203

Biochimica et Biophysica Acta, 381 ( 1 9 7 5 ) 2 0 3 - - 2 1 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s BBA 27572

THE HYDRATION OF PROTEOGLYCANS OF BOVINE CORNEA

F R E D E R I C K A. B E T T E L H E I M and B O A K E P L E S S Y *

Chemistry Department, Adelphi University, Garden City, N.Y. 11530 (U.S.A.) (Received J u l y 12th, 1 9 7 4 )

Summary The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a disaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.

Introduction In cells as well as in connective tissues, such as cornea, one of the most interesting problems is the nature of water which is associated with subcellular and macromolecular components. General reviews of this question [ 1--3 ] always encounter the difficulties of definitions. It is agreed that water exists in two states, " b o u n d " and " b u l k " water, but the definition of bound water as contrasted to bulk varies with the type of experiment performed. Most people use an operational definition, i.e. the bound water is that part of the water in the vicinity of macromolecules whose properties differ detectibly from the bulk water of the same system. The detection, however, depends on the technique used and leads to discrepancy between measured values of different techniques. One would like to think t h a t the bound water is localized on the macromolecules while the bulk water is mobile. However, the degree of ordering the

* Present address: Chemistry Department, Dillard University, New Orleans, La., U.S.A.

204 b o u n d water at a site varies as well as the degree of mobility of bulk water. Thus, no sharp " b o u n d a r y " exists in tissues between bound and bulk water, but a s m o o t h transition is expected. One of the most unambiguous ways o f detecting the result of macromolecule--water interaction is to follow changes in vapor pressure which is always decreasing when water " b i n d i n g " occurs. It has been alleged by m a ny authors [4--6] that the glycosaminoglycans of cornea are largely responsible for the water-binding capacity in this tissue. F u r t h e r m o r e , there are indications that keratan sulfate and chondroitin-4-sulfate play a different part in the h y d r a t i o n process [7--9]. The native state of glycosaminoglycans in cornea is in the form of proteoglycans. These proteoglycans are heterogeneous macromolecules carrying keratan sulfate and a chondroitin-4-sulfate side chain on a protein core in different p r o p o r t i o n s [ 1 0 ] . Therefore, it was decided to study the hydrat i on behavior of a n u m b e r of well-characterized proteoglycans of cornea to understand their role in water binding. F u r t h e r m o r e , we wanted to investigate to what e x t e n t the h y d r a t i o n behavior of proteoglycans differs from that of the corresponding glycosaminoglycans, namely, chondroitin-4-sulfate [ 1 1 ] , and keratan sulfate [ 1 2] . Materials and Methods

Procedure for Isolation The corneal proteoglycans were extracted into aqueous solution by the m e t h o d o f Sajdera and Hascall [ 1 3 ] . T w o hundred bovine eyes were obtained fr o m the slaughterhouse less than 24 h post m o r t e m and the cornea were immediately excised and the epithelium, endothelium and Descemet's membrane removed. Washing of the tissue was k ept minimal to avoid loss of the c o m p lex through dissolution into the wash water. The cornea were coarsely ground in a tissue grinder. The protein complexes were extracted with 4.0 M guanidinium chloride solution in 0.05 M Tris--HC1 buffer adjusted to pH 7.5. The ground tissue was magnetically stirred in approxi m at el y 5 ml of solution per cornea for 24 h at r o o m temperature. The slurry was centrifuged and the residual tissue was washed with one liter of solvent by additional stirring for 15 h at r o o m t e m p e r a t u r e . The supernatant was removed by centrifugation. The supernatant of bot h the initial ext r a c t and wash contained a fine gelatinous suspension n o t removed by centrifugation. This material was removed by filtration u n d er vacuum through a 2 cm cake of Celite on Whatman No. 42 filter paper [ 1 3 ] . The clear filtrates were dialyzed versus 20 liters of water at 18°C for 15 h and versus 0.05 M Tris--HC1 at pH 7.5 for an additional 5 h. A precipitate f o r m e d in the dialysis tubes, was removed by centrifugation and subsequently discarded. The supernatants were c o m b i n e d and stored at 4°C until applied to the column. The clear supernatants were c h r o m a t o g r a p h e d on a column of DEAESephadex (Pharmacia). The gel was swollen in water for 24 h prior to column preparation after which a 4 cm diameter by 45 cm length column was poured and gravity packed. The c ol um n was washed with water until no measurable ultraviolet absorption was not ed at 280 nm. The column was water jacketed and all subsequent operations were p e r f o r m e d at 4 ° C.

205 The corneal extracts were applied directly to the column. The initial elution was with water and was continued until no measureable optical density was noted at 280 nm. The fractions collected were discarded. Subsequent elutions were made with 0.15 M, 0.45 M, 0.6 M, 0.75 M, 1.5 M, 2.0 M, and 2.5 M sodium chloride solution in 0.05 M KHz PO4--Na2 HPO4 buffer at pH 6.9. Ten-ml fractions were collected at an initial rate of 1.5 ml/min. The column length decreased as the ionic strength of the eluting solvent increased. The fractions were monitored by the ultraviolet absorption at 280 nm and elution solvents were changed when the optical density was no longer significant. The fractions were subsequently analyzed for hexose and uronic acid content and those found with negative response to both tests were discarded. Two bands were found which were positive to all three tests, i.e., ultraviolet absorption, anthrone and carbazole. The fractions comprising each of these bands were pooled and labeled Fraction I and Fraction II. The proteoglycans were precipitated from solution by the addition of 0.5% w/v solution of cetylpyridinium chloride. The precipitates were harvested by centrifugation. Approximately 120 mg of material was collected in each fraction. Since the elution profile indicated that Fraction I, had a binodal distribution (Fig. 1) the freeze dried sample was redissolved in 0.05 M phosphate buffer and chromatographed on a DEAE-Sephadex A-50 column, 1.5 X 20 cm, preswollen in 1.0 M sodium chloride, 0.05 M phosphate buffer solution. The material was eluted with 1.5 M sodium chloride in 0.05 M phosphate buffer. The fractions were monitored by the ultraviolet absorption peak at 280 nm. Two fractions, Fraction Ia and Fraction Ib, were obtained and subsequently precipitated as the cetylpyridinium chloride salts. Portions of each fraction, were converted to the calcium salts by dissolving them in 2.5 M calcium chloride solution and precipitation from 70% ethanol solution.

A naly tical procedures The carbazole m e t h o d of Dische [15] was used for the determination of uronic acid content. Neutral sugars were analyzed by the anthrone reaction [16]. Total hexosamine c o n t e n t was determined by a modified Elson Morgan test of Antonopoulos, [17]. Total protein c o n t e n t was obtained by the m e t h o d of Lowry et al. [18]. Sulfate was analyzed turbidimetrically on hydrolyzed samples as described by Antonopoulos [17]. Amino acid analysis was performed on Beckman 120C amino acid analyzer after standard acid hydrolysis of the samples in 6 M HC1 in vacuo for 24 h at 110°C. Hexosamines were separated on the amino acid analyzer. In this case the samples were hydrolyzed for 3 h in 8 M HC1 at 95°C [19]. Molecular weight was determined by sedimentation equilibrium centrifugation in a Beckman Model E analytical centrifuge [20]. The sedimentation equilibrium studies were performed at three different concentrations and the partial specific volume of 0.68 ml/g was used for all three fractions.

Vapor sorption experiments Water vapor sorption studies were performed in a high vacuum sorption apparatus described previously [11]. The freeze-dried powdered samples ( ~ 3 0

206 rag) were placed in quartz pans that were suspended on quartz springs within the vacuum system. After the system was sealed and t h e r m o s t a t e d at 45°C, the system was evacuated and the pumping c o n t i n u e d 2--3 days at 10 -~ torr. For the removal of the last traces of water, the pumping was done at 10 -~ t orr using a mercury diffusion pump. The extension of the quartz spring, indicating the weight o f the sample, was read by a c a t h e t o m e t e r . The precision was -+0.05 rag. Usually, after 4 days, there was no change in the weight of the sample. However, the pumping cont i nued for a week and the weight recorded at the time was taken as dry weight. At that point the t h e r m o s t a t was adjusted to 29.6°C and the system was allowed to equilibrate. When the initial conditions for the system had been established, vapor was allowed to enter the system in small increments by opening the sorbate chamber. The small increments of pressure could be controlled by keeping the adsorption chamber closed from the system until the approxi m at e pressure desired had been reached and then allowing the vapor to be sorbed by the proteoglycans resting on the pans. The extension of the spring was m o n i t o r e d as a f u n ctio n o f time, until steady state condition was reached. However, final readings of the vapor pressure and spring extension were not taken for an additional 4 h during which time no measurable spring extension had occurred. Sorption was co n t i nue d until relative vapor pressures of a p p r o x i m a t e l y 0.6 had been reached. Desorption isotherms were obtained on the samples by reversing the adsorption procedure, i.e., removing vapor in small increments and recording the spring extension as a f unct i on of vapor pressure. Sorption isotherms were taken at two temperatures about 13°C apart. Desorption isotherms were obtained only at the higher temperature. Additionally, the runs at the higher t e m p e r a t u r e were made in duplicate, the reproducibility of the points being within the readability of the c a t h e t o m e t e r , + 0.05 rag. Results The separation of corneal proteoglycan extracts on DEAE-Sephadex column is given in Fig. 1. As is evident from the tests performed, fraction I a is a proteoglycan mainly with condroitin-4-sulfate chains. Fraction Ib is enriched in

0.6

2 5 M NaCl

0.75

'\,

\\

\ I00

200

500

ml

F i g . 1. E l u t i o n profile of corneal proteoglycans on a DEAE-Sephadex nm (protein); ...... , uronic acid; . . . . hexose.

column.

- -

-, a b s o r b a n c y

at 280

207 TABLE I

Fractions

Protein % Hexose % Uronic and % Hexosamine % Glucosamine/galactosamine* Hexose/uronic acid* Sulfate/hexosamine* Mr

Ia

Ib

II

40.6 1.9 16.3 19.4 0.13 0.11 0.88 128000

47.8 10.1 3.3 14.6 2.8 2.7 0.94 90000

41.4 8.1 5.2 17.0 1.78 ] .68 0.92 57000

* Molar ratio.

keratan side chains and Fraction II contains keratan sulfate and chondroitin-4sulfate side chains in 2 : 1 molar ratio. Detailed analytical data are given in Table I. The molecular weight obtained as weight average molecular weights, are relatively low as c om pa r ed to similar fractions from cartilage [ 1 2 , 1 9 ] . This is possible due to the fact t hat we e xt r act ed a coarse gring of corneal stroma with guanidium chloride instead of homogenizing the tissue in the solvent. The result was that only the lower molecular weight proteoglycans were solubilized and possibly we left the higher molecular weight c o m p o u n d s in the insoluble residue. This also accounts for our low yield o f 240 mg proteoglycan per 100 g wet cornea. Compared to this, Saliternik-Givant and Berman [21] obtained 44% yield o f ex tr action but after crystallization--fractionation, their yield was comparable to ours. Stuhlsatz, et al. [22] obtained 20% yield based on dry weight which correspond to a 4.4% yield on the basis of wet stroma. Antonopoulos et al. [19] e xt r act ed 1.5% proteoglycans from wet cornea. Table II presents the amino acid composition of our three fractions together with o th er data appeared in the literature. The first thing to notice is that all three o f our fractions have amino acid compositions similar to t hat of the u n f r a c t i o n a t e d corneal p r o t e o g l y c a n [19] and substantially different from the proteoglycan obtained f r om nasal cartilage [ 1 3 ] . The amino acid compositions of two proteoglycans, (one a corneal proteoglycan fraction similar to our Ia in glucosamine/galactosamine ratio and the o t h e r a fraction similar to our I b in glucosamine/galactosamine ratio) obtained by Stuhlsatz, et al. [ 2 2 ] , among others, f r o m bovine cornea gives the extremes of dispersion of amino acid c o m p o s itio n of the protein core and the values of our three fractions fall within these limits. In spite of the difference in acidic polysaccharide side chain composition between Ia and Ib, there is n o t much difference in the composition of the protein core. The slightly higher serine and t hreoni ne c o n t e n t of Ia m ay reflect the protein--polysaccharide linkage of chondroitin-4-sulfate chains through O-glycosidic linkage t o t he amino acids while the corneal keratan sulfates of Ib are linked via N-glycosyl linkage o f the amide group of aspargine [ 2 3 ] . Fraction II is more similar to Fraction Ia than to I b f r o m the point of view of acidic

208 T A B L E II AMINO ACID COMPOSITION

OF CORNEAL

PROTEOGLYCANS

P C S I , a c o r n e a l p r o t e o g l y c a n f r a c t i o n s i m i l a r to o u r I a in g l u c o s a m i n e / g a l a e t o s a m i n e t i o n s i m i l a r t o o u r I b in g l u c o s a m i n e / g a l a c t o s a m i n e ratio. PPCGu, proteoglycan

r a t i o ; P G I I A , a fracf r o m b o v i n e nasal

cartilage.

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valinc Methionine Isoleucine Leucine Tyrosine Phenylalanine

Ia

Ib

II

PCS1 [22]

PGIIA [22]

Unfractionated ~ [19]

PPCGu [13]

54 22 39 137 45 85 120 64 79 52

57 29 46 130 38 79 124 65 82 54

52 10 48 125 31 38

53 15 43 112 33 38

69 21 42 135 38 75 106 71 52 46 17 57 17 55 139 34 27

66 16 22 144 54 60 134 115 48 46 11 36 8 56 106 37 38

52 19 38 105 41 80 100 82 116 50 5 49 3 39 100 20 39

58 20 43 112 42 67 111 76 110 69 5 55 2 47 117 28 36

29 22 36 72 59 113 139 100 116 74 10 65 5 35 77 18 36

* Unfractionated

corneal extract.

p o l y s a c c h a r i d e c o m p o s i t i o n . This is also d e m o n s t r a t e d in t h e a m i n o acid c o m p o s i t i o n o f its p r o t e i n core. T h e o n l y e x c e p t i o n is t h e l o w glycine and alanine c o n t e n t o f F r a c t i o n II and t h e c o n s e q u e n t increase in l e u c i n e and i s o l e u c i n e c o n t e n t as c o m p a r e d to F r a c t i o n Ib. We m a y a s s u m e that o u r mild e x t r a c t i o n t e c h n i q u e resulted n o t o n l y in l o w m o l e c u l a r w e i g h t p r o t e o g l y c a n s but also in p r o t e o g l y c a n s , t h e side chains o f w h i c h are relatively short. This was b o r n o u t by the m e a s u r e m e n t of the m o l e c u l a r w e i g h t o f purified c o r n e a l keratan sulfate [ 1 2 ] that was f o u n d to be 7 0 0 0 . This is t h e l o w e n d o f t h e s p e c t r u m o f m o l e c u l a r w e i g h t s r e p o r t e d in t h e literature [ 2 4 ] . Similarly, w e m a y surmise that our c h o n d r o i t i n - 4 - s u l f a t e chains are also o n this end o f t h e s p e c t r u m . A s s u m i n g a m o l e c u l a r w e i g h t o f 14 0 0 0 for c h o n d r o i t i n - 4 - s u l f a t e and 7 0 0 0 for keratan sulfate, w e m a y visualize the average m o l e c u l e in each o f o u r f r a c t i o n s as f o l l o w s : Ia carries o n the average five c h o n d r o i t i n - 4 - s u l f a t e plus o n e keratan sulfate side chains o n a p r o t e i n core; Ib possesses o n t h e average five keratan sulfate side chains and o n e c h o n d r o i t i n 4-sulfate chain o n each p r o t e i n core; F r a c t i o n II has on t h e average, three keratan sulfate and o n e c h o n d r o i t i n - 4 - s u l f a t e chain o n t h e p r o t e i n core a prop o r t i o n w h i c h is c l o s e t o t h e overall dissaccharide ratio o f keratan sulfate to c h o n d r o i t i n - 4 - s u l f a t e in t h e u n f r a c t i o n a t e d s t r o m a . T h e w a t e r vapor s o r p t i o n i s o t h e r m s , e x h i b i t i n g hysteresis, are given in Fig. 2, at 2 9 . 6 ° C . It is clearly seen that p r o t e o g l y c a n Ib c o n t a i n i n g p r e d o m i n a n t l y keratan sulfate side chains sorbs m u c h m o r e w a t e r than either p r o t e o -

209 mg

T 40(:

rng g

500

600

200

o

"

0

400

c;

~

~//"

,/

/

i ......

0.2

0.4

0.6

P/~

0

0.2

0.4

0.6

P/~

Water vapor s o r p t i o n - - . , and d e s o r p t i o n . . . . . . . I s o t h e r m s at 2 9 . 6 ° C . * ~ , calcium salt o f p r o t e o g l y c a n Ia; ©c;, calcium salt of p r o t e o g l y c a n Ib; • e calcium salt of p r o t e o g l y c a n II. Fig. 2.

F i g . 3 . Water vapor s o r p t i o n i s o t h e r m s at 16"C o f calcium salts o f v ¢ , p r o t e o g l y c a n Ia; o o, p r o t e o g l y c a n Ib; • $ , p r o t e o g l y c a n If; e ~ , I a + Ib c o m b i n e d ; ~ e , keratan sulfate; • , chondroitin-4-sulfate.

glycan Ia or II. On the other hand, the amount of hysteresis exhibited by this sorbent is minimal. Proteoglycan Ia with predominantly chondroitin-4-sulfate side chains sorbs 22% more water than proteoglycan II, which has twice as much keratan sulfate than chondroitin-4-sulfate on the molar basis of the dissaccharidic repeating unit and which in its composition resembles the average acidic polysaccharide composition of the cornea. The hysteresis of proteoglycans Ia and II were similar. In Fig. 3, the water vapor sorption isotherms at 16.0°C are given for a number of sorbents beside the corneal proteoglycans. The relative sorptive capacities of proteoglycan Ia, Ib and II, are the same as at 29.6°C. What is interesting to note, is that the sorptive capacity of proteoglycan Ib having predominantly keratan sulfate side chains is very much the same as the sorptive capacity of keratan sulfate itself. On the other hand the sorptive capacity of proteoglycan Ia with predominantly chondroitin-4-sulfate side chains; is almost twice as high, as that of chondroitin-4-sulfate at higher relative humidities. The most interesting result is that the proteoglycan I and II fractions behaved almost identically regarding their hydration properties. It should be remembered that proteoglycan I comes off the column with 1.5 M NaC1 while proteoglycan II with 2 M NaC1 [10]. Proteoglycan I, essentially contained two fractions Ia and Ib, but its overal composition was very similar to that of II. D 2 0 vapor sorption studies on calcium salts of proteoglycans Ia and Ib and II, gave almost identical results to that of H2 O sorption. Discussion The cornea contains about 78% water and it is an elastic gel. Although the

210 total c o n c e n t r a t i o n of the proteoglycans in cornea is only 2%, there are ample evidences that th e y are largely responsible for its swelling as well as water re t e n tio n properties [ 4 , 6 ] . In tissues with such a high concentrations of solutes, the water uptake at a certain relative h u m i d i t y provide a simple scaling procedure to measure the h y d r a t i o n of the different macromolecules [25]. Any n u m b e r of theories accounting for vapor sorption isotherms propose that the molecules entering the solid matrix first, are the ones that are tightly held and therefore, identifiable with bound water [ 2 ] . The m o n o l a y e r c o n c e p t one one such t h e o r y , namely the Brunauer, E m m e t t and Teller (B.E.T.) model [ 2 6 ] , is used quite often also in swelling p o l y m e r networks [2,3]. However, the interpretation of the m o n o l a y e r is not water adsorbed on a physical surface in one molecular layer thickness, but one of tightly bound water molecules on molecular h y d r o p h y l i c domains, such as polar groups, ions, etc. [27]. Usually, it is f o u n d that the higher the water uptake at low relative humidity (within the B.E.T. mo n o lay er ) the higher the sorption of water vapor also at high relative humidities. This is definitely true in the case of our three proteoglycans. Comparing Fig. 1 and Fig. 2, with the B.E.T. m o n o l a y e r values given in Table III, one can state that the proteoglycan that binds greater amounts of water in its m o n o l a y e r will d e m o n s t r a t e higher state hydrations at high relative vapor pressure. Our presentation of the data in Figs 1 and 2, are only up to 0.6 P/Po, because between 0--0.6 relative h u m i d i t y we found that our sorption isotherms are reproducible within the error of the measurements. Above that range, the error in the reproducibility increases to + 5%. However, two samples I a and Ib in a final run, were carried up to P/Po = 0.95. We found that Ib sorbed 95% and I a 78% water at this relative humidity. Thus, our isotherms can be e x t e n d e d to the physiological range. In Table III, a second parameter is presented to describe the a m o u n t of b o u nd water sorbed by the proteoglycans. This parameter is derived from the plot o f negative differential e n t r o p y of sorption versus water uptake. Usually a m a x i m u m in this f unct i on arises f r om the fact the configurational e n t r o p y of water is the main c o n t r i b u t o r to this t h e r m o d y n a m i c p r o p e r t y , thus when the primary sorptive sites diminish (just before the c o m p l e t i o n of a monolayer) a m a x i m u m in negative e n t r o p y is expected. The m o n o l a y e r again means water molecules strongly b o u n d t o energetically most favorable (polar) sites [ 2 8 ] . It is true that the e n t r o p y of sorption derived from the t e m p e r a t u r e dependence

TABLE

III

Sample*

Amount of water strongly bound as a n a l y z e d b y :

(calcium salts)

Proteoglycan Ia Proteoglycan Ib Proteoglycan II Proteoglycan I K e r a t a n S u l f a t e [ 121 Chondroitin-4-sulfate

[ 11]

(g H 2 0 / g s o r b e n t ) . 1 0 2

B.E,T. [13]

AS maxima

Hysteresis

8.6 20.2 7.5 7.5 22.4 4.2

8.0 12.0 6.0 6.4 35.6 3.5

3.0 0.4 2.7 2.6 2.2 3.2

211 of the isotherms is a t h e r m o d y n a m i c function and applicable only to reversible processes. In swelling polymers with hysteresis, one deals with quasi-equilibrium states of an irreversible process. However, if not the numerical values, the positions of the maxima still can be used as an indicator for bound water or strongly sorbed monolayer water. This was demonstrated by comparing the" experimental calorimetric enthalpy and the calculated isosteric enthalpy of sorption functions which showed maxima at the same water uptake for different polymers [29]. One can see from Table III that the a m o u n t of water strongly bound to polymers follow the same order whether one uses the criterion of the B.E.T. monolayer or the m a x i m u m in the negative entropy of sorption function. Furthermore, the order among proteoglycans and glycosaminoglycans demonstrating hysteresis in their sorption behavior is exactly the opposite. It seems that a polymer matrix which binds large amounts of water strongly retains only a small part of this water irreversibly, while a polymer which sorbs smaller quantities of water on primary sites retains a larger portion of this irreversibly. This p h e n o m e n o n can be explained on the basis of the interaction between polymer chains in the solid and gel state. Whenever the intermolecular interaction between polymer chains is strong whether in hydrogen bonds or calcium bridges, a tight matrix results which has only a few polar sites for sorbing water in the monolayer. Furthermore these tight matrices will have resistance to free swelling, thus the water uptake even at high vapor pressures will be relatively low. On the other hand, once the water is sorbed strongly in the non-crosslinked polar sites, it will be retained there strongly, hence a large hysteresis. The opposite is true with open polymer networks. The infrequent interchain interaction allows a large a m o u n t of polar sites to sorb water strongly (in monolayer). The infrequent crosslinks allows also relatively free swelling. Thus one deals with large water uptake both in the form of bound water as well as bulk water filling the space of the open network. Since only small portions of this sorbed water molecules will occupy positions where they will be immobilized by 3--4 hydrogen bonds, there will be a small a m o u n t of hysteresis in these matrices. Thus the water sorptive and retentive capacity of the individual polymers reflects the type of tertiary and quaternary structure of these polymers in solids, gels and concentrated solutions. In this respect it is interesting to note that the overall sorptive capacity, and the a m o u n t of water strongly bound was very similar in proteoglycan Ib and in keratan sulfate. We interpreted the high sorptive capacity of calcium keratan sulfate as a result of an open network in which the Ca 2+ ions are in non-specific electrostatic binding rather than the chelating type [12]. Proteoglycan Ib is a polymer having approximately 48% of its material as protein core. There are almost three times more keratan sulfate repeating units as chondroitin-4-sulfate dissaccharidic units and on the average there are five keratan sulfate side chains and one chondroitin-4-sulfate side chain on each protein core. There are two possible reasons why proteoglycan I b has the same hydration property as keratan sulfate. (a) The protein core participates in the water

212

uptake even to a greater e x t e n t than the keratan sulfate side chains, thus compensating for the low water sorptive capacity of chondroitin-4-sulfate [ 1 1 ] . (b) The proteoglycan structure resembles a sort of comb, with a rigid central protein core and the side chain hanging from these cores. Even in the protein core participates only to a lesser degree in the water uptake [ 2 9 ] , the side chains within the proteoglycan are now separated further from each ot her than in a pure keratan sulfate gel. Thus, the keratan sulfate as well as the chondroitin-4-sulfate can sorb more water both in primary sites (bound water) and in interstices, than the individual glycosaminoglycans, in which the protein core did n o t separate them. Under these conditions, one visualizes that interactions between proteoglycan and proteoglycan molecules still exist but in the case o f such short side chains as keratansulfate these do not penetrate over the length o f the side chains. Both the intra- and inter-molecular interactions between keratan sulfate side chains decreases as com pared to those in pure keratan sulfate interactions, resulting in a rigid but much more open structure capable o f swelling and thus compensating for the relative low water binding of the protein core. We favor this second explanation. The same comparison can be made between chondroitin-4-sulfate and p r o t e o g ly can Ia regarding their h y d r a t i o n behavior. The latter p o l y m e r contains 40% protein in its core and in the side chains, nine times as much condroitin-4sulfate repeating units as keratan sulfate repeating units. The average structure of this proteoglycan is that of five chondroitin-4-sulfate side chains for each keratan sulfate side chain on the protein core; the chondroitin-4-sulfate chains being almost twice as long as the keratan sulfate chains. This proteoglycan, Ia, sorbs almost twice as much water, b o t h in the form of strongly b o u n d water ( " m o n o l a y e r " ) , and in the form of bulk water at high relative vapor pressures, as chondroitin-4-sulfate alone. This again can be explained on the basis of spacing the side chains on a rigid protein core. Chondroitin-4-sulfate o f all the other glycosaminoglycans [11,12] had the most tightly b o u n d matrix, possibly through localized Ca 2+ bridges as well as hydrogen bonding beside ion--dipole and dipole--dipole interactions. The spacing of the side chains along a protein core decreases the chondroitin-4-sulfate interactions with o th er side chains within the same proteoglycan. This leads to a more open n e t w o r k than in the case of chondroitin-4-sulfate chains alone and explains the greater sorptive and s om ew ha t lesser retentive capacity of proteoglycan Ia as co mp ar e d to chondroitin-4-sulfate. On the o th er hand, the longer chondroitin-4-sulfate chains can penetrate and interact with each ot her intermolecularly (i.e., between two proteoglycan molecules) to a greater e x t e n t than the shorter keratan sulfate side chains. Therefore, p r o teo gl yc a n Ia still has a much tighter matrix than proteoglycan Ib. This results in a higher water upt a ke by I b and a greater retentive power by Ia. The most interesting aspect of our study is that proteoglycan II which has a b o u t 1.7 times as m any keratan sulfate repeating units as chondroitin-4-sulfate repeating units and on the average three keratan sulfate side chains for each chondroitin-4-sulfate side chain on the protein core, has h y d r a t i o n properties similar to the proteoglycan with a p r e p o n d e r a n c e of chondroitin-4-sulfate side chains, I~. It seems t hat if a sufficient n u m b e r of long chondroitin-4-sulfate side chains are present in a proteoglycan (even if it is n o t the majority of the side

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chain components) their interaction with other side chains will result in a tight matrix. Proteoglycan II is the smallest among our isolated corneal proteoglycan molecules. This would indicate that the side chains are probably closer spaced in this polymer than in proteoglycan Ia and Ih. Thus, one expects m~re intraand inter-molecular interactions resulting in a tighter matrix. A tight matrix on the other hand will bind water to a smaller extent and retain it to a greater degree. That this is the proper explanation can be seen from the hydration behavior of the unfractionated proteoglycan I. In its overall composition, this material had the same keratan sulfate to chondroitin-4-sulfate repeating unit ratio as proteoglycan II. However, it was composed of two different proteoglycans (In and Ih) each with a quite distinct keratan sulfate/chondroitin sulfate ratio. In spite of that, the hydration behavior of proteoglycan I was almost identical to that of proteoglycan II. We assume as before, that the hydration properties are dependent on the openness or tightness of the polymer network. This then must mean that the tightness of a polymer network composed of these two acidic polysaccharides is determined by a critical mass of chondroitin-4-sulfate and is independent of the spatial arrangement of these side chains on the protein core. Therefore, we may conclude that in connective tissues, the proteoglycan molecules undergo hydration depending on their overall composition. The protein core participates in the water uptake to a small extent. If sufficient of chondroitin-4-sulfate chains are present they can interact with other side chains through Ca 2÷ bridges, hydrogen bonding, etc., to create a tight network. The result will be limited water uptake but high water retention in the polymer network. On the other hand, when chondroitin-4-sulfate is only a minor component of the side chains, the keratan sulfate side chains create an open network with large swelling capacity but very little retentive power. Acknowledgments This research was supported by a research grant of The National Eye Institute EY-00501-05 of the Public Health Service. We are grateful to Dr Leon Esterman of the Weizmann Institute of Science, Rehovot, Israel, for the mnlecular weight determinations. Our thanks are due also to Mr J o h n Moschers of the Biochemistry Department of New York Medical College, Valhalla, New York, for the amino acid analyses. References 1 C o n w a y , B.E. ( 1 9 7 2 ) Rev. M a c r o m o l . C h e m . 7, 1 1 3 - - 2 3 5 2 K u n t z , Jr, I.D. a n d K a u z m a n n , W. ( 1 9 7 4 ) Avd. P r o t e i n C h e m . 28, 2 3 9 - - 3 4 5 3 B e t t e l h e i m , F.A. ( 1 9 7 0 ) in Biological P o l y e l e c t r o l y t e s (A. Veis, e d . , ) Chap. 3, M. D e k k e r , Inc. N e w York 4 H e r i n g a , G.C., L e y n s , W.F. a n d W e i d i n g e r , A., ( 1 9 4 0 ) A c t a n e e r . M o r p h . 3, 1 9 6 - - 2 0 1 5 Pirie, A. a n d V a n H e y n i n g e n , R. ( 1 9 5 6 ) in B i o c h e m i s t r y o f t h e Eye, p. 144, Blackwell Scientific Publ., Oxford, U.K. 6 H e d b y s , B. ( 1 9 6 1 ) Exp. E y e Res. 1, 8 1 - - 9 1 7 A n s e t h , A. ( 1 9 6 9 ) E x p . E y e Res. 8, 2 9 7 - - 3 0 1 8 Praus, R. a n d D o h l m a n , C.H. ( 1 9 6 9 ) E x p . E y e Res. S, 6 9 - - 7 6

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