Micromechanical spectroscopy of cartilage proteoglycans: Hydration Alain Lamure Laboratoire de Physique des Solides Associt au C.N.R.S., Universitt Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Ctdex, France Marie-Franfoise Harmand INSERM U. 306, LIniversitt de Bordeaux 11, 146 Rue Lto Saignat, 33076 Bordeaux CMex, France Colette Lacabanne Laborafoirede Physique des Solides Associt au C.N.R.S., Universitt Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cidex, France Proteoglycan subunits extracted from calf cartilage have been studied with a high resolving power mechanical spectroscopy: the Thermostimulated Creep (TSC). The influence of hydration on TSC spectra shows the existence of two types of bound water: the weakly bound water increases the inertia of proteoglycan and stiffens their structure; the strongly bound water
is responsible to a compensation law indicating the existence of a resonance phenomenon at the physiological temperature. Because of the looseness of bonds in weakly bound water, an increase of the local pressure may induce, in viuo, a release of water i11 tissues. This hypothesis explains perfectly the role of a water pump of proteoglycans in cartilage.
INTRODUCTION
Proteoglycans have a very large number of structures and serve a variety of function in different tissues.’-” Cartilage has been used extensively as a source for proteoglycans. The chemical structure of a typical cartilage proteoglycan is exceedingly complex: glycosaminoglycan (GAG) chains essentially chondroitin surfates-are covalently bound to the core p r ~ t e i n ; ~ ” ~ 75% of those proteoglycans (PG) interact with hyaluronic acid-forming large aggregated4that are visible in electron micro~copy.’~,’~ Age-related variation in the size and chemistry of proteoglycans has been reported;’7-” pathological materials have also shown significant In the cartilage matrix, the collagen fibrils provide an interlaced network and the interspersed proteoglycan aggregates provide, within this network, a hydrated, viscous gel that absorbs compressive l ~ a d . ’ ~On , ’ ~a wet basis, the PG constitute approximately 10% w/w and on a dry basis, up to 50%.27 So, the mechanical properties of connective t i s s ~ e s ’ *are ~ ~highly ~ dependent upon the interaction and association of proteoglycans with water.30It is agreed that water exists in two states: bound and free ~ a t e r . ~One l - ~of~ the Journal of Biomedical Materials Research, Vol. 24, 735-747 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/060735-13$04.00
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LAMURE, HARMAND, AND LACABANNE
most unambiguous ways of detecting the result of macromolecule-water interaction is to follow changes in vapor pressure which are always decreasing when water "binding" occurs. Bettelheim et al.3',32may conclude from such experiments that, in connective tissues, the proteoglycan molecules undergo hydration depending on their overall composition. The core protein participates in the water uptake to a small extent. These authors maintain that, if sufficient chondroitin-4-sulfate chains are present, they can interact with other side chains through Ca2+bridges, hydrogen bond, 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-4sulfate is only a minor component of the side chains, the keratan sulfate side chains create an open network with large swelIing capacity but very little retentive power. More recently, the interaction between water and connective tissue polysaccharides was investigated by using the differential scanning calorimetry (DSC) (Wilfong D. L. and Hiltner A., personal communication). The polysaccharides were examined in the sodium salt, calcium salt, and hydrogen forms. Bound or nonfreezable water content was defined, in this work, as the amount of water which did not freeze when cooled below -50°C. Some of the polysaccharides have been found to exhibit more than one major endothermic peak which suggested different types of freezable and nonfreezable water associated with glycosaminoglycans s a l t ~ .Nevertheless, ~~*~ the published work does not allow precisely placing the origin at a molecular level of the observed energy losses. In order to determine those structure-mechanical properties relationships, we have undertaken a study by a micromechanical spectroscopy: thermostimulated creep (TSC) of proteoglycans in their hydrated condensed state. A characterization of the various hydration levels might be expected from a comparative TSC study of differently hydrated proteoglycans. The TSC technique that we proposed in 1977,35for the characterization of synthetic polymers is derived from the thermostimulated current spectroscopy which has been particularly successful in the area of biopolymers.36-39 Owing to the high resolving power of thermostimulated techniques, it becomes possible to define the various water-proteoglycan interactions by qualitative parameters such as their activation enthalpy.40For comparison, differential scanning calorimetry (DSC) experiments have also been performed on the same initial hydrated condensed proteoglycans. The micromechanical spectroscopy by thermostimulated creep (TSC) has been used in order to shed some light on that point. The aim of this work is to show the evolution upon hydration of molecular movements in proteoglycans.
EXPERIMENTAL
Materials Ethylene diaminetetraacetate acid (EDTA), guanidine-HCL (grade 1)were from Sigma Chemical (Saint-Louis, MO), CsC1, 5-aminohexanoic acid and
MICROMECHANICAL TSC OF PROTEOGLYCANS
737
benzamidine hydrochloride were obtained from Merck (Darmstadt, GDR); Sepharose 2B and Sepharose C1-2B were from Pharmacia Fine Chemicals (Uppsala, Sweden).
Preparation of proteoglycan aggregates and subunits Tissue preparation. Femoral head cartilage from calves 120 days of age were obtained fresh from the local slaughterhouse. The cartilage was dissected free from soft tissues and perichondrium frozen in liquid N2 and ground at liquid N, temperature in a Spex Freeze Mill (2 X 1 min). The resulting powder was stored at -80°C until extracted. Extraction ofproteoglycans. Cartilage powder was suspended in 10 x 10 mL of cold 4 M guanidinium chloride/100 mM phosphate buffer, pH 7.0, containing the protease inhibitors 100 mM EDTA, 100 mM 6-aminohexanoic acid and 5 mM benzaminidium ~hloride.~' Extraction was performed at 2°C for 24 h with gentle stirring. The residue obtained after centrifugation at 1508 was resuspended and again extracted for 24 h as above. The pooled extracts were centrifuged at 20,OOOg for 30 min at 4°C. The residue was washed with the extraction solvent and treated as the above extracts. The supernatants were added to the original extracts. Uronic acid assay4' of a papain digest of the residue showed that 88% of the uronic acid was extracted from the total cartilage. Purification of extracted proteoglycuns. The concentration of guanidinium chloride in the extracts was lowered to 0.4 M by dialysis against 9 volumes of 100 mM phosphate buffer, pH 7.0 (containing the protease inhibitors) for 48 h at 2°C. The density was adjusted to 1.60 g/mL by the addition of CsC1. Equilibrium density gradient centrifugation under "associative" conditions was performed in an 8 X 35 mL angle-head rotor in an MSE Super Speed 65 centrifuge, at 100,OOOg and 10°C for 48 h." After centrifugation, the bottom two-fifths of each tube containing the proteoglycan aggregates (A1 fraction according to the Hascall and Heinegard nomenclature4) was removed by gently aspirating from the bottom of the tube. In order to obtain proteoglycan subunits, A1 fraction was adjusted to 4 M in guanidinium hydrochloride by addition of 1 volume of 7.6 M guanidinium chloride in the same bufferprotease inhibitor solution used initially. The density-gradient centrifugation under "dissociative" conditions was performed under the centrifugal conditions described above. The gradient was then fractionated into four parts and the AID1 fraction, as originally described by Heinegard,@was recovered in the bottom one-fourth. Fraction AlDl was recycled in similar conditions. Highly purified proteoglycan subunits were recovered in fractions AlDlDl at the bottom one-fourth of this last gradient. The AlDlDl fractions were dialyzed at 2°C against double distilled water (without inhibitors) and freeze-dried, Characterization of proteogIycan subunits AlDlDl. The size distribution of proteoglycan monomers AlDlDl was determined by gel chromatography on a Sepharose CL2B (140 X 0.9 cm).*' In both associative (100 mM phosphate buffer, pH 7.0,2"C) and dissociative (4 M guanidinium chloride in the above
738
LAMURE, HARMAND, AND LACABANNE
-
buffer conditions) the AlDlDl fractions eluted as a single broad peak (Kav 0.30) which demonstrates an unimodal and polydisperse repartition of proteoglycan monomers (weight average molecular weight between 1 X lo6 and 3 X lo6 daltons). Protein,46hexuronic galactosamine, and glucosamine&were determined in the AlDlDl fractions. Protein to hexuronic acid ratio by weight was 0.70. The molar ratio of galactose to hexuronic acid and the ratio of glucosamine to galactosamine, which is representative of the keratan sulfate to chondroitin sulfate ratio, were respectively found to be 0.10 and 0.11. Similar findings have been reported for proteoglycans extracted from pig laryngeal cardage," calf nasal cartilage," as well as sheep (0.7 to 6 months) nasal cartilage.51
Sample preparation For Differential Scanning Calorimetry experiments, 3 to 10 mg of freezedried proteoglycan subunits was placed in aluminum pans. For Thermally Stimulated Creep (TSC) experiments on freeze dried proteoglycan subunits, the powder was deposited onto the glass braids used by Gillham for torsional braid analysis.52Such support have a null response in the frequency and temperature range where the thermostimulated creep experiments are performed. For deposition the same procedure was used for all the samples: 100 mg of powder were diluted into 0.1 mL of water at room temperature and the solution was deposited onto a uniformly rotating braid (at 25 rad/min). The sample was placed in the TSC cell where the temperature can vary from liquid nitrogen temperature to 200"C, under inert atmosphere. By loss of weight, the hydration levels of the deposited proteoglycans were estimated to be equal to 50% (by weight). Methods
Differential Scanning CalorimetrylDSC Differential Scanning Calorimehy was used to investigate the transition spectrum of proteoglycans. Thermograms were obtained with a PerkinElmer model DSCII. Standard heating rates were chosen as lO"C/min.
Thermally Sfimulated CreeplTSC Thermally Stimulated Creep was used to study the mechanical retardation spectrum of proteoglycans. Complex TSC spectrum. The principle of TSC is sketched on Figure l.35 A static shear stress cr = u0is applied to the solid state sample at the temperature T,; the corresponding strain will reach an equilibrium value yo. This configuration is frozen by decreasing the temperature to To, where the stress is cut off.
MICROMECHANICAL TSC OF PROTEOGLYCANS
739
Figure 1. Principle of Thermostimulated Creep. Variation versus time t of the stress cr, temperature T,strain y , and strain rate y.
The return to equilibrium of the sample is induced by a controlled increase of temperature. The strain y, the strain rate i, = dy/dt, and the temperature T of the sample are simultaneously recorded versus time t. The plot ? ( t ) generally passes through a maximum for a temperature TM. For the purpose of normalization toward the mechanical stress the reciprocal viscosity has been defined r)-' = y/vo. The peaks of the q-' (T) curve indicate mechanical energy losses due to molecular movements in the sample. Any modification of those movements, for example, any interaction of the macromolecule with water, will induce a modification of the TSC spectra. The TSC spectrum observed in proteoglycans is complex. The great advantage of the TSC technique is to allow experimental resolving of such complex modes into elementary processes: by elementary mode, we mean well described by the hypothesis of a single retardation time. Elementary spectra. Elementary TSC spectra can be experimentally isolated by the following pr~cedure.'~ The stress is applied at Tv,, for t = 2 min. Then the temperature is lowered up to T, = T,, - 10°C, where the stress is cut off and the temperature is kept constant for t. The usual cycle to obtain TSC allows the "elementary" TSC spectrum of the isolated process to be recorded. By shifting To, along the temperature axis, the whole TSC spectrum can be explored. Analysis of elementary TSC spectrum. The Kelvin-Voigt model is used for representing creep behavior. For isothermal creep recovery, the solution for the strain y is: t y = yo exp - 7
where T is the retardation time.
LAMURE, HARMAND, AND LACABANNE
740
In the thermally stimulated creep experiments, this relationship remains valid at a given temperature T. The retardation time is given by:
Temperature dependence of the refardation time. In macromolecular compounds, two types of equations have been proposed for fitting the experimental the Arrhenius-Eyring equation and the Fulcher Vogel equation. They may be introduced unsing, respectively, the thermally activated states theory and the free volume theory. In the thermally activated states theory, the mobile units jump from one equilibrium position to another by crossing a potential barrier defined by the activation enthalpy (AH). The retardation times follow an ArrheniusEyring equation:
(3
T ( T ) = T~~ exp -
where T~~ is the preexponential factor and k is the Boltzmann constant. is a linear function of T-'. Equations (1) and (2) Then, [log -y(T)]/[lj(T)[] give T~~ and AH from a single experiment. In the free volume theory, the mobility is governed by the available fractional free volume f. The basic hypothesis on the temperature dependence off is:
f
=
a(T - T,)
for T > T,
and f=O
for T < T,
where a is the thermal expansion off, T, is a critical temperature below which any molecular movement is frozen. The corresponding retardation times obey a Fulcher-Vogel equation:
T ( T ) = 7"" exp{a(T - Tm)}-'
(3)
where T, is the preexponential factor. If such a critical temperature T, exists, it linearizes the semi-logarithmic variation of Iy(T)/jQ')l with l/(T - T,) in TSC experiments. Then, T , and (Y can be deduced from Eqs. (1) and (3). RESULTS AND DISCUSSION
Differential scanning calorimetry The Differential Scanning Calorimetry (DSC) thermograms of freezedried proteoglycan subunits (AIDIDI)show the existence of two endothermic peaks around 100 and 200°C (Fig. 2). The peak located at 100°C disappears after initial heating, for 1 min, at 150°C of the samples. By analogy with
MICROMECHANICAL TSC OF PROTEOGLYCANS
741
Figure 2. Differential Scanning Calorimetry thermograms of cartilage proteoglycan subunits. Variation versus temperature f of the heat flow dH/dt.
thermal studies of glycosaminoglycans,55polypeptides and collagen,56r57 this peak has been assigned to the loss of bound water. As for the one located around 200"C, it has been attributed to the degradation of proteoglycans. This stability of proteoglycans till temperatures higher than 150°C has been confirmed by biochemical analysis after heat treatment: the proteoglycan molecular weights were tested by Sepharose CL-2B chromatography and the respective amounts of protein hexuronic acid and galactose were measured as described in "Methods." Neither their molecular weight nor their main composition were impaired by the thermal treatment. These observations allow us to determine the experimental protocol to be used for dehydrating this biopolymer: it can be heated, without damage, till 130°C, under inert atmosphere (helium).
Thermally stimulated creep (TSC)
Complex spectra The Thermostimulated Creep spectra of proteoglycan subunits are represented on Figure 3. After deposition on braids described in "Methods," the samples contain about 50% of water by weight: curve (a) corresponds to this initial, very hydrated state. The dehydrated state has been reached after heating the samples at 130°Cfor 1minute and pumping at 0.05 atm for 24 h; then, curve (c) is recorded. Curve (b) corresponds to an intermediate state with some 10% hydration; it corresponds approximately to the physiological concentration. All the spectra have been recorded in the same experimental conditions. The stress was applied for 2 min at the temperature (To= 75°C) indicated by a small arrow on the figure. Any y peak is indicative of mechanical energy losses due to macromolecular movements, i.e., to a higher flexibility of the material. Note that the reciprocal viscosity has been plotted in arbitrary units (a.u).
LAMURE, HARMAND, AND LACABANNE
742
t I----
20
45
PC)
- L A - -
51)
Figure 3. Complex Thermostimulated Creep spectra of hydrated proteoglycan subunits. Variation versus temperature f of the reciprocal of the viscosity 7) in arbitrary units (a.u). (a) -50% hydrated state; (b) intermediate state; (c) dehydrated state.
Spectrum (u) shows two resolved peaks: the low temperature one (LT) located at M"C, and the high temperature one (HT) located at 70°C. During dehydration, the HT mode is shifted towards low temperature (curve b) before disappearing (curve c), while the LT mode remains unchanged (curves u and c). This last mode involves more severe dehydrating conditions for disappearingm it has been assigned to strongly bound water. The former mode that is reversibly shifted along the temperature axis according to the hydration level, has been attributed to loosely bound water. Since, under hydration, this mechanical loss peak is shifted toward higher temperatures, the water molecules stiffen the proteoglycan structure. In collagen, a reciprocal effect is observed: bound water plasticizes molecular movements of polar side chains5' but a behavior analogous to the proteoglycan is observed in polypeptide^.^^^^^ To explain the stiffening of poly-L-proline under hydration, Guillet et al.37have proposed that two water molecules bridge adjacent peptidic groups. To explain the stiffening of proteoglycan subunits with hydration, we propose an analogous mechanism: several water molecules link glycosaminoglycan anionic groups. Because of the weakness of the hydrogen bonds involved for stabilizing such entities, these water molecules might be easily relased in a reversible manner under the effect of pressure. This in vitro model might explain the well known in vivo role of a water-pump insured by proteoglycans.
MICROMECHANICAL TSC OF PROTEOGLYCANS
743
Analysis of the TSC spectra For analyzing the TSC spectrum of dehydrated subunits, it has been experimentally resolved into elementary TSC spectra by applying fractional stresses.53Each elementary spectrum corresponds to the movements of a mobile unit that can be characterized by a retardation time from Eq. (1).The temperature variation of the retardation times associated with the various elementary peaks calculated from Eq. (l),have been reported on an Arrhenius diagram Ln T(T-') (cf. Fig. 4).The dots correspond to the experimental points, the dashed lines have been obtained by extrapolation. For peaks 1 to 6 and 11 to 22, the retardation times are well fitted by an Arrhenius-Eyring equation (Eq. (2)). The retardation times of peaks 11 to 22 have a particular behavior: they all take the same value T~ = 7.107s at a particular temperature T, = 38°C with corresponding error bars defined by:
5 x 107s < Tc (s) < lo8 s and 36°C < T , ("C) < 40°C In other words, the corresponding relaxation times follow a compensation equation: T = T~ exp
AH K
-(TI
-
Clearly, those data arise from relaxation processes which are associated with the same macroscopic event. Physical explanations of analogous compensation phenomena have been proposed: in apatites, the corresponding
Figure 4. Temperature variation of the mechanical retardation times T deduced from the analysis of elementary spectra.
744
LAMURE, HARMAND, AND LACABANNE
dielectric relaxation mode has been attributed to dipolar reorientation rein collagen, it sponsible for the structural monoclinic-hexagonal tran~ition;‘~ has been associated with the glass transition.6oThe above compensation phenomenon in proteoglycans has been assigned to cooperative movements involving loosely bound water. For the peaks 7 to 10, the In T ( T )variations versus T-’ are no longer linear (cf. Fig. 4).In that case, the retardation times have been found to follow a Fulcher Vogel equation (Eq. (3)).The various elementary processes isolated in this temperature range are characterized by parameters that remain of the same order of magnitude. The temperature dependence of those processes always reflects the redistribution of free volume giving rise in this particular case to relative motions of aggregates. CONCLUSION
The micromechanical spectroscopy by thermostimulated creep of hydrated proteoglycan subunits has shown the complexity of the waterproteoglycans interactions. Because of the very low frequency of this technique, the TSC peaks associated with the various types of bound water have been isolated. The observed stiffening of the glycosaminoglycan chains in hydrated proteoglycans might be due to a bridging of anionic groups of the main chains by loosely bound water. The superstructure of proteoglycans ensures the accessibility of the hydration sites for water and, under external pressure, water molecules might be released by breaking of the hydrogen bonds in a perfectly reversible manner. Proteoglycans appear as a natural buffer playing also the role of a water pump. After removal of the above-mentioned water, another retardation mode probably associated with strongly bound water is observed. Its resolution into elementary processes has revealed the existence of a significant amount of free volume: hydrated proteoglycans appear as a particularly loose structure. At higher temperature, another retardation mode is distinguished; the corresponding elementary processes have the same retardation time near the physiological temperature where a resonance phenomenon occurs. This peculiar behavior seems to reflect the specific role of water in biological materials.
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resolution of the retardation time spectrum in polymeric solids by a new method: thermostimulated creep,” 1. Mucromol. Sci. Phys., B13, 537-552 (1977). K. M. Ward, Mechanical Properties of Solids Poiymers, Wiley, London, 1971, pp. 133-165. J. Simon, M. Bihari-Varga, L. Erdey, and S. Gero, ”Thermal decomposition of glycosaminoglycans,” Acta Biochem. Biophys., 3, 365-374 (1978). D. Puett, ”DTA and heats of hydration of some polypeptides,” Biopoiymers, 5, 327-329 (1967). A. Lamure, N. Hitmi, E. Maurel, and C. Lacabanne, ”Etude du vieillissement du collagene par spectroscopie dielectrique tres basse frequence,” Innov. Tech. B i d . Med., 6, 381-392 (1985). A. Lamure, N. Hitmi, M. F. Harmand, E. Maurel, M. Th. Pieragg, and C. Lacabanne, “Etude des mouvements moleculaires dans les tissus calcifies par les techniques thermostimulees,” lnnov. Tech. Biol. Med., 4, 308-327 (1983). N. Hitmi:E. Lamure-Plaino, A. Lamure, C. Lacabanne, and R. A. Young, “Re-orientable electric dipole and cooperative phenomena in human tooth enamel,” Culcif. Tissue Intl., 38, 252-261 (1986). A. Lamure, N. Hitmi, M. F. Harmand, E. Maurel, and C. Lacabanne, “Etude des mouvements moleculaires dans le collagene et les proteoglycannes par spectroscopies dielectrique et mecanique,“ Bull. SOC. Chim.France, 4, 532-534 (1985).
Received March 15, 1989 Accepted November 30, 1989
is responsible to a compensation law indicating the existence of a resonance phenomenon at the physiological temperature. Because of the looseness of bonds in weakly bound water, an increase of the local pressure may induce, in viuo, a release of water i11 tissues. This hypothesis explains perfectly the role of a water pump of proteoglycans in cartilage.
INTRODUCTION
Proteoglycans have a very large number of structures and serve a variety of function in different tissues.’-” Cartilage has been used extensively as a source for proteoglycans. The chemical structure of a typical cartilage proteoglycan is exceedingly complex: glycosaminoglycan (GAG) chains essentially chondroitin surfates-are covalently bound to the core p r ~ t e i n ; ~ ” ~ 75% of those proteoglycans (PG) interact with hyaluronic acid-forming large aggregated4that are visible in electron micro~copy.’~,’~ Age-related variation in the size and chemistry of proteoglycans has been reported;’7-” pathological materials have also shown significant In the cartilage matrix, the collagen fibrils provide an interlaced network and the interspersed proteoglycan aggregates provide, within this network, a hydrated, viscous gel that absorbs compressive l ~ a d . ’ ~On , ’ ~a wet basis, the PG constitute approximately 10% w/w and on a dry basis, up to 50%.27 So, the mechanical properties of connective t i s s ~ e s ’ *are ~ ~highly ~ dependent upon the interaction and association of proteoglycans with water.30It is agreed that water exists in two states: bound and free ~ a t e r . ~One l - ~of~ the Journal of Biomedical Materials Research, Vol. 24, 735-747 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/060735-13$04.00
736
LAMURE, HARMAND, AND LACABANNE
most unambiguous ways of detecting the result of macromolecule-water interaction is to follow changes in vapor pressure which are always decreasing when water "binding" occurs. Bettelheim et al.3',32may conclude from such experiments that, in connective tissues, the proteoglycan molecules undergo hydration depending on their overall composition. The core protein participates in the water uptake to a small extent. These authors maintain that, if sufficient chondroitin-4-sulfate chains are present, they can interact with other side chains through Ca2+bridges, hydrogen bond, 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-4sulfate is only a minor component of the side chains, the keratan sulfate side chains create an open network with large swelIing capacity but very little retentive power. More recently, the interaction between water and connective tissue polysaccharides was investigated by using the differential scanning calorimetry (DSC) (Wilfong D. L. and Hiltner A., personal communication). The polysaccharides were examined in the sodium salt, calcium salt, and hydrogen forms. Bound or nonfreezable water content was defined, in this work, as the amount of water which did not freeze when cooled below -50°C. Some of the polysaccharides have been found to exhibit more than one major endothermic peak which suggested different types of freezable and nonfreezable water associated with glycosaminoglycans s a l t ~ .Nevertheless, ~~*~ the published work does not allow precisely placing the origin at a molecular level of the observed energy losses. In order to determine those structure-mechanical properties relationships, we have undertaken a study by a micromechanical spectroscopy: thermostimulated creep (TSC) of proteoglycans in their hydrated condensed state. A characterization of the various hydration levels might be expected from a comparative TSC study of differently hydrated proteoglycans. The TSC technique that we proposed in 1977,35for the characterization of synthetic polymers is derived from the thermostimulated current spectroscopy which has been particularly successful in the area of biopolymers.36-39 Owing to the high resolving power of thermostimulated techniques, it becomes possible to define the various water-proteoglycan interactions by qualitative parameters such as their activation enthalpy.40For comparison, differential scanning calorimetry (DSC) experiments have also been performed on the same initial hydrated condensed proteoglycans. The micromechanical spectroscopy by thermostimulated creep (TSC) has been used in order to shed some light on that point. The aim of this work is to show the evolution upon hydration of molecular movements in proteoglycans.
EXPERIMENTAL
Materials Ethylene diaminetetraacetate acid (EDTA), guanidine-HCL (grade 1)were from Sigma Chemical (Saint-Louis, MO), CsC1, 5-aminohexanoic acid and
MICROMECHANICAL TSC OF PROTEOGLYCANS
737
benzamidine hydrochloride were obtained from Merck (Darmstadt, GDR); Sepharose 2B and Sepharose C1-2B were from Pharmacia Fine Chemicals (Uppsala, Sweden).
Preparation of proteoglycan aggregates and subunits Tissue preparation. Femoral head cartilage from calves 120 days of age were obtained fresh from the local slaughterhouse. The cartilage was dissected free from soft tissues and perichondrium frozen in liquid N2 and ground at liquid N, temperature in a Spex Freeze Mill (2 X 1 min). The resulting powder was stored at -80°C until extracted. Extraction ofproteoglycans. Cartilage powder was suspended in 10 x 10 mL of cold 4 M guanidinium chloride/100 mM phosphate buffer, pH 7.0, containing the protease inhibitors 100 mM EDTA, 100 mM 6-aminohexanoic acid and 5 mM benzaminidium ~hloride.~' Extraction was performed at 2°C for 24 h with gentle stirring. The residue obtained after centrifugation at 1508 was resuspended and again extracted for 24 h as above. The pooled extracts were centrifuged at 20,OOOg for 30 min at 4°C. The residue was washed with the extraction solvent and treated as the above extracts. The supernatants were added to the original extracts. Uronic acid assay4' of a papain digest of the residue showed that 88% of the uronic acid was extracted from the total cartilage. Purification of extracted proteoglycuns. The concentration of guanidinium chloride in the extracts was lowered to 0.4 M by dialysis against 9 volumes of 100 mM phosphate buffer, pH 7.0 (containing the protease inhibitors) for 48 h at 2°C. The density was adjusted to 1.60 g/mL by the addition of CsC1. Equilibrium density gradient centrifugation under "associative" conditions was performed in an 8 X 35 mL angle-head rotor in an MSE Super Speed 65 centrifuge, at 100,OOOg and 10°C for 48 h." After centrifugation, the bottom two-fifths of each tube containing the proteoglycan aggregates (A1 fraction according to the Hascall and Heinegard nomenclature4) was removed by gently aspirating from the bottom of the tube. In order to obtain proteoglycan subunits, A1 fraction was adjusted to 4 M in guanidinium hydrochloride by addition of 1 volume of 7.6 M guanidinium chloride in the same bufferprotease inhibitor solution used initially. The density-gradient centrifugation under "dissociative" conditions was performed under the centrifugal conditions described above. The gradient was then fractionated into four parts and the AID1 fraction, as originally described by Heinegard,@was recovered in the bottom one-fourth. Fraction AlDl was recycled in similar conditions. Highly purified proteoglycan subunits were recovered in fractions AlDlDl at the bottom one-fourth of this last gradient. The AlDlDl fractions were dialyzed at 2°C against double distilled water (without inhibitors) and freeze-dried, Characterization of proteogIycan subunits AlDlDl. The size distribution of proteoglycan monomers AlDlDl was determined by gel chromatography on a Sepharose CL2B (140 X 0.9 cm).*' In both associative (100 mM phosphate buffer, pH 7.0,2"C) and dissociative (4 M guanidinium chloride in the above
738
LAMURE, HARMAND, AND LACABANNE
-
buffer conditions) the AlDlDl fractions eluted as a single broad peak (Kav 0.30) which demonstrates an unimodal and polydisperse repartition of proteoglycan monomers (weight average molecular weight between 1 X lo6 and 3 X lo6 daltons). Protein,46hexuronic galactosamine, and glucosamine&were determined in the AlDlDl fractions. Protein to hexuronic acid ratio by weight was 0.70. The molar ratio of galactose to hexuronic acid and the ratio of glucosamine to galactosamine, which is representative of the keratan sulfate to chondroitin sulfate ratio, were respectively found to be 0.10 and 0.11. Similar findings have been reported for proteoglycans extracted from pig laryngeal cardage," calf nasal cartilage," as well as sheep (0.7 to 6 months) nasal cartilage.51
Sample preparation For Differential Scanning Calorimetry experiments, 3 to 10 mg of freezedried proteoglycan subunits was placed in aluminum pans. For Thermally Stimulated Creep (TSC) experiments on freeze dried proteoglycan subunits, the powder was deposited onto the glass braids used by Gillham for torsional braid analysis.52Such support have a null response in the frequency and temperature range where the thermostimulated creep experiments are performed. For deposition the same procedure was used for all the samples: 100 mg of powder were diluted into 0.1 mL of water at room temperature and the solution was deposited onto a uniformly rotating braid (at 25 rad/min). The sample was placed in the TSC cell where the temperature can vary from liquid nitrogen temperature to 200"C, under inert atmosphere. By loss of weight, the hydration levels of the deposited proteoglycans were estimated to be equal to 50% (by weight). Methods
Differential Scanning CalorimetrylDSC Differential Scanning Calorimehy was used to investigate the transition spectrum of proteoglycans. Thermograms were obtained with a PerkinElmer model DSCII. Standard heating rates were chosen as lO"C/min.
Thermally Sfimulated CreeplTSC Thermally Stimulated Creep was used to study the mechanical retardation spectrum of proteoglycans. Complex TSC spectrum. The principle of TSC is sketched on Figure l.35 A static shear stress cr = u0is applied to the solid state sample at the temperature T,; the corresponding strain will reach an equilibrium value yo. This configuration is frozen by decreasing the temperature to To, where the stress is cut off.
MICROMECHANICAL TSC OF PROTEOGLYCANS
739
Figure 1. Principle of Thermostimulated Creep. Variation versus time t of the stress cr, temperature T,strain y , and strain rate y.
The return to equilibrium of the sample is induced by a controlled increase of temperature. The strain y, the strain rate i, = dy/dt, and the temperature T of the sample are simultaneously recorded versus time t. The plot ? ( t ) generally passes through a maximum for a temperature TM. For the purpose of normalization toward the mechanical stress the reciprocal viscosity has been defined r)-' = y/vo. The peaks of the q-' (T) curve indicate mechanical energy losses due to molecular movements in the sample. Any modification of those movements, for example, any interaction of the macromolecule with water, will induce a modification of the TSC spectra. The TSC spectrum observed in proteoglycans is complex. The great advantage of the TSC technique is to allow experimental resolving of such complex modes into elementary processes: by elementary mode, we mean well described by the hypothesis of a single retardation time. Elementary spectra. Elementary TSC spectra can be experimentally isolated by the following pr~cedure.'~ The stress is applied at Tv,, for t = 2 min. Then the temperature is lowered up to T, = T,, - 10°C, where the stress is cut off and the temperature is kept constant for t. The usual cycle to obtain TSC allows the "elementary" TSC spectrum of the isolated process to be recorded. By shifting To, along the temperature axis, the whole TSC spectrum can be explored. Analysis of elementary TSC spectrum. The Kelvin-Voigt model is used for representing creep behavior. For isothermal creep recovery, the solution for the strain y is: t y = yo exp - 7
where T is the retardation time.
LAMURE, HARMAND, AND LACABANNE
740
In the thermally stimulated creep experiments, this relationship remains valid at a given temperature T. The retardation time is given by:
Temperature dependence of the refardation time. In macromolecular compounds, two types of equations have been proposed for fitting the experimental the Arrhenius-Eyring equation and the Fulcher Vogel equation. They may be introduced unsing, respectively, the thermally activated states theory and the free volume theory. In the thermally activated states theory, the mobile units jump from one equilibrium position to another by crossing a potential barrier defined by the activation enthalpy (AH). The retardation times follow an ArrheniusEyring equation:
(3
T ( T ) = T~~ exp -
where T~~ is the preexponential factor and k is the Boltzmann constant. is a linear function of T-'. Equations (1) and (2) Then, [log -y(T)]/[lj(T)[] give T~~ and AH from a single experiment. In the free volume theory, the mobility is governed by the available fractional free volume f. The basic hypothesis on the temperature dependence off is:
f
=
a(T - T,)
for T > T,
and f=O
for T < T,
where a is the thermal expansion off, T, is a critical temperature below which any molecular movement is frozen. The corresponding retardation times obey a Fulcher-Vogel equation:
T ( T ) = 7"" exp{a(T - Tm)}-'
(3)
where T, is the preexponential factor. If such a critical temperature T, exists, it linearizes the semi-logarithmic variation of Iy(T)/jQ')l with l/(T - T,) in TSC experiments. Then, T , and (Y can be deduced from Eqs. (1) and (3). RESULTS AND DISCUSSION
Differential scanning calorimetry The Differential Scanning Calorimetry (DSC) thermograms of freezedried proteoglycan subunits (AIDIDI)show the existence of two endothermic peaks around 100 and 200°C (Fig. 2). The peak located at 100°C disappears after initial heating, for 1 min, at 150°C of the samples. By analogy with
MICROMECHANICAL TSC OF PROTEOGLYCANS
741
Figure 2. Differential Scanning Calorimetry thermograms of cartilage proteoglycan subunits. Variation versus temperature f of the heat flow dH/dt.
thermal studies of glycosaminoglycans,55polypeptides and collagen,56r57 this peak has been assigned to the loss of bound water. As for the one located around 200"C, it has been attributed to the degradation of proteoglycans. This stability of proteoglycans till temperatures higher than 150°C has been confirmed by biochemical analysis after heat treatment: the proteoglycan molecular weights were tested by Sepharose CL-2B chromatography and the respective amounts of protein hexuronic acid and galactose were measured as described in "Methods." Neither their molecular weight nor their main composition were impaired by the thermal treatment. These observations allow us to determine the experimental protocol to be used for dehydrating this biopolymer: it can be heated, without damage, till 130°C, under inert atmosphere (helium).
Thermally stimulated creep (TSC)
Complex spectra The Thermostimulated Creep spectra of proteoglycan subunits are represented on Figure 3. After deposition on braids described in "Methods," the samples contain about 50% of water by weight: curve (a) corresponds to this initial, very hydrated state. The dehydrated state has been reached after heating the samples at 130°Cfor 1minute and pumping at 0.05 atm for 24 h; then, curve (c) is recorded. Curve (b) corresponds to an intermediate state with some 10% hydration; it corresponds approximately to the physiological concentration. All the spectra have been recorded in the same experimental conditions. The stress was applied for 2 min at the temperature (To= 75°C) indicated by a small arrow on the figure. Any y peak is indicative of mechanical energy losses due to macromolecular movements, i.e., to a higher flexibility of the material. Note that the reciprocal viscosity has been plotted in arbitrary units (a.u).
LAMURE, HARMAND, AND LACABANNE
742
t I----
20
45
PC)
- L A - -
51)
Figure 3. Complex Thermostimulated Creep spectra of hydrated proteoglycan subunits. Variation versus temperature f of the reciprocal of the viscosity 7) in arbitrary units (a.u). (a) -50% hydrated state; (b) intermediate state; (c) dehydrated state.
Spectrum (u) shows two resolved peaks: the low temperature one (LT) located at M"C, and the high temperature one (HT) located at 70°C. During dehydration, the HT mode is shifted towards low temperature (curve b) before disappearing (curve c), while the LT mode remains unchanged (curves u and c). This last mode involves more severe dehydrating conditions for disappearingm it has been assigned to strongly bound water. The former mode that is reversibly shifted along the temperature axis according to the hydration level, has been attributed to loosely bound water. Since, under hydration, this mechanical loss peak is shifted toward higher temperatures, the water molecules stiffen the proteoglycan structure. In collagen, a reciprocal effect is observed: bound water plasticizes molecular movements of polar side chains5' but a behavior analogous to the proteoglycan is observed in polypeptide^.^^^^^ To explain the stiffening of poly-L-proline under hydration, Guillet et al.37have proposed that two water molecules bridge adjacent peptidic groups. To explain the stiffening of proteoglycan subunits with hydration, we propose an analogous mechanism: several water molecules link glycosaminoglycan anionic groups. Because of the weakness of the hydrogen bonds involved for stabilizing such entities, these water molecules might be easily relased in a reversible manner under the effect of pressure. This in vitro model might explain the well known in vivo role of a water-pump insured by proteoglycans.
MICROMECHANICAL TSC OF PROTEOGLYCANS
743
Analysis of the TSC spectra For analyzing the TSC spectrum of dehydrated subunits, it has been experimentally resolved into elementary TSC spectra by applying fractional stresses.53Each elementary spectrum corresponds to the movements of a mobile unit that can be characterized by a retardation time from Eq. (1).The temperature variation of the retardation times associated with the various elementary peaks calculated from Eq. (l),have been reported on an Arrhenius diagram Ln T(T-') (cf. Fig. 4).The dots correspond to the experimental points, the dashed lines have been obtained by extrapolation. For peaks 1 to 6 and 11 to 22, the retardation times are well fitted by an Arrhenius-Eyring equation (Eq. (2)). The retardation times of peaks 11 to 22 have a particular behavior: they all take the same value T~ = 7.107s at a particular temperature T, = 38°C with corresponding error bars defined by:
5 x 107s < Tc (s) < lo8 s and 36°C < T , ("C) < 40°C In other words, the corresponding relaxation times follow a compensation equation: T = T~ exp
AH K
-(TI
-
Clearly, those data arise from relaxation processes which are associated with the same macroscopic event. Physical explanations of analogous compensation phenomena have been proposed: in apatites, the corresponding
Figure 4. Temperature variation of the mechanical retardation times T deduced from the analysis of elementary spectra.
744
LAMURE, HARMAND, AND LACABANNE
dielectric relaxation mode has been attributed to dipolar reorientation rein collagen, it sponsible for the structural monoclinic-hexagonal tran~ition;‘~ has been associated with the glass transition.6oThe above compensation phenomenon in proteoglycans has been assigned to cooperative movements involving loosely bound water. For the peaks 7 to 10, the In T ( T )variations versus T-’ are no longer linear (cf. Fig. 4).In that case, the retardation times have been found to follow a Fulcher Vogel equation (Eq. (3)).The various elementary processes isolated in this temperature range are characterized by parameters that remain of the same order of magnitude. The temperature dependence of those processes always reflects the redistribution of free volume giving rise in this particular case to relative motions of aggregates. CONCLUSION
The micromechanical spectroscopy by thermostimulated creep of hydrated proteoglycan subunits has shown the complexity of the waterproteoglycans interactions. Because of the very low frequency of this technique, the TSC peaks associated with the various types of bound water have been isolated. The observed stiffening of the glycosaminoglycan chains in hydrated proteoglycans might be due to a bridging of anionic groups of the main chains by loosely bound water. The superstructure of proteoglycans ensures the accessibility of the hydration sites for water and, under external pressure, water molecules might be released by breaking of the hydrogen bonds in a perfectly reversible manner. Proteoglycans appear as a natural buffer playing also the role of a water pump. After removal of the above-mentioned water, another retardation mode probably associated with strongly bound water is observed. Its resolution into elementary processes has revealed the existence of a significant amount of free volume: hydrated proteoglycans appear as a particularly loose structure. At higher temperature, another retardation mode is distinguished; the corresponding elementary processes have the same retardation time near the physiological temperature where a resonance phenomenon occurs. This peculiar behavior seems to reflect the specific role of water in biological materials.
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P. Speziale, M. S. Speziale, L. Galligani, and C. Balduini, ”Interactions between different corneal proteoglycans,” Biochem J., 173, 935-939 (1979). R. L. Stevens, P. G. Dondi, and H. Muir, ”Proteoglycans of the invertebra1 disc,” Biochem. J., 179, 573-578 (1979). P. J. Reihanian, A. M. Jamieson, J. Blackwell, L. H. Tang, and L. Rosenberg, “Structural characterization of proteoglycan subunit from nasal septum by laser light scattering,” ACS Symp. Ser., 150, 201-211 (1981). A. Oldberg, E. G. Hayman, and E. Ruoslahti, “Isolation of a chondroitin sulfate proteoglycan from a rat yolk sac tumor and immunochemical demonstration of its cells surface localization,” J . Bid. Chem., 256, 10847-10852 (1981). P. J. Roughley, D. McNicol, V. Santer, and J. Buckwalter, ”The presence of a cartilage like proteoglycan in the adult human meniscus,” Biochem. J., 197, 77-83 (1981). B. G. Salisbury and W. D. Wagner, ”Isolation and preliminary characterization of proteoglycans dissociatively extracted from human aorta,” J. Bid. Chem., 256, 8050-8057 (1981). R. Kapoor, C. F. Phelps, L. Coster, and L. A. Fransson, “Bovine aortic chondroitin sulfate and dermatan sulfate containing proteoglycans,” Biochem. I., 197, 259-268 (1981). L. Kjellen, I. Petterson, and Hook, ”Cell surface heparan sulfate: an intercalated membrane proteoglycan,” Proc. Nutl. Acad. Sci. U S A , 78, 5371-5375 (1981). J.F. Kennedy, Proteoglycuns- Biological and Chemical Aspects in Human Life. Elsevier, AmsterdamlOxfordiNew York, 1979, pp. 1-28. V. C. Hascall and J. H. Kimura, in Methods in Enzymology, V. Ginsburg (ed.), Academic Press, New York, 1982, pp. 82, 769-800. S. J. Perkins, A. Miller, T. E. Hardingham, and H. Muir, ”Physical properties of the hyaluronate binding region of proteoglycan from pig laryngeal cartilage,” J. MoZ. Biol., 150, 69-95 (1981). D. Heinegard, S. Lohmander, and J. Thyberg, ”Cartilage proteoglycan 175, 913-919 (1978). aggregates,” Biochem. I., L. Rosenberg, From L. Roden, The Biochemistry of Glycoproteins and Proteoglycans, W. J. Lennarz (ed.), Plenum Press, New YorWLondon, 1980, pp. 289-290. S. Inerot, D. Heinegard, L. Audell, and S. E. Olsson, ”Articular-cartilage proteoglycans in aging and osteoarthritis,” Biochem. I., 169, 143-156 (1978). M.T. Bayliss and Y. Ali, “Age related changes in the composition and structure of human articular-cartilage proteoglycans,” Biochem. I., 176, 683-693 (1978). E. J. Thonar and M. B. Sweet, ”Maturation-related changes in proteoglycans of fetal articular cartilage,” Arch. Biochem. Biophys., 208, 535547 (1981). H. G. Garg and D. R. Swam, “Age related changes in the chemical composition bovine articular cartilage,” Biochem. J., 193, 459-468 (1981). T. Reid and M. H. Flint, ”Changes in glycosaminoglycan content of healing rabbit tendon,” J. Embyol. Exp. Morphol., 31, 489-495 (1974). I. R. Dickson and P. J. Roughley, “A comparative study of the proteoglycan of growth cartilage of normal and rachitic chicks,” Biochem. J , , 17l, 675-682 (1978). J. Steinberg, C. D. Sledge, J. Noble, and C. R. Stirrat, “A tissue culture model of cartilage breakdown in rheumatoid arthritis,” Biochem. J., 180, 403-412 (1979). T.R. Oegema, ”Delayed formation of proteoglycan aggregate structures in human articular cartilage disease states,“ Nature, 288, 583-585 (1980).
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LAMURE, HARMAND, AND LACABANNE 25. L. C. Rosenberg, S. Pal, and J. A. Buckwalter, ”Structural changes related to malignancy in proteoglycans from cartilage neoplasms,” Alabama J. Med. Sci., 17, 283-292 (1980). 26. M. B. Mathews and L. Decker, ”Comparative studies of water sorption of hyaline cartilage,’’ Biochim. Biophys. Acfa, 497, 151-159 (1977). 27. A.Maroudas, Adult Articular Carfilage, 2nd Ed., M. A. R. Freeman (ed.1, Pitman Medical, London, 1979, pp. 215-290. 28. G. E. Kempson, H. Muir, S. A.V. Swanson, and M.A. R. Freeman, ”Correlations between stiffness and the chemical constituents of cartilage on the human femoral head,” Biochim. Biophys. Acta, 215, 70-77 (1970). 29. G. Matsumura, ”Rheological studies of proteoglycan,” A.C.S. Symp. Ser., 150, 213-227 (1981). 30. M. B. E. Sweet, E. J. Thonar, and A. R. Immelman, ”Regional distribution of water and glucosaminoglycan in immature articular cartilage,” Biochim. Biophys. Acta, 500, 173-186 (1977). 31. F. A. Bettelheim and B. Plessy, “The hydration of proteoglycans of bovine cornea,” Biochim. Biophys. Acta, 381, 203-214 (1975). 32. F. A. Bettelheim and D. Goetz, ”Distribution of hexosamines in bovine cornea,” Invest. Ophthalmol., 15, 301-304 (1976). 33. Y. Ikada, M. Suzudi, and H. Iwata, “Water in mucopolysaccharides, water in polymers,” A.C.S. Symp. Ser., 127, 287-305 (1980). 34. W.T. Winter, S. Arnott, D. H. Isaac, and E. D.T. Atkins, ”Chondroitin 4-sulfate: the structure of a sulfated glycosaminoglycan,” J. Mol. Biol., 325, 1-19 (1978). 35. J. C. Monpagens, D. G. Chatain, C. Lacabanne, and P. Gautier, ”A new method for the study of molecular motion in polymeric solids: thermally stimulated creep,” J. Polym. Sci. Phys. Ed., 15, 767-772 (1977). 36. J. Guillet, Ph.D. Thesis, University of Lyon (1975). 37. J. Guillet, G. Seytre, D. G. Chatain, C. Lacabanne, and J. C. Monpagens, ”T.S.C. and dielectric study of multiple relaxation in Poly-L-proline,” J. Polym. Sci. Phys. Ed., 15, 541-554 (1977). 38. N. Hitmi, E. Maurel, M.Th. Pieraggi, and C. Lacabanne, Zst Intern. Conf. on Conduction and breakdown in solid dielectrics, Toulouse (1983). 39. N. Hitmi, Ph. D. Thesis, University of Toulouse (1983). 40. A. Lamure, Thesis, University of Toulouse (1983). 41. T.R. Oegema, V. C. Hascall, and D. D. Dziewiatkowski, ”Isolation and characterization of proteoglycans from the swarm rat chondrosarcorma,” J. Biol. Chem., 250, 6151-6159 (1975). 42. T. Bitter and S. W. Sajdera, “A modified uronic acid carbazole reaction,” Anal. Biochem., 4, 330-334 (1962). 43. V.C. Hascall and S. W. Sajdera, ”Protein polysaccharide complex from bovine nasal cartilage,” J. Biol. Chem., 244, 2384-2396 (1969). 44. V.C. Hascall and D. Heinegard, ”Aggregation of cartilage proteoglycans,” J. Biol. Chem., 249, 4232-4241 (1974). 45. M. F. Harmand, R. Duphil, and P. Blanquet, ”Proteoglycan synthesis in chondrocyte cultures from osteoarthrotic and normal human articular cartilage,” Biochem. Biophys. Acfa, 7l7, 190-202 (1982). 46. D.H. Lowry, N. J. Rosebrough, A . L. Fan, and R. J. Randall, ”Protein measurement with the Folin phenol reagent,” ]. Biol. Chem., 193, 265275 (1981). 47. J. R. Helbert and K. D. Brown, ”Color reaction of anthrone with monosaccharide mixtures and oligo-and polysaccharides containing hexuronic acides,” Anal. Chem., 29, 1464-1466 (1957). 48. C. P. Tsiganos and H. Muir, ”Studies on protein polysaccharides from pig laryngeal cartilage,” Biochem. J., 113, 879-894 (1969). 49. T. E. Hardingham and H. Muir, “Hyaluronic acid in cartilage and proteoglycan aggregation,” Biochw. ]. , 139, 565-581 (1974).
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Received March 15, 1989 Accepted November 30, 1989
