, the transmttted beam is deflected 1"6' a tilted rear windo~ t o pre~,ent reflections ~,hich could lead to un~,anted ~ a t t e r i n g . The total path length of the b e a m in the cell i~, i(I.t# cm. T h e volume o f ~ l u t i o n illuminated t'6 the laser beam and seen ~ the detector ~ a s
154 limited by the small acceptance anglc cf the detector opening, and was approx. 5 mm ~" when the detcctor was positkmed at 9(F from the tram;mitted beam. The oxygen tension of the solution could be changed within a few rain ~'y pumping mixtures of a i r / 5 ~ CO, and nitrogen/5% CC', through a bundle of small porous polyethylene tubes immtrsed in the solution (type X 10, 0.(13 p,m pore size, Cellgard, Charlotte, NC:. A stopper isolated the cuvette from the room atmosphere. A gold-plated coil of copper tubing connected to a water bath maintained the temperature at 37 + I° C. A Clark oxygen electrode was used to record the oxygen tension. Concentrated salt solutions were injected into the cuvette through a small hole in the center of the stopper. In preparation for the scattering experiments the apparatus was calibrated by removing the scattering cell and positioning the detector directly in line with the laser beam. A 1/4-wave plate and a linear polarizer were placed in front ,,,f the detector to produce a reference beam. This setup mimics a sample that transforms all incoming light into right circularly polarized light. Subsequent measurements were recorded as a percentage of this value. Next the cuvette was replaced and filled with a suspension of 0.091 ~,m latex spheres (0.002% by weight, in 0.58% N H a O H to prevent aggregation). Because the latex spheres are non-chiral and show no preference for scattering right circular polarized light, their Sn4 signal was used as a baseline for Saa measurements of blood. After recording the S~4 signal, the latex suspension was removed and 75 ml of stock solution was transferred to the cuvette. The solution was continuously stirred by a vacuum-driven magnetic stirrer and equilibrated to 37°C. The solution was equilibrated with A i r / 5 % CO 2 and the oxygen electrode was calibrated to 100%. Then the blood solution (1% h e m a t o c r i t ) w a s removed from refrigeration, shaken and a few hundred microliters were transferred to the cuvette {see Sample preparation section below). The optimum blood concentration in order to prevent multiple scattering was found to be near 0.005% Hat (hematocrit) from measurements of the concentration dependence of the S~4 signal. For non-normalized $2~ measurements the concentration of hemoglobin was critical and enough blood was added each time to bring the total hemoglobin concentration in the cuvette to 1.5 mg/dl. The Sn4 signal was recorded either by scanning the detector through all scattering angles, or by holding the detector arm fixed at a certain angle and measuring the signal over time. Data were collected and stored by computer. The time resolution of the setup was limited to 1 s by the data acquisition unit.
Sample preparation Blood was drawn into Vacutaincr tubes containing
E D T A or A C D (acid citrate dextrose) from homoz3'gous sickle cell patient's, courtesy of Aita Bates hospital. Berkeley and Children's Hospital. Oakland. The blood was kcpt overnight on ice and prepared the following morning. The blood was washed three limes in PBS (phosphate-buffered sMinc. 150 mM NaCI, 10 mM sodium phosphate buffer, pH 7.4, 290 mOsm) and resuspended at 1% by volume {1% Hot) in the stock solution until ready to be used in the ncphelometer. The hemoglobin concentration was measured by methemoglobin absorpt!on at 540 nm (prepared using Drabkin's reagent). The stock solution was prepared according to Ohnishi [14], and contained 108 mM NaCI, 6 mM KCI, 1.2 mM MgCI 2, 27 mM NaHCO3, 2.4 mM N a H 2 P O 4. 1 mM adenine, 1 mM inosine, 5 mM glucose. 30 mM sucrose, 2(~1 u n i t s / l i t e r penicillin G and 200 m g / l i t e r streptomycin. During the experiments the blood suspension was perfused with mixtures of air and nitrogen containing 5e/~ CO,. Under these conditions the pH of the suspension was 7.3. The blood was used within 24 h of having been drawn. Sickle trait blood and normal blood were prepared by the same method.
Density gradients Two mcthods were used to separate red cells by density. In one method, cells were prepared into two populations using continuous gradients of PercolI-Renografin as described by Vettorc ct al. [15] and modified by Ohnishi et al. [14]. The density gradient solution consisted of 53% by volume Pcrcoll (Pharmaeia, Piscataway, N J), 187; by volume Renografin-60 (megfamine diatrizoate 60%. Squibb, NY), 27 mM N a H C O 3, 1 mM MgCI, and 1 mM glucose. {I.4 ml of 25q4 Hot washed blood was placed on 5.0 ml of gradient solution and spun in a fixed-angle rotor (Sorvall SS-34) at 20000 × g for 15 rain. The dense fraction was removed from below by vacuum aspiration. Small samples of both fractions were fixed in 0.25% glutaraldehyde for light microscopic observation. Cell densities were approximated by running normal blood and a set of Pharmacia density marker beads in separate tubes containing the same gradient. Using the density profile of the normal blood and a typical dcnsity profile for normal blood from the htcrature [16], the density marker beads were recalibrated for this gradient solution. Then the markers were used to approximate the sickle cell density profile. This method calculated the maximum density of the light fraction to bc 35 g / d l and the minimum density of the dcnse fraction to be 37 g/dl. in the second method, separation was performed on Stractan discontinu, as gradients (sold as Lare7 by Larcx International, Tacoma, WA) as developed oy Corash ct al. [17] and perfected by Clark ct al. ([18], for a detailed description of thc density gradient preparation see ref. 19). One ml of 2 4 ~ Hct washed blood was
I55 placed on top of a total of 16 ml of Stractan layers of different densities and spun in a swinging>bucket rotor (Sorvall SW-27) at 20001) rpm for 45 min. Layers wcre removed from below by a vacuum aspirator. Small samples of each layer were fixed in ().25c,~ glutaraldehyde for light microscopic observation. Oxygen dissocialiotl measltre,,e, ts Oxygen dissociation curves of sickle cells were measured at 37°C and pH 7.4 by spectrophotometry. Ceils were diluted to approx 0.1% hematocrit and pumped (variable speed peristaltic pump, model #505-121~, Harvard Apparatus, South Natick, MAY continuously through a gas exchanger, an oxygen electrode and a spe~:trophotometer cuvctte in series. The gas exchanger consisted of a bundle of microporous hollow capillary. fibers (type X-20, Celgard, Charlotte, NC), which allow rapid exchange with gases flowing over them [20,21]. Oxygen content of the gases was varied continuously by using flowmcters to mix oxygen/55~- C O , with nitrog e n / 5 % C O , . Oxygen tension in the blood suspension was measured with a polarographic oxygen electrode (model # D O - 1 6 6 F T , Lazar Research. Los Angeles, CA). Optical densities, measured at 540, 560 and 576 nm with a Cary model 210 spectrophotometer, were used to compute the percentage oxyhemoglobin as outlined by Benesch et al. [22]. Differences between pairs of optical densities at different wavelengths were taken to eliminate the portion of the optiea! density due to light scattering. This method is possible because light scattering is essentially independent of ~vavclength in the range of measurcments [23]. Using extinction coefficients given by Benesch et al. [22]. the percent oxyhemoglobin can be calculated from the 576 and 560 nm absorption peaks: '~4oxyhem'~globin= 69.2{( A,-! )/( AA I~,~)} ~ 30.8 where AA = A : , 7 ~ n m - ms(~onm and A A , , ~ is the 1.A at tl)11% oxygen saturation A similar calculation was made with thc 5411 and 56t) nm pair, and the results were averaged. Results and Discussion
Certain criteria must be met by any technique which purports to measure hemoglobin polymer inside cells. In our case these include demonstrations that: (1) The amount of polymer dctected in sickle cells increases with decreasing oxygen tension; more specifically polymer increases with decreasing oxygen saturation of hemoglobin. (2) Any S14 signal shown by normal (HbA) cells is independent of oxygen tension and oxygen saturation. (3) For any given oxygen tension, the polymer signal should increase as the concentration of Hb in-
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creases. This implies that (a) for any given oxygen tension, denser cells ~ontain more polymer than lighter ceils and (bY shrinking the cells osmotically should increase the amount of polymer. (4) For a given amount ef polymer formation, signals obtained should be indepcndent of the rate at which the polymer was produced land hence intlependent of the distribution of polymer sizes [24]). (5) Theory on simplified systems composed of randomly oriented helices predicts that the polymer signal (S j4) is maximal at a scattering angle = 120° and that the signal is largest when the wave length of light approaches the pitch of the po;ymer helix
[81. Added confidence m our particular method would accrue if our S~4 measurements agree with these predictions. The following resu[ls provide evidence that these criteria are fulfilled by circular polarized light scattering. De-oa-vgenated sickle cell blo~;d shows a large S~. o,~3'gep~at('d blood does not Using a fixed scattering angle ol ~ 120:. we used the SI4 signal as a function of time during de-oxygenation and re-oxygenation. As the oxy'gen tension decreased, the S H signal increased, and vice-versa (Fig. 2a). In Fig. 2b we plot the Si4 signal from Fig. 2a directly as a function of ox~,'gen tension. In most experiments we measured normalized S~4, (i.e. the true Sl~ has been divided by Sii). Thi~ provides a convenient way to obtain and compare measurements because the results are independent of Hb concentration, In other cases P was more convenient to use non-normalized S,4. Results were qualitatively similar in both cases. The non normalized Si4 signal is shown in Fig. 3. Here we plot the oxygen tension as hemoglobin oxygen saturation. The relationship between oxygen tension and hemoglobin oxygen saturation was determined spectrophotometrically as described in Materials and Methods.
156 1.6 2.o[
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Fig. 2 (a) Change in the S14 signal caused by deox3,genation-oxygenation cycle. Measurements were taken at 120° scattering angle. S=4 signal was normalized to the maximum Sll signal. The smooth curve shows simultaneous measurement of oxygen tension in the cuvette. (b). Sj4 signal versus oxygen tension for dense cells ( > 37 g/d]). Solid curve shows deox3'genatiom broken curve shows subsequent reoxygenation. These curves were taken from the data in Fig. 2a.
T h e s e findings a g r e e with the results o f N M R m e a s u r e m e n t s o f i n t r a c e l l u l a r p o l y m e r r e p o r t e d by N o g u c h i ct al. [25]. T h e i r m e a s u r e m e n t s w e r e in a g r e e m e n t with t h e o r e t i c a l c a l c u l a t i o n s b a s e d on an allosteric m o d e l o f h e m o g ! e b i n ox3"gen b i n d i n g d e v e l o p e d by M i n t o n [26]. Both t h e i r d a t a a n d the m o d e l p r e d i c t t h a t even at high oxygen t e n s i o n s h e m o g l o b i n p o l y m e r can be f o u n d in sickle cells. O u r results c o n f i r m these findings; Fig. 3 clearly i n d i c a t e s that a significant a m o u n t of p o l y m e r r e m a i n s even at high oxygen s a t u r a t i o n . F o r c a l i b r a t i o n we u s e d N o g u c h i ' s c a l c u l a t i o n s o f p o l y m e r fraction as a function o f H b O 2 fraction a n d total H b c o n c e n t r a t i o n . W e a s s u m e that St4 is p r o p o r tional to p o l y m e r fraction a n d e v a l u a t e the p r o p o r t i o n ality c o n s t a n t by c o m p a r i n g the signal with the calculated value at 0 % O z. I( o u r a s s u m p t i o n s are correct, 1.6 °-t--
t h e n t h e two sets o f d a t a s h o u l d a g r e e n o t o n l y at 0 % 0 2, b u t at all o t h e r H b O 2 fractions. Fig. 4 shows t h e predicted agreement. Sl4 response to H b S polymer is m a x i m a l near a scanning angle o f 120 ° F r o m a t h e o r e t i c a l s t a n d p o i n t , t h e p r e f e r e n t i a l scattering o f right c i r c u l a r p o l a r i z e d light by h e m o g l o b i n S p o l y m e r is s u p p o r t e d by the c a l c u l a t i o n s o f B u s t a m a n t e et al, [8]. T h e y show t h a t d i l u t e s o l u t i o n s o f r a n d o m l y o r i e n t e d helical a r r a y s o f p o i n t a n i s o t r o p i c s c a t t e r e r s s h o u l d exhibit a m a x i m a l S~4 signal at a p p r o x i m a t e l y 120 ° from the f o r w a r d d i r e c t i o n .
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Fig, 3. SI4 signal versus hemoglobin fractional saturation for dense cells ( _>37 g/dl). Hemoglobin fraction saturation values were determined as in Materials and Methods. St4 signal was not normalized to the max mum Stl signal and is therefore expressed in arbitrary units. The smooth curve was interpolated from the data points shown.
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Angle (degrees) Fig. 5. Si4 signal of oxygenated and deoxygenated sickle blood versus scattering angle. The upper aqd lower curves show deoxygenated and
oxygenated blood, respectively (0 and 100% of atmospheric oxygen). The oxygenated curve did trot differ from the baseline obtained with latex particles. Data wa~, collected at 1.3 seconds per degree at 5(10mOs. T o d e t e r m i i t e w h e t h e r the S~4 signal was a valid m e a s u r e ot h e m o g l o b i n S polymer, we c o m p a r e d scatt e r i n g data from n o r m a l a n d sickle blood. A n g l e scans o f S~4 were t a k e n before a n d after de-oxygenating n o r m a l a n d sickle red cells with n i t r o g e n / 5 % CO_,. Sickle cells showed a large increase in S ~ signal u n d e r d e o x y g e n a t e d c o n d i t i o n s with a m a x i m u m signal n e a r the p r e d i c t e d 120 ° (Fig. 5). N o r m a l b l o o d showed n o c h a n g e in Sin signal u p o n deoxygenation. T h e small n o n - z e r o signals seen u n d e r fully o x y g e n a t e d condit i o n s were d u e to strains in the optics a n d r e m a i n e d c o n s t a n t t h r o u g h o u t the e x p e r i m e n t s . A s u s p e n s i o n of latex s p h e r e s (which should exhibit no S~a signal) gene r a t e d a n identical ba',eline.
Sz4 response increases as the wacelength approaches the pitch o f the helix
Lake City, U T ) indicates a strong wavelength d e p e n d e n c e of the S=4 signal (Fig, 6). B u s t a m a n t e et al.'s m o d e l predicts that the wavelength d e p e n d e n c e of the S. 4 is sharply r e s o n a n t with the helix pitch. The sharp increase in SI4 signal toward shorter wavelengths app a r e n t in Fig. 6 suggests such a resonance. A s s u m i n g the helix pitch is 3(100 A as indicated by the electron micrographs of Dykes et al. [27], a n d taking into acc o u n t the i n c r e a s e d index of refraction inside the cells (for p r o t e i n , n = 1.47), we arrive at a r e s o n a n t wavelength in good a g r e e m e n t with o u r SI4 d a t a (3000 × 1.47 = 441 nm). D u e to i n s t r u m e n t a t i o n limitations, we were u n a b l e to show that 441 n m was an actual m a x i m u m ; m e a s u r e m e n t s at s h o r t e r wavelengths using a Hg-vapor arc lamp could definitively answer this q u e s t i o n . W h i l e it is gratifying to find the good a g r e e m e n t of o u r m e a s u r e m e n t s as well as o t h e r e x p e r i m e n t a l d a t a with B u s t e m e n t e et al.'s calculations, it is i m p o r t a n t to realize that we have e x t r a p o l a t e d the theory from its i n t e n d e d d o m a i n of dilute, uniformly dispersed solutions a n d applied it to o u r case of scattering from dilute dispersions of optically d e n s e material intracellular h e m o g l o b i n [28].
S H response increases with increasing Cell density (Hh concentration) i n a second series of e x p e r i m e n t s we s t u d i e d effects o f cell density o n polymerization. D e n s e cells, which c o n t a i n higher c o n c e n t r a t i o n s of h e m o g l o b i n t h a n light cells, are m o r e susceptible to polymerization w h e n placed u n d e r oxygen deficits or hypertonic stress. Using P e r c o l l - R e n o g r a f i n density g r a d i e n t c e n t r i f u g a t i o n , cells were f r a c t i o n a t e d into two p o p u l a t i o n s : a d e n s e fraction, whose h e m o g l o b i n c o n c e n t r a t i o n s were estim a t e d to be g r e a t e r than 37 g / d l , a n d a light fraction,
D a t a t a k e n at different w a v e l e n g t h s using an argonion laser (model 5500A, Ion Laser Technology, Salt
2.4 O
o
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. . . . . .
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. . . . . . . . . . . . . . . . .
460
470
480
490
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Wavelength, nm Fig. 6. Si4 signal versus wavelength. S=4 signal of deoxygenated dense cells ( >_37 g/dl) was measured with a HeCd laser (442 i:m) and an argon-ion laser (457, 465,488, 496, 501 and 514 nm).
0
10 20 30 40 50 60 70 Oxygen, % of atmospheric oxygen Fig. 7, S~4 signal versus oxygen tension for den~ ( >_37 g/dl) and light ( _
1...-
Hemoglobin polymerization in sickle cells studied by circular polarized light scattering C o r n e l i u s T . G r o s s z..~, H u g h S a l a m o n
3 A r i o n J. H u n t
~, R o b e r t I. M a c e y
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F r a n k O r m e ~ a n d A l e x a n d r e T. Q u i n t a n i l h a t.2.3 : ,4pp/ied Science Ditt~ion. Lawren~'e Berkel~ Laborator~ Berkeh% CA ¢U.S.A ~ 2 Center fi~r X-ray Optie.~, l_awrence Berkeley Laboratory, Berkeley C.4 ¢U.S.A. J and ~Department off Cell attd ,~,l~decular Biok~ga'. Dt~ i.~icn~ of Btophy.sies and ('eli Ph~'~tolox'y, £.'m~ er~tt) of ('al(fornta~ Berkeley. ('A ¢U..~.A.
(Rccci~cd 28 February 19911
Ke} ~ords: ltemoglobin S: Polymerization: Light Scattering: Sickle cell
We have studied intracellular polymerization of hemoglobin S in suspensions of small populations of sickle cells using circular polarized light scattering. We argue that the preferential scattering of right circular polarized light (as expressed by measurements of the Sl4 Mueller scattering matrix element) directly reflects the a m o u n t of polymer inside cells. This technique has made it possible to investigate the effect of oxygen tension, cell density and osmotic stress on intraeellular hemoglobin polymerization. Using S l ~ to determine hemoglobin polymer, we show that the polymer increases with deoxyhemoglobin concentration, that cells containing higher hemoglobin concentrations show significantly more polymer than cells containing less hemoglobin, and tha'. polymerization occurs in sickle-trait cells in hypertonic solutions as the oxygen tension in the suspension is reduced. We also present kinetic measurements of polymerization, including that induced by osmotic shock. Finally, we demonstrate that the total light scattered (St~ Mueller scattering matrix element) that is routinely measured simultaneously with St4 can be used to estimate the percent of reduced ~deoxy) Hb in the sample. These experiments demonstrate the potential of this technique to monitor hemoglobin polymerization simultaneously with oxygen dissociation under a wide variety of physiological conditions.
Introduction
The polymerization of hemoglobin S plays a key role in the pathology of sickle cell anemia [1], The presence of intracellular hemoglobin (Hb) polymer reduces the deformability of sickle cells and is a major cause of blood vessel blockage in sickle cell disease. Since Hb polymerization is believed to be the first step in the development of pathologs' it is desirablc to be able to ,letcct and predict this polymerization within cells under various physiological conditions. Aside from a few notable exceptions [2-5], most extensive studies of hemoglobin polymerization have been limited to measurements on cell-free solutions of hemoglobin. In this paper, we present a practical new circular polarized light scattering technique for studying ooth the equilib-
Correspondence: R.I. Macey. Department o1 ('ell and Molecular Biology. DiviMon of Biophysics and Cell Phy~,iolog~,. Uni~cr,,ily of Calilornia. Berkeley, CA 94720. U.S.A.
rium concentration and kinetics of formation of intracellular hemoglobin polymer in very dilute suspensions of sickle cell, ~Vtth this technique it is possible to measure the effect of oxygen tension, cell density, osmotic stress, temperature, acidity, electrolytes and drugs on the polymerization process. Other researchers have u ~ d polarized light ~ a t t e r ing to study biological macromolecules. Crossed polarizers have been used to detect hemoglobin S polymer, and linear dichrc, i~m s:udies of single ,sickle cells have produced images of hemoglobin polymer orientation [5,6]. Several researchers [7,8] have suggested circular polarized light .scattering for detection of helical structures. Maestre e~ al. [9] have reported circular polarized light .scattering signals from the helical octopus sperm head. Our method is based on the tendency of right helical polymers to scatter right circular polarized light when the wavelength of the light approaches the pitch of the helix [7,8]. Our results show that ~ a t t e r e d light signals conform to a number of criteria for polymer measurements, it, addition we present preliminary
153 data on kinetics o f polymerization, on polymer fl~rmation in sickle trait cells, as well as evidence that the total scattering can be used to measure ,~x3/gen saturation simultaneously with polymerization on the same dilute sample of blood. An abstract describing .some of these results has a p p e a r e d [ 10].
equation h3 a gcncr:d~z~..d ~,cattering r~mHrix ~cprc~,cR~ins the blood suspension gi~c~ the final ~,c~ttcred li~hl: s..s.~s~ S~ ~'~-~ iS
Materials and Methods
T h e light ~ a t t e r i n g a p p a r a t u s ( n e p h e l o m e t e r ) used for these m e a s u r e m e n t s was developed by H u n t [I 1] and consists of a laser ( H e C d 441.6 nm, model 405(I. Liconix, M o u n t a i n View, C A ) ~ h o s c light is directed t h r o u g h a polarizer and a photoelastic m o d u l a t o r ( P E M - 8 0 Hinds International. Portland. O R ) into a d a r k e n e d c h a m b e r containing the sample cuvette and the d e t e c t o r (photomultiplier tube, Vincent As~,~3ciates. Rochester, N Y ) m o u n t e d on a rotating arm (Fig. 1). T h e photoelastie modular'or consists o f a fused silica block mechanically driven by an adjacent piezoelectric quartz crystal at 50 kHz. T h e periodic compressions of the silica m o d u l a t e the polarization of the laser beam. As a result, the light which impinges on the sample cuvette has a complex polarization which c h a n g e s periodically at 50 kHz. T o describe this process mathematically, it is convenient to divide ar, y light beam into four c o m p o n e n t s . each normali2cd to unity and g r o u p e d together as a vector. T h e first c l e m e n t is the total intensity of the beam. T h e second element is the intensity of horizontal minus vertical polarized light; the third is the intensity o f + 4 5 ° minus - 4 5 ° polarized light; a n d the fourth is the intensity o f right minus left circular lx~larized light. A n y complex mixture of polarized light can be r e p r e s e n t e d by such a vcctor (for a complete discu,,sion o f this representation see Ref. 12). in ot, r case. the light beam incident on the photoelastic m o d u l a t o r is horizontally polarized: its fl)ur c o m t x m e n t s are I. I. 0,0. Using the Stokes formalism, the action of the pholoelastic m o d u l a t o r can be completely described by the following four-by-four Mueller matrix v, hich o p e r a t e s on the incident light b e a m vector.
I0
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- Mn ,6 (I
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Light scattering apparatus
0
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p h o t o e l a s t i c nla~dulah~r x l i n e a r p ~ l a r i z t r d b ~ ' j m ,,~ m ~ d u l J , c d
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This matrix takes account o f the phase retardation = A In ~ t intn~luced by, the m(rdu[ator, where A = the p h a ~ retardation at m a x i m u m c~'stal compression and oJ = 51) kHz. Multiplying the m o d u l a t e d b e a m from the a b t ~ c
].~-. - S-.~o~- ~'~- S:~ ",in ,~ i
~Jmp~c)
•
m t , d u l a t c d h c d m = ,.ca~!cr~.'d light
St4 is the scattering matrLx element used to measure hemoglobin polymer. It relates the circular polarization c o m p o n e n t of the m o d u l a t e d beam to the unlx~larized c o m p o n e n t of the ~'attered light. It can be i n t e ~ r e t e d as the t e n d e n c y of the sample to ~ ' a t t c r right circular over !eft circular polarized light. T h e S~.~ matrix element is determined by measuring the first e l e m e n t of the vector on the right in the abo~e equation, v, hich r c p r c ~ n t s the l/me ~.aDing intensity of the unpolarized ~ a t t e r e d light+ Substituting for the phase r e t a r d a t m n and expanding the trigonometric terms as sums ot- Bessel functions gives [13]: Inlen',,it~
:
S:~Sj:IJ.(-11-2Jq.4)cL~.2.,t-
J,(,.t) is eliminated by setting .4
)
t o 134: (J;~(t3~':)= 0. w e Re[ 13). A lock-in analyzer (model 52(M, Princeton Applied Research. Princeton. N J) .,,clectivcly detects the 51} kHz c o m p o n e n t (sin a,t c o m p o n e n t ) of the signal, which is then directly prol'~rtional to S~. The D C c o m p o n e n t of the signal, on the o t h e r hand. is the total ~ a t t e r i n g . or S,~. in ~his paper "S~=" rotors either t o Sj4 or to the ratio Sis di~.ided by S~+. rcsi~'cliveb called +non-normalized" and "normalized S ~,'. T h e r a t i o o f S++ to the total ~+attered light is m a r c con+enicnt because it is independent of fluctuations m beam intensit5 o r n u m b e r of scattering particles. It is obtained with the aid of a feedback kwap which adjusts the high xoltage on the photomuhiplier tube to maintain a constant current from the delector, thus effeetl~'ely normalizing Ihc S~.~ signal to the total ~.'attercd light. This ratio ts al.,,,o referred to as circular inten.,,it~ differential ~ a t t e r i n g (CIDS). T o measure the non-normalized S~.: signal, or to measure the St, signal by itself. the feedback l t ~ p could be switched off. With either oi~tical setup, a positive Ss4 signal reflects a preference for scattering right circular polarized light. "l'hc sample cuvette is an annealed silica eTlinder (7.0 e m × 4.b cm diameter) ~hich stands on an adjustable platform. A flat entry, window" for incident light avoids outicai elfects from the cur~'ed cuvette walls. In addition, the transmttted beam is deflected 1"6' a tilted rear windo~ t o pre~,ent reflections ~,hich could lead to un~,anted ~ a t t e r i n g . The total path length of the b e a m in the cell i~, i(I.t# cm. T h e volume o f ~ l u t i o n illuminated t'6 the laser beam and seen ~ the detector ~ a s
154 limited by the small acceptance anglc cf the detector opening, and was approx. 5 mm ~" when the detcctor was positkmed at 9(F from the tram;mitted beam. The oxygen tension of the solution could be changed within a few rain ~'y pumping mixtures of a i r / 5 ~ CO, and nitrogen/5% CC', through a bundle of small porous polyethylene tubes immtrsed in the solution (type X 10, 0.(13 p,m pore size, Cellgard, Charlotte, NC:. A stopper isolated the cuvette from the room atmosphere. A gold-plated coil of copper tubing connected to a water bath maintained the temperature at 37 + I° C. A Clark oxygen electrode was used to record the oxygen tension. Concentrated salt solutions were injected into the cuvette through a small hole in the center of the stopper. In preparation for the scattering experiments the apparatus was calibrated by removing the scattering cell and positioning the detector directly in line with the laser beam. A 1/4-wave plate and a linear polarizer were placed in front ,,,f the detector to produce a reference beam. This setup mimics a sample that transforms all incoming light into right circularly polarized light. Subsequent measurements were recorded as a percentage of this value. Next the cuvette was replaced and filled with a suspension of 0.091 ~,m latex spheres (0.002% by weight, in 0.58% N H a O H to prevent aggregation). Because the latex spheres are non-chiral and show no preference for scattering right circular polarized light, their Sn4 signal was used as a baseline for Saa measurements of blood. After recording the S~4 signal, the latex suspension was removed and 75 ml of stock solution was transferred to the cuvette. The solution was continuously stirred by a vacuum-driven magnetic stirrer and equilibrated to 37°C. The solution was equilibrated with A i r / 5 % CO 2 and the oxygen electrode was calibrated to 100%. Then the blood solution (1% h e m a t o c r i t ) w a s removed from refrigeration, shaken and a few hundred microliters were transferred to the cuvette {see Sample preparation section below). The optimum blood concentration in order to prevent multiple scattering was found to be near 0.005% Hat (hematocrit) from measurements of the concentration dependence of the S~4 signal. For non-normalized $2~ measurements the concentration of hemoglobin was critical and enough blood was added each time to bring the total hemoglobin concentration in the cuvette to 1.5 mg/dl. The Sn4 signal was recorded either by scanning the detector through all scattering angles, or by holding the detector arm fixed at a certain angle and measuring the signal over time. Data were collected and stored by computer. The time resolution of the setup was limited to 1 s by the data acquisition unit.
Sample preparation Blood was drawn into Vacutaincr tubes containing
E D T A or A C D (acid citrate dextrose) from homoz3'gous sickle cell patient's, courtesy of Aita Bates hospital. Berkeley and Children's Hospital. Oakland. The blood was kcpt overnight on ice and prepared the following morning. The blood was washed three limes in PBS (phosphate-buffered sMinc. 150 mM NaCI, 10 mM sodium phosphate buffer, pH 7.4, 290 mOsm) and resuspended at 1% by volume {1% Hot) in the stock solution until ready to be used in the ncphelometer. The hemoglobin concentration was measured by methemoglobin absorpt!on at 540 nm (prepared using Drabkin's reagent). The stock solution was prepared according to Ohnishi [14], and contained 108 mM NaCI, 6 mM KCI, 1.2 mM MgCI 2, 27 mM NaHCO3, 2.4 mM N a H 2 P O 4. 1 mM adenine, 1 mM inosine, 5 mM glucose. 30 mM sucrose, 2(~1 u n i t s / l i t e r penicillin G and 200 m g / l i t e r streptomycin. During the experiments the blood suspension was perfused with mixtures of air and nitrogen containing 5e/~ CO,. Under these conditions the pH of the suspension was 7.3. The blood was used within 24 h of having been drawn. Sickle trait blood and normal blood were prepared by the same method.
Density gradients Two mcthods were used to separate red cells by density. In one method, cells were prepared into two populations using continuous gradients of PercolI-Renografin as described by Vettorc ct al. [15] and modified by Ohnishi et al. [14]. The density gradient solution consisted of 53% by volume Pcrcoll (Pharmaeia, Piscataway, N J), 187; by volume Renografin-60 (megfamine diatrizoate 60%. Squibb, NY), 27 mM N a H C O 3, 1 mM MgCI, and 1 mM glucose. {I.4 ml of 25q4 Hot washed blood was placed on 5.0 ml of gradient solution and spun in a fixed-angle rotor (Sorvall SS-34) at 20000 × g for 15 rain. The dense fraction was removed from below by vacuum aspiration. Small samples of both fractions were fixed in 0.25% glutaraldehyde for light microscopic observation. Cell densities were approximated by running normal blood and a set of Pharmacia density marker beads in separate tubes containing the same gradient. Using the density profile of the normal blood and a typical dcnsity profile for normal blood from the htcrature [16], the density marker beads were recalibrated for this gradient solution. Then the markers were used to approximate the sickle cell density profile. This method calculated the maximum density of the light fraction to bc 35 g / d l and the minimum density of the dcnse fraction to be 37 g/dl. in the second method, separation was performed on Stractan discontinu, as gradients (sold as Lare7 by Larcx International, Tacoma, WA) as developed oy Corash ct al. [17] and perfected by Clark ct al. ([18], for a detailed description of thc density gradient preparation see ref. 19). One ml of 2 4 ~ Hct washed blood was
I55 placed on top of a total of 16 ml of Stractan layers of different densities and spun in a swinging>bucket rotor (Sorvall SW-27) at 20001) rpm for 45 min. Layers wcre removed from below by a vacuum aspirator. Small samples of each layer were fixed in ().25c,~ glutaraldehyde for light microscopic observation. Oxygen dissocialiotl measltre,,e, ts Oxygen dissociation curves of sickle cells were measured at 37°C and pH 7.4 by spectrophotometry. Ceils were diluted to approx 0.1% hematocrit and pumped (variable speed peristaltic pump, model #505-121~, Harvard Apparatus, South Natick, MAY continuously through a gas exchanger, an oxygen electrode and a spe~:trophotometer cuvctte in series. The gas exchanger consisted of a bundle of microporous hollow capillary. fibers (type X-20, Celgard, Charlotte, NC), which allow rapid exchange with gases flowing over them [20,21]. Oxygen content of the gases was varied continuously by using flowmcters to mix oxygen/55~- C O , with nitrog e n / 5 % C O , . Oxygen tension in the blood suspension was measured with a polarographic oxygen electrode (model # D O - 1 6 6 F T , Lazar Research. Los Angeles, CA). Optical densities, measured at 540, 560 and 576 nm with a Cary model 210 spectrophotometer, were used to compute the percentage oxyhemoglobin as outlined by Benesch et al. [22]. Differences between pairs of optical densities at different wavelengths were taken to eliminate the portion of the optiea! density due to light scattering. This method is possible because light scattering is essentially independent of ~vavclength in the range of measurcments [23]. Using extinction coefficients given by Benesch et al. [22]. the percent oxyhemoglobin can be calculated from the 576 and 560 nm absorption peaks: '~4oxyhem'~globin= 69.2{( A,-! )/( AA I~,~)} ~ 30.8 where AA = A : , 7 ~ n m - ms(~onm and A A , , ~ is the 1.A at tl)11% oxygen saturation A similar calculation was made with thc 5411 and 56t) nm pair, and the results were averaged. Results and Discussion
Certain criteria must be met by any technique which purports to measure hemoglobin polymer inside cells. In our case these include demonstrations that: (1) The amount of polymer dctected in sickle cells increases with decreasing oxygen tension; more specifically polymer increases with decreasing oxygen saturation of hemoglobin. (2) Any S14 signal shown by normal (HbA) cells is independent of oxygen tension and oxygen saturation. (3) For any given oxygen tension, the polymer signal should increase as the concentration of Hb in-
"-
---1 He-Cd Laser 441.6 ~m
1-
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Sample cuvette \
-'\ Polarization
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..~
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creases. This implies that (a) for any given oxygen tension, denser cells ~ontain more polymer than lighter ceils and (bY shrinking the cells osmotically should increase the amount of polymer. (4) For a given amount ef polymer formation, signals obtained should be indepcndent of the rate at which the polymer was produced land hence intlependent of the distribution of polymer sizes [24]). (5) Theory on simplified systems composed of randomly oriented helices predicts that the polymer signal (S j4) is maximal at a scattering angle = 120° and that the signal is largest when the wave length of light approaches the pitch of the po;ymer helix
[81. Added confidence m our particular method would accrue if our S~4 measurements agree with these predictions. The following resu[ls provide evidence that these criteria are fulfilled by circular polarized light scattering. De-oa-vgenated sickle cell blo~;d shows a large S~. o,~3'gep~at('d blood does not Using a fixed scattering angle ol ~ 120:. we used the SI4 signal as a function of time during de-oxygenation and re-oxygenation. As the oxy'gen tension decreased, the S H signal increased, and vice-versa (Fig. 2a). In Fig. 2b we plot the Si4 signal from Fig. 2a directly as a function of ox~,'gen tension. In most experiments we measured normalized S~4, (i.e. the true Sl~ has been divided by Sii). Thi~ provides a convenient way to obtain and compare measurements because the results are independent of Hb concentration, In other cases P was more convenient to use non-normalized S,4. Results were qualitatively similar in both cases. The non normalized Si4 signal is shown in Fig. 3. Here we plot the oxygen tension as hemoglobin oxygen saturation. The relationship between oxygen tension and hemoglobin oxygen saturation was determined spectrophotometrically as described in Materials and Methods.
156 1.6 2.o[
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' - ~ 100
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Oxygen tension
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Oxygen % of atmospheric oxygen
Time, minutes
Fig. 2 (a) Change in the S14 signal caused by deox3,genation-oxygenation cycle. Measurements were taken at 120° scattering angle. S=4 signal was normalized to the maximum Sll signal. The smooth curve shows simultaneous measurement of oxygen tension in the cuvette. (b). Sj4 signal versus oxygen tension for dense cells ( > 37 g/d]). Solid curve shows deox3'genatiom broken curve shows subsequent reoxygenation. These curves were taken from the data in Fig. 2a.
T h e s e findings a g r e e with the results o f N M R m e a s u r e m e n t s o f i n t r a c e l l u l a r p o l y m e r r e p o r t e d by N o g u c h i ct al. [25]. T h e i r m e a s u r e m e n t s w e r e in a g r e e m e n t with t h e o r e t i c a l c a l c u l a t i o n s b a s e d on an allosteric m o d e l o f h e m o g ! e b i n ox3"gen b i n d i n g d e v e l o p e d by M i n t o n [26]. Both t h e i r d a t a a n d the m o d e l p r e d i c t t h a t even at high oxygen t e n s i o n s h e m o g l o b i n p o l y m e r can be f o u n d in sickle cells. O u r results c o n f i r m these findings; Fig. 3 clearly i n d i c a t e s that a significant a m o u n t of p o l y m e r r e m a i n s even at high oxygen s a t u r a t i o n . F o r c a l i b r a t i o n we u s e d N o g u c h i ' s c a l c u l a t i o n s o f p o l y m e r fraction as a function o f H b O 2 fraction a n d total H b c o n c e n t r a t i o n . W e a s s u m e that St4 is p r o p o r tional to p o l y m e r fraction a n d e v a l u a t e the p r o p o r t i o n ality c o n s t a n t by c o m p a r i n g the signal with the calculated value at 0 % O z. I( o u r a s s u m p t i o n s are correct, 1.6 °-t--
t h e n t h e two sets o f d a t a s h o u l d a g r e e n o t o n l y at 0 % 0 2, b u t at all o t h e r H b O 2 fractions. Fig. 4 shows t h e predicted agreement. Sl4 response to H b S polymer is m a x i m a l near a scanning angle o f 120 ° F r o m a t h e o r e t i c a l s t a n d p o i n t , t h e p r e f e r e n t i a l scattering o f right c i r c u l a r p o l a r i z e d light by h e m o g l o b i n S p o l y m e r is s u p p o r t e d by the c a l c u l a t i o n s o f B u s t a m a n t e et al, [8]. T h e y show t h a t d i l u t e s o l u t i o n s o f r a n d o m l y o r i e n t e d helical a r r a y s o f p o i n t a n i s o t r o p i c s c a t t e r e r s s h o u l d exhibit a m a x i m a l S~4 signal at a p p r o x i m a t e l y 120 ° from the f o r w a r d d i r e c t i o n .
c
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Fig, 3. SI4 signal versus hemoglobin fractional saturation for dense cells ( _>37 g/dl). Hemoglobin fraction saturation values were determined as in Materials and Methods. St4 signal was not normalized to the max mum Stl signal and is therefore expressed in arbitrary units. The smooth curve was interpolated from the data points shown.
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Fraction HbO 2 Fig. 4. Comparison of St4 signal with NMR measurements of intracellular Imlymer [25]. Si, data was taken from Fig. 3. We assume that St4 is proportional to polymer fraction and evaluate the proportionality constant by comparing the signal with the calculated value at 0% 02 .
157 -
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150
Angle (degrees) Fig. 5. Si4 signal of oxygenated and deoxygenated sickle blood versus scattering angle. The upper aqd lower curves show deoxygenated and
oxygenated blood, respectively (0 and 100% of atmospheric oxygen). The oxygenated curve did trot differ from the baseline obtained with latex particles. Data wa~, collected at 1.3 seconds per degree at 5(10mOs. T o d e t e r m i i t e w h e t h e r the S~4 signal was a valid m e a s u r e ot h e m o g l o b i n S polymer, we c o m p a r e d scatt e r i n g data from n o r m a l a n d sickle blood. A n g l e scans o f S~4 were t a k e n before a n d after de-oxygenating n o r m a l a n d sickle red cells with n i t r o g e n / 5 % CO_,. Sickle cells showed a large increase in S ~ signal u n d e r d e o x y g e n a t e d c o n d i t i o n s with a m a x i m u m signal n e a r the p r e d i c t e d 120 ° (Fig. 5). N o r m a l b l o o d showed n o c h a n g e in Sin signal u p o n deoxygenation. T h e small n o n - z e r o signals seen u n d e r fully o x y g e n a t e d condit i o n s were d u e to strains in the optics a n d r e m a i n e d c o n s t a n t t h r o u g h o u t the e x p e r i m e n t s . A s u s p e n s i o n of latex s p h e r e s (which should exhibit no S~a signal) gene r a t e d a n identical ba',eline.
Sz4 response increases as the wacelength approaches the pitch o f the helix
Lake City, U T ) indicates a strong wavelength d e p e n d e n c e of the S=4 signal (Fig, 6). B u s t a m a n t e et al.'s m o d e l predicts that the wavelength d e p e n d e n c e of the S. 4 is sharply r e s o n a n t with the helix pitch. The sharp increase in SI4 signal toward shorter wavelengths app a r e n t in Fig. 6 suggests such a resonance. A s s u m i n g the helix pitch is 3(100 A as indicated by the electron micrographs of Dykes et al. [27], a n d taking into acc o u n t the i n c r e a s e d index of refraction inside the cells (for p r o t e i n , n = 1.47), we arrive at a r e s o n a n t wavelength in good a g r e e m e n t with o u r SI4 d a t a (3000 × 1.47 = 441 nm). D u e to i n s t r u m e n t a t i o n limitations, we were u n a b l e to show that 441 n m was an actual m a x i m u m ; m e a s u r e m e n t s at s h o r t e r wavelengths using a Hg-vapor arc lamp could definitively answer this q u e s t i o n . W h i l e it is gratifying to find the good a g r e e m e n t of o u r m e a s u r e m e n t s as well as o t h e r e x p e r i m e n t a l d a t a with B u s t e m e n t e et al.'s calculations, it is i m p o r t a n t to realize that we have e x t r a p o l a t e d the theory from its i n t e n d e d d o m a i n of dilute, uniformly dispersed solutions a n d applied it to o u r case of scattering from dilute dispersions of optically d e n s e material intracellular h e m o g l o b i n [28].
S H response increases with increasing Cell density (Hh concentration) i n a second series of e x p e r i m e n t s we s t u d i e d effects o f cell density o n polymerization. D e n s e cells, which c o n t a i n higher c o n c e n t r a t i o n s of h e m o g l o b i n t h a n light cells, are m o r e susceptible to polymerization w h e n placed u n d e r oxygen deficits or hypertonic stress. Using P e r c o l l - R e n o g r a f i n density g r a d i e n t c e n t r i f u g a t i o n , cells were f r a c t i o n a t e d into two p o p u l a t i o n s : a d e n s e fraction, whose h e m o g l o b i n c o n c e n t r a t i o n s were estim a t e d to be g r e a t e r than 37 g / d l , a n d a light fraction,
D a t a t a k e n at different w a v e l e n g t h s using an argonion laser (model 5500A, Ion Laser Technology, Salt
2.4 O
o
,p-
1.9
1.9
X
~" 1.4
j-
O O
"- 0.9
1.4 X A .r--
~
0.9
0
0.4
0
-0.1
0.4 A -0.1
. . . . . .
440
450
,
,,
,
. . . . . . . . . . . . . . . . .
460
470
480
490
~,.
v.
500
. . . . .
510
O..
520
Wavelength, nm Fig. 6. Si4 signal versus wavelength. S=4 signal of deoxygenated dense cells ( >_37 g/dl) was measured with a HeCd laser (442 i:m) and an argon-ion laser (457, 465,488, 496, 501 and 514 nm).
0
10 20 30 40 50 60 70 Oxygen, % of atmospheric oxygen Fig. 7, S~4 signal versus oxygen tension for den~ ( >_37 g/dl) and light ( _
