1948
AND SANFORD H. EHRLICH FREDERICK A. BETTELHEIM
method. The study further suggests that the polyglutamic acid forms a number of anhydrides between two adjacent carboxyl groups during thorough drying under a high vacuum. The poly-y-ammonium-L-glutainate is a more hydrophilic substance than the polyglutamic acid and appears to exhibit much the same hydrophilic character as does poly- y-sodium-1,-glutamate.
1701* 67
DISCUSSION D. GRAHAM (E. I. du Pont de ?;emours and Company).-To what extent may bulk sorption (involving actual penetration of t h e solid) be involved? The slow desorption suggests this possibility. L. H. REYERSON.-iTe have no evidence that bulk sorption is involved in these studies. Swelling n a s not observed.
WATER VAPOR SORPTION OF nrUCOPOLYSACCNARInES’ BY FREDERICK ,4.BETTELHEIM AND SANFORD €1. EHRLICH~ Chemiszry Department, Adetphi College, Garden City, A’. Y . Received illarch 20, 1963 Water and deuterated water vapor sorption isotherms were obtained on calcium salts of chondroitin sulfates A, B, and C, as well as on heparin. The sorptive capacity and extent of hysteresis were analyzed and correlated with the thermodynamic functions of sorption and with the B.E.T. “monolayer.” Calcium chondroitin sulfate A has the largest interchain interaction in the solid state and its water sorptive capacity is the smallest among these polyelectrolytes. Calcium chondroitin sulfate C, on the other hand, has almost free smelling properties with minimal hysteresis efYect. The water sorptive and retention capacities were correlated with molecular structure.
Studies performed on polymers in dilute solutions and extrapolated to infinitely dilute solution yield parameters describing the behavior of a single molecule. While this itself is an important characterization, it, may not be very significant for polymers in a condensed phase. An alternative approach adopted in this Laboratory aims to characterize the polymers in the solid state and observe the t8ransitionfrom solid to gel to concentrated solution. The work reported here is part of this program. The mucopolysaccharides selected for this study were isomeric chondroitin sulfates A, B, and 6, and heparin, The primary structure of these polymers has been largely established in the past ten yearsa-* (see Fig. 1). What uncertainty remains is with respect to the heparin structure where the exact position of an 0-sulfate group is still unknown. Since all these polymers are amorphous, the isomeric chondroitin sulfates and to a lesser degree heparin provide an interesting example of the steric effect of the different polar groups on the water sorption capacity of the polymer. Mucopolysaccharides are important components of animal connective tissuesg where they occur mostly in a complex form chemically linked and associated with proteins.10-18 These tissues are fully hydrated; meta(1) Supported in part b y Grant C-03984 BBC of the National Cancer Institute, Public Health Service. (2) Based in part on a thesis submitted S. H. Ehrlich t o the Graduate School of -4delphi College for the partial fulfillment of the Ph.D. requirements. (3) E. Davidson, P. Hoffman, K. Meyer, and A. Linker, B i o c h i m . B i o p h y s . A c t a , 21, 506 (1956). (4) P. Hoffman, A. Linker, P. Sampson, K. Meyer, and E . Korn, ibid., 26, 658 (1957). ( 5 ) P. Hoffman, A. Linker, and K. Meyer, Federation Proc., 17, 1078 (1958). (6) P. Stoffyn and R. Jeanlos. J. Biol. Chem., 235, 2507 (1959). (7) M. Wolfrom, D. Weisblat, J. Karabinos, UT.McIXelly, and J. McLean, J . Am. Chew. Soc., 66, 2077 (1943). (8) I. Danishefsky, H. Eiber, a n d J. Carr, A r c h . Biochem. Biophys., 90, 114 (1960). (9) K . Meyer, “The Chemistry of Mesodermal Ground Substances,” Harvey Lecture Series LI, 1955-1956, Academic Press, iSew York, N. Y., 1957. (10) M. Schubert, Federation Proc., 17, 1099 (1958). (11) M. B. Mathens a n d I. Lozaityte, A r c h . Biochem. B i o p h y s . , 74, 155 (1958). (12) S. M. Partridge, H. F, D$vis,.and G. 6. Adair, Biochem. J., 79, 15 (19613.
bolic products are passing through them, and among the components of the tissues the mucopolysaccharides are largely responsible for the retention of mater; hence the hydrated state of the tissues. The water sorptive capacities of these mucopolysaccharides may be the clue to their physiological function, although one has to be aware of the fact that the water sorption pattern of these nzucopolysaccharides might be diff ereiit in their more complex form associated with proteins. Experimental A. Material.-The samples used in these experiments were generously supplied by researchers investigating the physicalchemical properties of these compounds. Preparation of chondroitin sulfate A isolated from bovine trachea has been described.14+ Chondroitin sulfate B, lot no. R01-2232/715 obtained from F. Hoffman-La Roche and Co., Basel, Switzerland, through the courtesy of Dr. A. Winterstein, has also been des ~ r i b e d . 1 ~Chondroitin sulfate C, lrindly furnished by Dr. M . R. R l a t h e ~ s ,was ~ ~ obtained ,~~ from human chordoma. A sample of heparin (3208) lot no. D-3835 was received from Mann Research Laboratories, Inc., New York 6, N. Y . All preparations were the calcium salts of the respective polymers. The samples were ground to a fine white powder. B. Sorption Apparatus and Procedure. 1. Sorption Apparatus.-Four quartz helix-springs purchased from Microchemical Specialties Co., Berkeley, California and used in the gravinietric determinations had a sensitivity of 1 mm. per mg. The quartz helix had a 20-cm. extension and hence a maximum load capacity of 200 mg. The extension ITVas essentially linear over the working range and was calibrated over a 100-mg. range. A loop a t the end of the quartz helix provided the support for the sample pan. The deep quartz pan accommodated a 0.5-g. load of 1 ml. in volume. A Model M911 cathetometer (Gaertner Scientific Corporation, Chicago, Illinois), having an accuracy of zk0.05 mm., permitted a precise measurement of vertical spring extension during sorp~ a 10-1. tion. The temperature was controlled to z l ~ 0 . 0 5and ballast bulb was attached to the manifold to smooth out pressure and temperature fluctuations. balance case containing the 2 . Sorption Procedures.-The quartz helix balance was attached to the vacuum manifold and submerged in the thermostat. (13) h l . B. Mathevs, Biochim. B i o p h y s . Acta, 68, 92 (1962). (14) J. Einbinder and M. Schubert, J. Bid. Chem.. 191, 591 (1951). (15) F. A. Bettelheim a n d D. Philpott, N a t u r e , 188, 654 (1960). (16) R. Marbet a n d 8. Winterstein, H e h . C h i m . A c t a , 34, 367 (1956). (17) A. Dorfman a n d &.I.B. Matheirs, A r c h . Biochem. Biophys., 42, 41 (1953). (18) A I . B. 3latiiews, private conirnu~iicatiun.
Uct., 1963
1949
\vL4TER VAPOR SORPTION O F ~lVCOPOLYSACCI-ISRIDE8
I-
z
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k
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'/PO,
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HEPARIN
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Fig. 1.-Structure
of mucopolysaccharides.
An initial reading of the index fiber was noted without samples in the pans. The samples were degassed a t 40" until a constant extension had been maintained for 24 hr. a t approximately mm. pressure. A. second reading was then obtained to determine the dry weight of the sample. The adsorption-desorption isotherms were determined a t temperatures approximately 10" apart. The temperature of the bath, after degacsing, was lowered to the desired experimental temperature. Small increases of pressure were recorded by visually observing the mercury height differential in the manometer. The purified adsorbate water and, in subsequent experiments, 99.89% deuterated watw, obtained from Biological Radiation Laboratory, Richmond, California, was degassed during a series of freezings and thawings cn a Dry Ice-acetone bath while pumping down to a pressure of mm. The vapor was allowed to be sorbed by the adsorbent resting in the pans. The rate of eorption was determined by observing the extension of the helix as a function of time. When equilibrium was established, the vapor pressure and equilibrium position of the index arm were recorded. Equilibrium q-as reached after 3-5 hr. depending on the relative vapor pressure but the final reading was taken after an additional 4-8 lir. during which time no change in weight could be observed.
Results and Discussion Water sorption on polyelectrolytes is a complex phenomenon. We cannot talk about adsorption on the surfaces because water vapor can fully penetrate the polymer matrix whether it is a powder or a film. On the other hand, the different polar groups of the
Fig. 2-3.-Water vapor sorption isotherms of mucopolysaccharides a t 30": ,sorption isotherms; - - - -, desorption isotherms. CS-A, -B, and -C represent calcium chondrcitin sulfate A, B, and C, respectively.
polymers provide energetically different preferential sites for the water; hence the more general term of sorption seems to be proper. Resides having many different polar groups for water vapor sorption through hydrogen bonding, dipole-dipole, ion-dipole interactions, etc., the polymers themselves are in a dynamic state. Polymer chains in the solid state are bound inter- and possible intramolecularly by similar forces as above in addition to the ionic calcium bridges. During sorption, swelling occurs which can open up previously unavailable sites for further water vapor sorption. -41~0water vapor can break existing hydrogen bonds between polymer chains and establish new ones. At higher coverages subsequent electrostatic repulsion of the anionic sites of the polymer may cause further expansion of the "surface." Since the sorbent polymer is in such a dynamic state the question may arise whether the points on an isotherm represent a truly equilibrium state. Even after one proves, as it will be shown below, that the sorption and desorption isotherms can be fully reproduced by cyclic repetitions and, therefore, equilibrium conditions can be assumed, the complexity of the sorbent polymer matrices will allow only a semiquantitative treatment. The sorption isotherms of HsO and DzO on isomeric chondroitin sulfates and heparin are given in Fig. 2-5.
H. EHRLICH FREDERICK ,4.BETTELHEIM AKD SANFORD
1950
M O L E S h$/
I I-
*
M O L E H E P A R I k R E P E A T I N G UNIT
2 I
I
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Vol. 67 3
4
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I
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-as0
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M O L E S II,O/MOLE
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40
80
120
I60
200
rng. H 2 0 / g . ADSORBENT. 0'
I
I
I
0.2
04
0.6
I
08
Fig. 4-5.-Deuterated .vr-ater sorption isotherms of mucopolysaccharides at 24": __- , sorption isotherms; - - - -, desorption isotherms. CSA, CSB, and C8C represent calcium chondroitin sulfate -4,B, and C, respectively.
I n spite of the hysteresis of the isotherms, the sorption part was fully reproducible after degassing and returning to the original zero point. Repeated isotherms a t different temperatures were performed on all samples, with a reproducibility error of f1%. Generally speaking, the water sorptive capacities of the polymers were higher than the DzO sorptive capacities. Individually a t low vapor pressures, the heparin sorbed the greatest amount of mater, followed by chondroitin sulfates C > B > A. At higher vapor pressures chondroitin sulfate C contained more water vapor than heparin per unit weight of sorbent. On the basis of moles of water per moles of disacchariderepeating-unit of the polymer chain, the sorptive capacity order was C > heparin > B > A. It is interesting to note that the width of the hysteresis loop displayed exactly the reverse order. I n a swelling polymer the hysteresis cannot be interpreted as a result of capillary condensation. CassielQ has shown that in swelling gels the amount of hysteresis reflects the mechanical constraints measurable by the elastic properties of the material. Hence the interpretation of this phenomenon may be that calcium chondroitin sulfate A is the most tightly bound polymer with respect to intermolecular interactions. Probably frequent interchain links by calcium bridges and hydrogen bonds do not allow an easily swelling process. (19) A.
B. D. Cassie,
12'rans. Faraday Soc., 41, 458 (1045).
I
Fig. 6.-Differential standard entropy function of water vapor sorption on mucopolysaccharides a t different coverages.
Hence a relatively smaller number of water molecules can be sorbed in the matrices of this polymer a t equivalent vapor pressures compared to the other mucopolysaccharides. Furthermore, these sorbed water molecules will be more tightly bound because of the mechanical constraint exerted upon them by the neighboring chains, hence the large hysteresis effect. This same interpretation can be advanced also for the other polymers. Calcium chondroitin sulfate C, which has the largest sorptive capacity, also shows the least hysteresis. As a matter of fact, no hysteresis loop appears in the higher vapor pressure range. This is indicative of the ease of swelling of calcium chondroitin sulfate C with subsequent large water uptake. Seemingly, the polymer chains can separate easily and in this way open up all the polar sites for water sorption which will result in an early solvation process of the chains. Therefore, those solvating water molecules can be removed without hysteresis effect. Since the only difference between chondroitin sulfate A and C is the position of the sulfate group (C, and c6) it is obvious that the sulfate on c6 is more available for water vapor sorption. This conclusion can be reached also upon inspecting molecular models. Furthermore, since a sulfate group in C6 enlarges the width of the polymer chain to a greater degree than a t Cd it is less likely that the chondroitin sulfate C chains can be tightly bound to each other. This results in a greater swelling power. The difference between chondroitin sulfate A and B i s the steric position of the carboxyl group. O b the
WATERVAPORSORPTION OF NUCOPOLYSACCHARIDES
Oct., 1963
1951
M O L E S H ~ O / M O L E H E P A R I N REPE:ATING U N I T .
K CAL MOLE
-As0 e.u.
M O L E S ~ I ~ O / M O L Ec s R E P E A T I V G UNIT.
I
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REPEATING UNIT,
I
I
2
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3
4
24
60
0
40
80
mg. H,O/g.
120
160
I
200
0
ADSORBENT,
Fig. 7.-Isosteric heats of water vapor sorption of mucopolysaccharides a t different coverages.
basis of sorptive capa,city and hysteresis effect one may suggest that the carboxyl group in the L-iduronic acid moiety provides a more available sorptive site than in D-glucuronic a,cid. Heparin is an interesting case. It obviously has more polar groups than chondroitin sulfate C and still it sorbs somewhat less water. The explanation here again may be that heparin has a large number of interchain links which restricts its swelling similarly to that of chondroitin sulfate A; on the other hand the larger number of polar groups partly compensates for this restriction. The DzO sorption isotherms generally showed about 20y0 lesser sorptive capacity of the polymers than the water isotherms a t the same temperature. The order of sorptive capacity has changed ; the highest is chondroitin sulfate C followed by B and A and finally hieparin. The width of the hysteresis loop, howerer, retained the same order as in water, indicating again that hysteresis reflects more the mechanical constraints present in the smelling gel than the availability of polar groups. The relatively bad performance of heparin toward D20 as compared to HzO deserves some comments. One can assume as a first approximation that HzO and DzO molecules can hydrogen bond in two ways: (a) with their oxygen to the hydrogen of a hydroxyl or amide group 012 the polymer or (b) with their protium and deuterium to the oxygen atoms of the polymer molecules. It will be demonstrated later by the differential thermodyhamie data that the hydrogeh bollding of deuterium i e ener@&icallyless favwabk than protium.
Rig. O,O/g.
ADSORBENT,
Fig. 8.-Differential standard entropy function of deuterated water vapor sorption of mucopolysaccharides a t different coverages.
In that case heparin, which has the greatest number of availableoxygensites, would suffer the greatest reduction in sorptive capacity going from protium to deuterium. The over-all reduction in the sorptive capacity of the isomeric chondroitin sulfates going from HzO to DzO can be also explained by the same mechanism. Additional evidence for the energetically less favorable 0-D . . . 0-R hydrogen bond can be found in the fact that very little deuterium-hydrogen exchange was found in our polymers, definitely within the limits of error of our gravimetric determinations. Using sorption isotherms a t different temperatures which were fully reproducible, the isosteric heats of sorption were calculated from the Clausius-Clapeyron equation. The standard differential free energg values were obtained a t 30.0' for HzO and 32.6' for DzO from the corresponding isotherms and the differential entropy values were calculated from the above data. 23 These thermodynamic functions are plotted against coverage, as shown in Fig. 6-9. In general, the differential entropy and enthalpy curl-es followed each other in all the polymers. The absolute values of both enthalpy and entropy functions indicate a highly localized sorption of HzO and DzO on these polymers. However, the values for DzO sorption were lower than for H 2 0 sorption, especially for heparin. This would reinforce the argument presented above that the hydrogen bonding by deuterium is energetically less favorable than by protium and, therefore, the localization of the DzO molecule through
.
(20) P. A. Bsttslhaim and D. E. Volman, J ,Polymer Sa.,2 4 , 445 (1957)
FREDERICK A. BETTELHEIM AND SANFORD H. EHRLICH
1952
- 5 He
K CAL" MOLE
I
I
MOLES
D,O/MOLE
I
I
I
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REPEATING UNIT. I
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3
4
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,i
the (b) type of hydrogen bonding (see above) is less than that of HgO. The magnitude of the maxima in the entropy and enthalpy functions have the following order for HtO sorption: A > heparin > B > C. The entropy function in a swelling polymer must include the change in configurational entropy for both sorbate and sorbent molecule in addition to the entropy value due to the loss of degrees of freedom in the translational and rotational modes of motion of the sorbate. Therefore entropy values are expected in excess of those calculated from the translational and rotational contributions. (Vibrational entropies can be neglected a t room temperatures.20) In view of this fact, one would expect a greater entropy change when the water molecule is sorbed on a polymer which swells with greater difficulty. As Hill, et al., pointed out,21a maximum in the entropy function is indicative of strong binding to the surface and a sharp restriction of the number of possible configurations. I n a polymer which swells with greater diffirulty, the number of available sites is more restricted and probably the sorbed water molecule will come in contact with more polar sorbent groups. For this reason one also would expect maxima in the enthalpy functions. The entropy and enthalpy functioiis of chondroitin sulfate A and heparin hare a greater maximum than chondroitin sulfate B and C. On the basis of the above argument this would indicate that the chondroitin sul(21) T. Hill, P. Emmett, a n d C. Joynw. J . Am. Chem. S o c . , 73, 5102 (1951).
Vol. 67
fate A and heparin chains have strong interchain attractions in the solid state. This conclusion was also reached on the basis of the magnitude of the hysteresis in these substances. Similarly, the largest maximum in the thermodynamic functions from the DzO isotherms is in the case of heparin. This polymer indicated a tightly bound matrix also in the case of mater sorption. There, however, it was compensated by the larger number of polar groups available per repeating unit. According to our argument above these additional polar sites (Le., the additional sulfate groups) will participate to a lesser degree in the D20 binding because of the energetically less favorable 0-D . . . . 0-R hydrogen bonding. Therefore, the larger mechanical constraints in swelling will be less conipelisated by the additional sulfate groups in DzO sorption than in H2O sorption. More than one peak appears in all the thermodynamic functions. A maximum in the entropy function is indicative of the restriction of possible configurations. One could also expect that a surelling process opens up new sites. hccordingly, the appearance of more than one peak in the thermodynamic functions can be interpreted as a sign of stepwise smelling process. A further interpretation of the multiple peaks and their relative magnitude can be as follom: if upon swelling new polar sites open up whicli were iiot hydrogen bonded previously (i.e., interchain hydrogen bonds) one would expect a greater second maximum than the first; on the other hand, if only partial swelling occurs and the subsequent sorption process involves, first, the cleavage of existing hydrogen bonds between chaiiis and then establishment of new ones, one would expect a lesser second maximum in the thermodynamic functions. The occurrence of the thermodynanlic peaks a t the corresponding coverages is summarized in Table I. One can see that chondroitin sulfate A has the smallest amount of coverage before swelling occurs and then a n equal amount of water is sorbed again. In spite of the smallest amount of total coverage in the monolayer, chondroitin sulfate A demonstrates the largest relative increment upon swelling. (Monolayer in this terminology means coverage of the available polar sites with sorbate molecules.) The interpretation of the position of the maxima of the thermodynaniic functions is, therefore, in agreement with those proposed on the basis of the magnitude of the thermodynamic functions, hysteresis, and sorptive capacity considerations. Similarly, the relatively slight increase in sorptive rapacity of chondroitin sulfate C on secondary sites indicates again the relative free swelling character of this polymer. I n addition, this reinforces the arguTABLE I EXTEKT O F S O R P T I O N , hIOLESoF W A T E R / > l O L C S REPEATING UNIT
Sorbent
ChondroitinsulfateA Chondroitin sulfate B ChondroitinsulfateC Heparin
OF DIS4VCHARIDh
At
At
primary sites
secondary sites
Total
B.E.T. monolayer moles HzO/rnoles disaccharide repeating unit
0 5 0 5 0 25 1.0
1 0 1 5 1 75 2.5
7.2 1 9 2 2 2 4
0 5 1 1.5 1.5
Oct., 1963
WATER VAPOR SORPTION O F MUCOPOLYSACCHARIDES
ments advanced by the interpretation of lack of hysteresis in the higher pressure range. The large sorptive capacity of this polymer coupled with the relatively mediocre soription o n primary and secondary sites indicates a greabt amount of solvation of the polymer chain. With respect to heparin the large number of polar sites accommodate the same nuniber of water molecules in the primary sites as in C but heparin shows a greater number of new polar sites opened up by swelling, again in agreement with our previous interpretations. The total amount of water sorbed in the “monolayer” ( L e . , primary and secondary sites) is given in column 4. It is interesting to correlate this with the monolayer from the B.E:.T. interpretation of the isotherms. It is understood that B.E.T. theory cannot be applied to a swelling non-homogeneous surface in which stronger-than-van der Waals forces bind the sorbate. Still the monolayer concept, if it is given only in terms of moles of HzO/mole of repeating unit, is applicable to a certain degree.22 It is evident from Table I that the order of the moiiolayer coverage in the different polymers is the same when obtained from the thermodynamic interpretation and from the B.E.T. theory. The latter values are somewhat higher in most of the cases.
Conclusions The combined results of sorptive capacity, hysteresis effects, thermodynamic functions of sorption, and B.E.T. monolayer indicate that calcium chondroitin sulfate A has the least capadcitytoward water vapor sorption among the investigated mucopolysaccharides. It is (22) L. Pauling, J . Am. Chem. Soc., 67, 555 (1945).
1953
proposed that this effect is mainly due to the strong interchain interactions of this polymer in the solid state and consequently a restricted capacity of swelling. Chondroitin sulfate B which differs from A only in the steric position of a carboxyl group has greater accessibility to water vapors and also less restriction in smelling. Chondroitin sulfate C absorbs the greatest amount of water per repeating unit and has the least restriction to free smelling, going over to a solvation process a t relatively low vapor pressures. These properties of the isomeric chondroitin sulfates may have physiological significance with respect to aging problems, especially in the light of the established composition pattern of tissues such as skin. It was shown by Loemi and MeyerZ3that the embryonic skin has a mucopolysaccharide composition of 20% of chondroitin sulfate C and only 5-12% chondroitin sulfate B. In adult skin the chondroitin sulfate C is only 0.1% of the total mucopolysaccharides while chondroitin sulfate €3 constitutes 64yG. These figures indicate that the change in composition of mucopolysaccharides with age results in a decrease of water holding capacity of tissues. Heparin can sorb a large number of water molecules per repeating unit due to the numerous polar groups available and a t the same time has a restricted capacity for swelling not unlike chondroitin sulfate A. The DzO sorption isotherms showed a lesser sorptive capacity in general and especially in the case of heparin. The thermodynamic data indicate that the hydrogen bonding of D20 is energetically less favorable than that of HzO. (23) G . Loewi and K. Meyer, Baochim. Bzophys. Acta, 27, 456 (1958).
