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Studies of Elastin Coacervation by Quasielastic Light Scattering BY A. M. JAMIESON,B. SIMIC-GLAVASKI, K . TANSEY AND A. G . WALTON Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106, U.S.A.

Received 1 1tlz December, 1975 The coacervation behaviour of elastin appears to be an example of a mixed coacervation, intermediate between the classes of unicomplex and simple coacervations defined by Bungenberg de Jong. Studies of the angular and temperature dependence of the Rayleigh spectrum of the light scattered by aqueous solutions of tropoelastin are reported. Analysis of these data permit a thermodynamic interpretation of tropoelastin coacervation based on the theoretical model of Veis and Aranyi. Above the coacervation temperature, the kinetic analysis of change in volume of coacervate drops, determined from the change in diffusion coefficient with time, indicates a nucleation-controlled growth process rather than a simple coalescence phenomenon. The implications of these results for current theories of elastin structure are discussed.

Elastin is one of the principal protein components of the connective tissue of the body and is found predominantly in those tissues which require the properties of mechanical extensibility and flexibility. Although the amino acid sequence of elastin has been partially determined,' the nature of the tertiary organization of the elastin macromolecules in the fibres is still unresolved. The most popular current models are the globular cluster model of Partridge,2 the cross-linked random coil " rubber " of Flory et aZ.,3and the linked " oiled coil " arrangement of Sandberg et aL4 Both the " solubilized " elastin obtained by digestion and hydrolysis of elastic tissue fragments and the soluble elastin precursor, tropoelastin, obtained from copper deficient swine undergo a liquid-liquid phase separation phenomenon at elevated temperatures which appears at first sight to be a typical example of the unicomplex coacervation class defined by Bungenberg de J ~ n g driven ,~ by the intermolecular electrostatic interactions of the amphoion. However, certain features of the coacervation 8 , such as its moderate insensitivity to pH, ionic strength and polymer concentration suggest also that hydrophobic interactions play a strong role and, therefore, the system may be more accurately represented as mixed coacervation with both simple and complex behaviour. The thermodynamic basis for coacervation processes has been extensively explored. For non-ionic systems, Bamford and Tompa loshowed that coacervation is predicted as an extreme case of the general class of critical phase separations using the FloryHuggins expression for free energy of a polymer solution.

where N' is the total number of lattice sites, ri the number of sites occupied by the i*h polymer molecule, q i the volume fraction of the ith molecule and k the Boltzman constant. The dependence of the Huggins interaction parameter x on T may be written

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M. JAMIESON, B . S I M I C - G L A V A S K I , K . T A N S E Y A N D A . G . W A L T O N

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x = xs-tp/Tcr

195 (2)

where xs is the entropic contribution and /I/T,, is a contribution arising from the heat of dilution. T,, is the temperature of the phase transition and deviates from the Flory temperature 0 as a function of polymer molecular weight." In most systems, the parameter p is positive and precipitation occurs at the lower critical point by cooling the system until x > 1/2-(1/2-~J(l-Q/Tcr). In a few systems, p i s negative and phase separation occurs at an upper critical point by heating the system. For polyelectrolytes, Voorn l 2 modified the Flory-Huggins theory replacing the Huggins interaction term x12with an electrostatic interaction term a in the expression for the free energy of mixing F,,, : I'

I

I

J

(3) (4)

where ai is the charge density of the it'' molecule. Eqn (4) can be rigorously applied only to solutions at relatively high concentrations of either salt or polymer, where a model of polymer random coils in a continuous distribution of charges can be assumed. Veis et aZ.I3recognized these limitations and proposed an alternative treatment for complex coacervation of polyelectrolytes from dilute solution at low ionic strength. This analysis was used with some success to account for their experimental data on gelatin-gelatin complex coacervation. The complete form of eqn (3) was used and both solute-solvent interactions and solutesolute interactions were found to be of comparable importance to electrostatic forces through the parameters xi and rsolute,respectively. For coinplex coacervation between polyelectrolytes P+ and Q-, under these conditions, Veis l 3 proposes the scheme [P+Q-, aggregate^],^,

[P'Q',

aggregatesIdii+ [P',

random coil+Q-, random COilIcoac. (6)

The first irreversible reaction occurs instantaneously on dissolving the polymer. The second reaction represents the formation of two phases, a dilute solution of aggregates and a concentrated solution of each polymer species in a random coil conformation (the coacervate phase). The driving force for the phase separation comes from the positive entropy change due to dilution of the electrostatically bound aggregates of low configurational entropy and also the formation of the concentrated phase, which can be regarded as a typical solution of random chain polymers of high configurational entropy. This situation will obtain only in dilute solutions. At higher concentrations, the Voorn-Overbeek treatments may be more applicable. The technique of quasielastic laser light scattering 14* l5has found especially fruitful application in studying the dynamics of density and concentration fluctuations in fluids near the critical phase separation point.lG In binary mixtures, far from the critical point, the light scattered from concentration fluctutions has a Lorentzian spectral distribution with halfwidth at halfheight r D given by: where is the mutual diffusion coefficient and K is the scattering vector. This relation holds both for binary mixtures of small molecules l7 and for solutions of polyrners.l8

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196

E L A S T I N COACERVATION B Y Q U A S I E L A S T I C L I G H T S C A T T E R I N G

At the critical demixing point, the well-known phenomenon of critical opalescence occurs, the intensity of the scattered light increases dramatically and the linewidth of the spectrum of this light now has a more complex dependence on the scattering vector,17*l8

where is a temperature-dependent correlation length which describes the development of long-range interactions between like molecules at the critical point. From the temperature dependence of both D and 5 as the system approaches the critical temperature T,, it turns out that 16-18

where y and o are critical exponents defined in terms of the " scaling law " concept, and I in the range of molecular forces.lg Eqn (9) describes the rapid narrowing of the Rayleigh linewidth due to the critical " slowing down " of concentration fluctuations as T, is approached. The above considerations are confined to systems with a lower consolute temperature (Tone-phase > TJ. For polymer solutions, as well as for the few cases of binary mixtures of small molecules, which possess an upper consolute temperature, a different analysis may be appropriate. The tropoelastin system discussed here is one example of this class and the completely different system polyethyleneglycol dioxane 2o is another. These solutions appear to have the common feature that they exhibit a large negative excess entropy of mixing.21 Thus at lower temperatures a low entropy, structured solution is stable, and the phase separation at higher temperatures is driven entropically by the formation of a higher entropy, two phase system.20 This also seems to be true in the dilute solution coacervation of gelatin-gelatin discussed by Veis. Indeed, it should be pointed out that even in systems with a lower consolute temperature, deviations from the behaviour represented by eqn (9) have been found 22 which appear to be due to the inherent configurational entropy of macromolecular chains. As one raises the temperature in a system exhibiting an upper consolute temperature, it seems plausible that a spectral broadening of the Rayleigh scattering can occur due to the disintegration of structured entities and development of the higher entropy phase. Because, however, of the decreasing mutual solubility of the components and the macroscopic separation of droplets of each phase at the critical temperature, each of which can also exhibit translational diffusive degrees of freedom, there will also certainly be a narrow spectral component due to scattering from these interfaces. In our earlier heterodyne studies of hydrolysed elastin coacer~ation,~ we observed that the phase separation appeared to involve, first, an essentially irreversible aggregation process, then a reversible change (corresponding to visible coacervation) in which the particle size seemed to decrease! Based on these data we proposed a mechanism for elastin coacervation similar to that of Veis. At lower temperatures, the elastin molecules form aggregates which we call micellar, since the driving force must be the poor solubility of the apolar amino acids present. As the temperature increases, the solubility decreases and the aggregates increase in size. At some critical size, we postulate that a nucleation process occurs involving formation of a new phase in which the elastin chains are in a " random coil " conformation, the driving force being the increase in total entropy of the system. The physical model we envisage is shown in fig. 1. The Rayleigh spectral distribution of light scattered by the macro-

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197

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molecular species in a coacervating elastin solution is therefore of the form A*D,ic2 Be D2u2 C.D3x2 s(''w) = w 2 + ( D l ~ 2 ) 2 + w z + ( D 2 ~ z ) 2 + ~ 2 + ( D 3 u 2 ) 2 where D1 is the aggregate diffusion coefficient in the dilute phase and D2 is the diffusion coefficient of the coacervate drops and D3 is the diffusion coefficient of random chains inside the coacervate phase. Obviously, D1 and D2 < D3. The result is that a broad spectral component will appear as the phase separation takes place. However, the spectral weighting coefficients A and B $- C so the integrated intensity of the

Reversible

@

@ Ccacxvation

@ @

FIG.1.-Molecular model for elastin coacervation involving a transition from dense micellar aggregates to drops of random protein coils in a theta solution. The process is ultimately reversible but the nucleation controlled kinetics implies a hysteresis period between forward and reverse processes.

broad component is very small. In this paper we describe quasielastic light scattering studies of a coacervating solution of tropoelastin which apparently displays this behaviour. A further application of quasielastic laser light scattering to particle aggregation studies arises from the fact that with modern electronic photodetection methods,23it is possible to obtain good spectra and, therefore, accurate values of in as little as a few minutes. If the solutions are sufiiciently dilute that the Stokes-Einstein relation for b can be applied

This means that kinetic studies of changes in the particle radius R can be carried out. We will report kinetic measurements of the development of coacervate droplets of hydrolysed elastin above the critical temperature. EXPERIMENTAL The sample of tropoelastin used in this study was kindly donated by Dr. L. B. Sandberg, Department of Surgery and Pathology, University of Utah Medical Center, Salt Lake City. Characterization of this material has been described in the literature.6 The tropoelastin was used in our experiments without further chemical purification. All spectral measurements were carried out in aqueous solution at 0.12% w/v of polymer. The solution was buffered with a Tris buffer at pH 8.2 and 0.094 M NaCl so that the ionic strength was 0.1. This system was chosen so that the intensity of coacervation was not too large, hence minimizing experimental problems in scattering measurements from turbidity. Solutions were clarified for optical studies by ultracentrifugation at 15 0oO rpm for 1 h at 5 "C. The optical mixing spectrometer used in these investigations has been well described e l ~ e w h e r e . ~It~consists, presently, of a Coherent Radiation Model 52B Arf laser, thermoregulated scattering cell, EM1 9656 KR phstotube with Keithley 244 high voltage supply, Keithley 104 widebend amplifier and SAICOR SA1-52 real time 400 point spectrum analyser and digital integrator. Computer analysis of the spectra are performed by a Digital Equipment Corporation PDP8/L cornputer/AX08 interface. Scattering intensity (turbidity) is monitored by a millivoltmeter. " Equilibrium " spectra were recorded in the heterodyne configuration as described e1se~here.l~Kinetic experiments were carried out using homodyne detection on 1 mg/ml solutions of the a-fraction of hydrolysed elastin at pH = 4.5.

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198

ELASTIN COACERVATION BY QUASIELASTIC LIGHT SCATTERING

RESULTS

The angular dependence of the Rayleigh spectrum from 8 = 30 to 130 was studied at 14.8 "C. The linewidth of the spectrum was determined by a single Lorentzian computer fit to squared data points. An example is shown in fig. 2. The varia-

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O

I

I

5

0

I

I

10

15

O

I

20

frequency I k Hz FIG.2.-Single Lorentzian fit to points on heterodyne power spectrum of 0.12%. Tropoelastin solution at 8 = 135 T = 14.8 "C, pH = 8.2 and p = 0.1 Avl/z = (1/2n)(Dt)lc2= 1.59 kHz.

.

O,

tion of linewidth with the square of the scattering vector 7c is shown in fig. 3. From cm2 s-l. As the these data, the diffusion coefficient was calculated to be 1.9 x molecular weight of tropoelastin is 68 000,6 this certainly means that the polymer is highly aggregated, even at these ionic strengths, since the mean hydrodynamic diameter of the elastin particles is -220 A. The good linear rc2-dependenceof the linewidth evident in fig. 3 indicates that there is no significant departure of the aggregates from spherical symmetry (i.e., the aggregates are not fibrous). The viscosity-corrected temperature dependence of the Rayleigh linewidth through the coacervation temperature enables us to determine the variation in particle size R,shown in fig. 4. As the temperature is raised, a rapid, irreversible aggregation takes place as evi-

1875

t

1250N

I \

s

a 625 -

I

0

FIG. 3.-Quadratic

0.1

I

0.3

I

1

0.5 sin28/ 2

1

0.7

0.9

Ic-vector dependence of Rayleigh spectral line-width of tropoelastin solution indicating spherical aggregate geometry.

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A . M. JAMIESON, B . SIMIC-GLAVASKI, K . TANSEY AND A. G . WALTON

199

denced by the increase in particle size until larger aggregates, some 400 A in diameter, are formed. At some critical aggregate size Y*, separation into two phases takes place. At this point, a broad spectral component appears, paralleling previous observations of the coacervation of hydrolysed e l a ~ t i n . ~The tropoelastin data presently reported, however, clearly show that there is also a narrow spectral component. Sample spectra are shown in fig. 4. From two-Lorentzian computer analysis the broad component corresponds to a diffusion coefficient of 2.90 x lo-' cm' s-'.

Two Phases

1

15

FIG.4.-hcrease

I

I

I

25

I

35

in aggregate size as temperature rises to coacervation point.

This broad component is apparently related to the coacervate phase, since the integrated intensity of the component which is very small increases as coacervation proceeds. The narrow component corresponds in the intial stages of coacervation cm' s-l. to a diffusion coefficient of 1 x A series of kinetic homodyne experiments on the intense narrow spectrum of scattering from aggregates in a bovine ligamentum nuchae hydrolysed elastin preparation at several temperatures above the coacervation temperature were made. Small sample volumes (-1 ml) were used so that thermal equilibration was rapid. The change in mean volume of the aggregates with time determined by the Stokes-Einstein relation is shown in fig. 5.

. .

--x

m .

.-m

L

k.

al

L

.-c Is,

.-

z

KHz

+ L 0

-m 1

0

I

2.5

1

5.0

I

5.0

frequency I kHz

FIG.5.-Heterodyne square root power spectra of light scattered from tropoelastin solutions above T,. As the temperature rises a broad spectral wing appears which we propose is due to protein random coils in the coacervate.

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E L A S T I N COACERVATION B Y QUASIELASTIC L I G H T S C A T T E R I N G

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DISCUSSION

For the tropoelastin system, the excess chemical potential of the polymer is determined from eqn (3) with ol= 1 = r1 and xlz = xzl = x, to be

Differentiating twice with respect to

vl,we obtain

Since the tropoelastin system studied here is very dilute (CT = 0.12% w/v), we may set ql = 0.999 = 1 and eqn (13) becomes 2Xc+(ll-,-1)+(3/4)ac(l--a)

= 0.

(14)

This equation defines the limit of stability of the homogeneous tropoelastin solution. Our results support the concept that the coacervation of tropoelastin is driven by the positive entropy change resulting from disintegration of specific intermolecular aggregates in the dilute phase and formation of a concentrated phase of polymer in a random coil conformation. At low temperature, the light scattering data indicate that tropoelastin exists in aqueous solution as isotropic aggregates. These aggregates are held together by both electrostatic and hydrophobic interactions and, because of the high proportion of non-polar amino acids in the protein,6 are labelled micellar by analogy with block copolymer aggregation p h e n ~ m e n a . ~As ~ the temperature is raised, the size of these aggregates increases and the second term in eqn (14) approaches -1 until a critical mass is reached. At the same time, the third term in eqn (l4), which is small and positive, decreases. At the critical temperature defined by eqn (14) and (2) a new phase is formed, the coacervate, which is a solution of the protein in a random coil conformation. The new diffusive freedom of the polymer in the coacervate is the origin of the broad component which appears in the Rayleigh spectrum. At the same time, the dilute phase of dissolved aggregates will be depleted. Scattering from these aggregates will still give rise to a narrow spectral component. Superimposed on this, however, will be another narrow component, due to the scattering from the interfaces of droplets of the new phase which have their own slow diffusional mobility. Our data lack sufficient resolution in the very low frequency regions to distinguish these two narrow components. This sequence of events is represented by the reaction: Tropoelastin

[Tropoelastin, micellar agg.]

(15) Tropoelastin, mic. agg. -+ [Tropoelastin, mic. agg.]dil. [Tropoelastin, independent random coil],,,,. (16) --f

+

In consideration, finally, of the particle volume changes above T, leading to the development of a dispersed coacervate phase, we will consider two models : (a) The aggregates nucleate rapidly into coacervate droplets and subsequent coalescence occurs, the kinetics of which may be followed in the spectrometer. (b) The process of coacervation involves a slow phase change in each of the aggregate clusters, causing swelling, and little or no subsequent coalescence occurs.

In the coalescence model (a) the growth rate, based on elementary binary collision theory 25 predicts the number of particles at time t to be

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A . M. JAMIESON, B. SIMIC-GLAVASKI, K. TANSEY A N D A. G . W A L T O N

n, =

201

n0

1+4nDTnot-

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Inserting We obtain n

--

3n0r

- 3q+2kTnot

If the total volume of coacervate v = noVo= nrijt where and P, the average volume at time t

ijo

is the average initial volume

ij,+-2kTvt/3q. (19) Thus experimental plots of the data should show ijt linear in t with constant intercept and slope related v and T. Fig. 6 shows that V t is approximately linear in t in the ijt =

0

FIG.6.-Variation

10

20 time /min

30

of the average volume of elastin aggregates with time at different temperatures.

initial stages of particle growth; however, the slope is drastically affected by T. It seems then that the growth is too catastrophic to be accounted for by simple agglomeration and is much more reminiscent of a nucleation process. If the process of coacervation is one of conversion of aggregates into coacervate droplets, this process might be regarded as similar to the familiar droplet nucleation experiment .26 If there are no aggregates of initial volume To (average). Then at time t we may have a statistical number of aggregates “ nucleated ” into coacervate. Assuming only one nucleating event per aggregate and rapid subsequent coacervation within any one aggregate -1n-

nrr

= JVot no where n” is the number of non-nucleated (coacervated) particles.

--In n”/no = Qvot exp-AG*/kT

(21) where i2 is a kinetic constant, involving chain reordering and AG* is the free energy barrier to nucleation. Now for a constant total number of particles (no)

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202

ELASTIN COACERVATION B Y QUASIELASTIC L I G H T SCATTERING

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where i j f and ijo are the final and initial average volume of particles (i.e., coacervated and non-coacervated). Then if i j f B ij,,

n"/n0

-1

-i j t / i i f .

(23)

A plot of ln(l-ijt/ijf) against t gives J, the rate of nucleation. Then

InJ = lnR-(16/3)7t03v2Tc2/kTAS~AT2 (24) where v is the molecular volume, T, the coacervation temperature, AS, the entropy of coacervation, AT = T-Tc and o is the coacervate droplet interfacial free energy.26 Plots of ln(l-ijt/ff) may be obtained only very approximately from the present data, thus the further analysis of InJ against 1/TAT2is particularly imprecise. Nevertheless, the catastrophic temperature dependence of 1nJ is approximately verified if we choose Tc = 25 "C as shown in fig. 7. With improved data it should be possible to obtain from eqn (24) a better estimate of Tc and either o or ASc if the other can be measured independently. It is to be expected that the interfacial energy will be very small, perhaps less than 1-5 erg/cm2.

5

15

25

35

time /min

FIG.7.-Plot

of ln(fina1 volume-volume at time t ) against time. The slope of each line is v,J.

These experiments have some important biological implications for the morphogenesis and structure of elastin fibres which are worth noting here. Since elastin (or tropoelastin) coacervation can occur at physiological pH and temperature, this biological precipitation process has been proposed as the initial step in the deposition of elastin fibres in vivo after secretion of tropoelastin from the fibroblast.8 A subsequent step is the covalent linking of the aggregated tropoelastin chains. If this assumption is correct, then the entropy-driven nature of the phase separation strongly suggests that the elastin chains are in a random coil conformation with high configurational entropy. This deduction like the n.m.r. data of Torchia and pie^,^^ therefore, favours the Hoeve-Flory model of a cross-linked rubber over the alternatives proposed by Partridge and Sandberg.3

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A. M. JAMIESON, B. SIMIC-GLAVASKI,

K. TANSEY AND A. G. WALTON

203

1.0

0.1

U '

1.67

0.33

20

r-'AJ-* x loL

FIG. &--Plot

of In (average initial volume x rate of nucleation) against T-'(T- Tc)-2,assuming T, = 25 "C. The slope cca2/ASz.

However, these conclusions seem to be at odds with the circular dichroism data of Urry and coworkers ** which indicate an increase in order during coacervation of elastin and model polypeptides as evidenced by development of a CD spectrum indicating as much as 50% a-helical secondary structure. The entropy changes we propose during coacervation refer to the total entropy of the chains corresponding to increase in translational and rotational as well as configurational degrees of freedom. Our observations, therefore, would not preclude an increase in order in localized sections of the chain (e.g., in the alanine-rich crosslink regions 29). Another interesting biological possibility arises from the observation, that on prolonged heating, a slow coalescence of the coacervate drops to form a dense gel-like viscous mass takes place,8 involving exclusion of water from the coacervate. This slow process may mimic biological ageing of elastic tissue which is known to involve loss of We wish to acknowledge the partial financial support of this work by the U . S . National Institutes of Health under grants NIAMD17110 and NICHD 00669.

'

J. A. Foster, E. Bruenger, W. R. Gray and L. B. Sandberg, J. Biochem., 1973, 248,2876. S. M. Partridge in Chemistry and Molecular Biolggy of the Intercellular iMntrix, ed. E. A. Balazs (Academic Press, London, 1970), Vol. 1, pp. 593-616. C . A. J. Hoeve and P. J. Flory, Biopolynaers, 1974, 13, 677. W. R. Gray, L. B. Sandberg and J. A. Foster, Nature, 1973, 246, 461. S. M. Partridge, Adv. Protein Chem., 1962, 12,227. L. B. Sandberg, N. Weissman and D. W. Smith, Biochem., 1969, 8, 2940; L. B. Sandberg, N. Weissman and W. R. Gray, Biochem., 1971, 10, 52. H. G. Bungenberg de Jong in Colloid Science, ed. H. R. Kruyt (Elsevier Publishing Co., Amsterdam, 1949), Vol. 2, Chap. VIII. G . C. Wood, Biochem. J., 1958, 69, 539. A. M. Jamieson, C. E. Downs and A. G. Walton, Biochim. Biophys. Acta, 1972, 271, 34.

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H. Bamford and H. Tompa, Trans. Faraday SOC., 1950, 46, 310. P. J. Flory in Principles of Polymer Chemistry (Cornell University Press, Ithaca, N.Y., 1953). l2 M. J. Voorn, Fortschr. Hochpo1ym.-Forsch., 1959, 1, 192. l3 A. Veis and C. Aranyi, J . Phys. Chem., 1960,64,1203; A. Veis, 1960,65,1798; A. Veis, 1963, 67, 1960. l4 H. A. Cummins and H. L. Swinney, Progr. Opt., 1970, 8, 133. l5 A. M. Jamieson and A. R. Maret, Chem. SOC. Revs., 1973,2, 325. l6 B. Chu, Ann. Rev. Phys. Chem., 1970, 21, 145. l7 B. Chu and F. J Schoenes, Phys. Rev. Letters, 1968, 21, 6. l8 N. Kuwahara, D. V. Fenby, M. Tamsky and B. Chu, J . Chem. Phys., 1971, 55, 1140. l9 L. P. Kadanoff, W. Gotze, D. Hamblen, R. Hecht, E. A. S. Lewis, V. V. Palcianskas, M. Ray1 and J. Swift, Rev. Mod. Phys., 1967, 39, 395. 2o G. N. Malcolm and J. S . Rowlinson, Trans. Faraday SOC., 1957, 53, 921. 21 J. S. Rowlinson, Liquids and Liquid Mixtures (Butterworth, London, 1969), Chap. 5. 22 S. P. Lee, W. Tscharnuter, B. Chu and N. Kuwahara, J. Chem. Phys., 1972, 57, 4240. 23 B. Chu, Laser Light Scattering (Academic Press, N.Y., 1974), Chap. VII, p. 152. 24 C. Price, J. D. G. McAdam, T. P. Lally and D. Woods, Polymer, 1974, 15, 228. 25 S. Chandrasekhar, Rev. Mod. Phys., 1943, 15, 59. 26 A. G. Walton in Nucleation, ed. A. Zettlemoyer (Dekker, N.Y., 1969), p. 242. 27 D. A. Torchia and K. A. Piez, J. Mol. Biol., 1973, 76, 419. 2s B. C. Starcher, G. Saccomani and D. W. Urry, Biochim. Biophys. Acta, 1973, 310,481 ; B. A. Cox, B. C. Starcher and D. W. Urry, J . Biol. Chem., 1974, 249, 997. 29 D. B. Wender, L. R. Treiber, H. B. Bensusan and A. G. Walton, Biopolymers, 1974, 13, 1929. 30 R. Ross, J. Histoehem. Cytochem., 1973, 21, 199.

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lo C .

Studies of elastin coacervation by quasielastic light scattering.

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