Phase separation in aqueous solutions of lens y - crystallins : Special role of ys ( ternary mixture / cataract )

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens 'y-crystallin proteins. Specifically, we have examined two ternary systems containing ys-namely, yIVa with ys in water and yll with ys in water. For each system, the phase-separation temperatures (Tph(4)))a as a function of the overall protein volume fraction ¢b at various fixed compositions a (the "cloud-point curves") were measured. For the yiva, ys, and water ternary solution, a binodal curve composed of pairs of coexisting points, (4l, a') and (4 ll,a"), at a fixed temperature (20°C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloudpoint curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that ys-crystallin may play an important role in maintaining the transparency of the lens. Phase separation of the eye lens cytoplasm has been implicated in cataract formation (1), where opacification of the eye lens results from disturbances in the uniform spatial distribution of the lens proteins (2, 3). The lens proteins primarily involved in phase separation are the -y-crystallins (4), a family of lensspecific monomeric proteins. Previous studies (5-7) have focused on phase separation in binary aqueous solutions of pure, individual members of the calf lens -y-crystallin family. These proteins fall into two groups: the "high-Tc" (T38°C) proteins 'Yllla (,yc) and 'YIV, (YE) and the "low-Tc" (T5°C) proteins YII (YB) and Ylll.b (YD). Here Tc is the critical temperature of a binary solution, which is also the maximum phaseseparation temperature (Tph) on the coexistence curve in the temperature and volume fraction phase diagram. The critical volume fractions (4)c) of all these y-crystallins studied are approximately the same (1c = 0.21 + 0.02) (7). The lens cell, however, contains multiple protein species (8). Thus it is important to understand the phase-separation behavior of multicomponent aqueous solutions of lens proteins. We have therefore begun a systematic study of the phase-separation properties of protein ternary solutions each consisting of two species of lens proteins in water. The phase boundary for liquid-liquid phase separation in a ternary solution is a coexistence surface in the threedimensional temperature (T), overall protein volume fraction [4 = (NA + NB)Qlp/V ], and composition [a = NA/(NA + NB)] phase diagram. Here NA and NB are the numbers of protein molecules of type A and type B, and V is the total volume of the solution. The effective volume flp of a single protein molecule is assumed to be the same for both species. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. coexistence surface T = Tph(q), a) separates the one-phase regime (T > Tph) from the two-phase regime (T < Tph). If one starts with a one-phase sample of fixed volume fraction and composition and lowers the temperatures to below Tph, then two coexisting phases, designated as (4)1, a') and ()11, all), appear. Such coexisting phases are represented by pairs of points at the same temperature on the coexistence surface. The line connecting each pair of coexisting points is called a tie-line. All coexisting points at this temperature constitute the binodal curve. There is a special line on the coexistence surface formed by points representing pairs of coexisting phases infinitely close in both composition and volume fraction. At each point on this line, coexisting phases become indistinguishable. This line is the so-called "critical line," and we will refer to any point on this critical line as a critical point. We have previously published (9, 10) our experimental and theoretical study of the coexistence surface Tph(4, a) for aqueous ternary solutions consisting of various combinations of the proteins YlIlla YlVa, YI1j and Ylllb. For such ternary solutions, the coexistence surface has a simple feature: the constant a sections (Tph(4))a of the coexistence surface (the "cloud-point curves") retain their shape and can be obtained by a vertical displacement (along the T axis) of the coexistence curves of the corresponding binary solutions. In addition, the critical points of the ternary mixture at all compositions a remain at the same concentration as the critical volume fractions of the binary solutions. We have found that both of these features result from the fact that the interaction energies among the y-crystallins mentioned above differ by less than kBT, where kB is the Boltzmann constant. Such proteins may be designated as "similar proteins." In this paper we examine protein ternary solutions that include an important member of the y-crystallin family, Yscrystallin. Ys is a monomeric protein of a molecular mass and radius about the same as those of the other y-crystallins-i.e., 21 kDa and 24 A, respectively. Examination of binary aqueous solutions of pure y,-crystallin showed that, under the conditions used to study other pure y-crystallins, ys solutions do not phase separate at temperatures as low as 10°C. In addition, our separate studies show that ys suppresses the aggregation of yii-crystallin and 'Yva-crystallin (C.L., J.P., A.L., O.O., and G.B.B., unpublished data). In both bovine and human lenses, the synthesis of ys-crystallin increases with age. In contrast, other y-crystallins are mainly expressed prenatally, and their syntheses decrease progressively during the development of lens (11-14). These properties of ys-crystallin lead us to hypothesize that ys may play an important role in maintaining the transparency of the lens. We therefore undertook an investigation of the effect of Ys on the phase-separation temperature of other lens proteins in mixtures where increasing amounts of ys are present. We present here our studies of two ternary solutions composed of Abbreviations: DTT, dithiothreitol; Tc, critical temperature; Tph, phase-separation temperature. *To whom reprint requests should be addressed.