The Polyelectrolyte Properties of C. P. Winlove K. H. Parker A. R. Ewins N. E. Birchler Physiological Flow Studies Unit, Centre for Biological and Medical Studies, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom
E
l
a
H
lastin
The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.
Introduction Elastin is widely regarded as a particularly inert protein. form of both rings and fibrils obtained by grinding dried samHowever, accumulation of lipids and calcium in the elastic ples in order to assess the effects of the method of preparation lamellae is one of the earliest manifestations of atherosclerosis on charge structure. Since radiotracer studies suggest that [1,2] and the performance of elastin used in vascular prostheses stretching elastin increases the number of sites of ion binding is frequently compromised by similar interactions [3]. There [15] experiments were also performed on rings of elastin subject have been a number of studies on the binding of lipids [4], to mechanical strain. The enthalpies of ionization of the charge ions [5-9], and macromolecules [10] including enzymes [11] to groups were determined directly by means flow microcalosoluble and fibrous elastin in vitro, but in very few cases have rimetry. the sites and mechanisms of interaction been determined. Even We investigated ionic interactions by comparing titration the most extensively investigated problem, calcium binding, is curves obtained in media of different salt composition, by still not fully resolved. It has been shown that calcium interacts measuring the displacement of hydrogen ions by salts added both electrostatically and, under appropriate conditions, at a to the bathing solution and by measuring the uptake of raneutral site [8, 9] but the relative importance of ionic and diolabelled ions by elastin. The potentiometric and radiotracer neutral site binding under physiological conditions is unclear. techniques are complementary in the ranges of ionic concenThe present study was undertaken in the belief that the trations over which they are applicable and in the information reactivity of elastin might be clarified by a better understanding they provide. The former experiments reflect largely electroof the nature and density of charged groups on fibrous elastin, static interactions [16] whilst the latter experiments also detect their reactivity towards small ions and their role in determining nonelectrostatic interactions. the structure of fibrous elastin. We also investigated the role of electrostatic interactions in Biochemical analysis shows that the elastin molecule con- determining the conformation of the matrix using a simple tains both anionic and cationic groups but the degree of ion- picnometric technique. Changing the pH or ionic composition ization of these groups and their accessibility in elastin fibers . of the bathing solution changes the conformation of soluble has been established only for elastin derived from the liga- elastin [17] and by establishing whether similar changes occur mentum nuchae [12-14]. Our potentiometric titration meas- in the fibrous protein we should obtain insight into the interurements were performed on porcine arterial elastin isolated actions occurring between elements of the elastin matrix. by alkali, formic acid, or cyanogen bromide extraction in the Methods Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENGINEERINO. Manuscript received by the Bioengineering Division June 28, 1991; revised manuscript received March 17, 1992. Associate Technical Editor: M. Friedman.
Materials. Elastin was prepared by sodium hydroxide extraction or cyanogen bromide digestion of the thoracic aortas of young pigs as detailed elsewhere [18] or by formic acid
Journal of Biomechanical Engineering
AUGUST 1992, Vol. 114/293
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extraction [19]. The material was returned to neutral pH with HCI or NaOH and washed very extensively with deionized water before use. The elastin was used either as rings 1-2 cm in width or after drying, grinding to a fine powder consisting of particles 200-800 fim in diameter and rehydrating. The amino acid composition of a sample of the alkali digest was determined by Dr. J. Powell, Department of Biochemistry, Charing Cross Hospital. Reagents were prepared by standard methods using deionized water and Analar grade chemicals. Titration Experiments. pH measurements were performed with a glass electrode (Orion, model 8103SC) connected to an electrometer (Keithley 614). The electrode was calibrated in standard buffer solutions (pH 2.00 and 12.00, Aldrich Chemicals, pH 4.01, 7.00, 9.19 Orion Research) before and after each series of measurements. The preparation to be titrated, consisting of a sample of elastin with a dry weight of approximately 500 mg and 20 ml of degassed solution, was placed in a glass vial in a water bath thermostated to 25.0 ± 0.20°C and stirred by a gentle stream of nitrogen bubbles. Before the experiment, the elastin was hydrated and equilibrated with several changes of solution for at least 48 h until a constant pH was attained. Titrant was added by micropipette and pH was recorded after equilibration. Equilibration was almost instantaneous at the extremes of pH but between pH 6 and 8 the voltage usually overshot initially and equilibration could take up to 25 min. We were unable to establish whether this behavior reflects differences in the accessibility of ionic groups or their rate of reaction or is due to conformation changes in the elastin. To study the effects of strain, a small rig was constructed containing two parallel glass rods 5 cm long and 3 mm in diameter whose separation could be varied by a locking screw mechanism. An elastin ring was looped over the rods and extended to the chosen strain relative to a reference length which was defined as the position at which the sides of the elastin ring first became parallel. Only the glass part of the apparatus came into contact with the solution during a measurement. In order to investigate the effects of changes in the ionic composition of the bathing solution on hydrogen ion binding by elastin, pH changes resulting from the addition of 20 ^1 aliquots of 0.01 M salt solution were measured. Calibration experiments were conducted using buffer alone. Titration curves were computed from the equation: K([acid] - [base] + [OH"] - [H + ]) *~ J*ll-[OH"]) where Hb is the number of moles of H bound per g dry weight of elastin, V is the volume of the bathing solution, W is the dry weight of elastin and [acid], and [base] are the moles of acid or base added per unit volume of the solution. [H + ] = 10" pH and [OH"] = 10" 1 4 + p H are the measured concentrations of H + and O H " ions in the solution. (Corrections were made for the activity coefficients of the hydrogen and hydroxyl ions, determined from the titration curves of water blanks under the assumption that the coefficients were unaffected by the presence of protein [20].) We have assumed that after the initial washing procedure the protein was at its isoionic point and adopted this as the reference state [21]. In deionized water, the isoionic point was pH = 6.93 ± 0.82 averaged over all alkali extracted preparations. At this pH, a variation of 0.82 pH units is equivalent to an Hft of about 0.1 /xmoles/g dry weight which represents a negligible uncertainty in the ordinate of the titration curve. The difference in isoionic point in the various salt solutions, discussed below, was also not significant in this context. Radiotracer Uptake Experiments. Rings of elastin approximately 1 cm wide were weighed in air and then suspended 294 / Vol. 114, AUGUST 1992
in 200 ml of the required medium containing the radiolabelled ion and incubated at room temperature for 4 h with constant stirring. Preliminary experiments showed that this period was sufficient to attain equilibrium. After the incubation period the tissue was either counted immediately or washed in several changes of buffer until no more radioactivity appeared in the washing solution. Samples for 7-counting were counted immediately together with aliquots of the incubation medium and then dried to constant weight. Samples for ^-counting were solubilized in Optisolv (Pharmacia) and HiSafe 3 (Pharmacia) was used as scintillant for both tissue and medium samples. Control experiments were undertaken to provide a correction factor for the tissue quenching of ^-activity. Because the quench correction was rather large, particularly for 45Ca, some experiments were repeated using only a 10 ml volume of tracer and uptake was calculated from the loss of radioactivity from the bathing medium. Both procedures gave comparable results. Solutions containing 22 NaCl, Na 36 Cl, Na 125 I, Na 2 35 S0 4 , and 45 CaCl2 at concentrations of 0.15M, 0.05M, and 0.01M and pHs (adjusted by addition of acid or alkali and unbuffered) of 2.5, 7.0, and 10.5 were employed. A series of experiments was also undertaken with rings of elastin extended to 30 percent strain in the apparatus described above. Tracer uptake, U, was defined as: counts/g dry weight counts/^mole of ion in the medium where the dry weight was determined directly for the experiments on -/-emitting isotopes and for the /3-emitters was calculated from the wet weight and mean water content of matched specimens. For unwashed tissue the measured radioactivity arose largely from tracer contained within the water spaces of the elastin. Because of electrostatic interactions the ionic composition of this fluid was not necessarily identical to that of the bathing solution and it was impossible to distinguish between ions "free" in this water space and those "bound" to the elastin fibers. It is tempting to refer to the tracer remaining after extensive washing as being "irreversibly" bound. However, this definition is also imprecise because radioactivity in the washing tended only asymptotically to zero. These difficulties confound comparisons with other data in the literature and dictate that uptake values be regarded only as comparative indicators of the behavior of different ions. In an attempt to obtain a more rigorous measure of interactions, ground elastin was also used as a substrate for affinity chromatography [32]. A suspension of elastin fibers was packed into a 12 cm by 1 cm column [Pharmacia]. The column was equilibrated with eluate at a flow rate of 8 ml/hr generated by a peristaltic pump [LKB] at the column outlet. 1-2 ml aliquots of radiolabelled solution of known concentration and specific activity were applied to the column. Eluate was collected and counted. The quantity of radioactivity retained on the column after eluate activity had fallen to background was taken to be the irreversible binding capacity of the elastin. The amounts of radioactivity removed by washing with solutions of different ionic composition or pH were also measured. At the end of a series of experiments, the elastin was quantitatively recovered from the column and its dry weight determined so that binding could be expressed as /u,moles/g dry weight. Calorimetry. Approximately 50 mg of dry, ground elastin were packed in the chamber of a flow microcalorimeter (Microscal, Model 3S) as described elsewhere [23]. Solution flowed through the chamber at a rate of 3.3 ml/h and the heat change following a step change in solution pH was recorded. In order to avoid the effects of changes in ionic composition which would accompany the use of pH buffer solutions these experiments were performed using degassed, deionized water whose pH was adjusted by small additions of NaOH or HCL. Transactions of the ASME
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The enthalpy changes resulting from changes in Na + or Cl~ concentration were negligible in comparison with the heats of ionization [23]. Enthalpies of ionization were derived from the areas under the heat-time curves. Density Measurements. A ring of elastin was attached to a clip on one arm of a lever arm balance. The sample was immersed in a beaker of deionized water and its apparent weight was measured (M„) after equilibration. The beaker of water was then removed and the specimen was carefully blotted dry and weighed in air ( M J . The elastin ring was then reimmersed in a beaker filled to the same level with the test solution, allowed to equilibrate and the weighing procedure was repeated (Mj). When the required cycle of solution changes was completed the elastin ring was immersed in deionized water to allow any solutes to diffuse out and then dried for 72 hr at 60 °C and its dry weight was measured (Me). The densities of the test solutions (pw and ps) were measured with a specific gravity bottle. All weighings in solution were corrected for the buoyancy of the clip assembly. The volume (Ve) and density (pe) of the elastin, the initial water space (V„) and the solution space after equilibration with test solution (Vw) were calculated from the following formulae: Ve=(Me-Mw)/pw Vw=(Ma-Me)/Pw pe =
pwMe/(Me-Mw)
Ms-Me + peVe V =(Pw-Ps)
Results (1) Analysis of Titration Curves. Our titration curves (Fig. 1) were similar to those obtained for ligamentum elastin [12-14]. We normally confined measurements to the range pH 2 to 11.5 where the curves were reversible. Most curves reached a plateau at pH « 2 but occasionally we found an increase in Hb at low pH due either to strongly acidic or weakly basic groups or degradation of the substrate. Though earlier work on collagen favoured the former explanation [24], we found that long exposure to very low pH does solubilize elastin. We could not detect a plateau at the basic end of the curve.
-400
Fig. 1 Titration curves of alkali extracted aortic elastin in different salt solutions. (o )-deionized water, (o )-.15 M NaC1, ( • )-.15 M CaCI2, (o )-.15 M Nal.
Journal of Biomechanical Engineering
The attribution of different portions of the titration curve to specific groups has been discussed by many authors [e.g., 20,25]. We assume that the region from pH 2 to 6.25 represents e-carboxyl groups, pH 6.25 to 8 corresponds to imidazole groups, and that from pH 8 to 11 all basic groups other than guanidyl are titrated. With these assumptions there is satisfactory agreement between titration results and biochemical analysis for alkali extracted elastin. The amino acid composition was very similar to that previously reported for this type of preparation {26]. Titration showed 120 ± 1 5 /xmoles carboxyl groups/g dry weight and the total of glutamine/glutamic acid and asparganine/aspartic acid was 200 /xmoles/g dry weight. Given the uncertainty about the forms of glutamate and aspartate this agreement seemed satisfactory and suggested that only a few of the carboxyl groups are masked in the fibrous protein and that, as for collagen, there are few a-carboxyl groups. However, compensatory intrafibrillar interactions between carboxyl and amino groups have been suggested to occur in ligament elastin [12] and we cannot discount this possibility in our preparation. Between pH 6.25 and 8 only the imidazole groups of histidine are titrated and their number, 17 ± 4 /xmoles/g dry weight is reasonably consistent with the 30 detected by amino acid analysis. The greatest problem is the interpretation of the portion of the curve beyond pH 8. The pKs of the e-amino groups of lysine, the phenolic groups of tyrosine and the a-amino and imino groups of proline and hydroxyproline lie close together and overlap the guanidyl groups of arginine [20]. The problem of attribution has been discussed for collagen [21], and our difficulties were further compounded because the number of titratable groups was very sensitive to the ionic strength of the medium. Adopting the usual upper bound of pH 11.5 for the titration of e-amino and phenolic groups gave more charges than detected biochemically but with an end point of 10.5 gave good agreement with the total tyrosine and lysine content (19 biochemically against 16 ± 4 from titration). Over the chosen range of carboxyl group titrations, a plot of log (a/1 - a ) versus pH, where a is the degree of dissociation, was linear (Fig. 2) which provides some confidence in the attribution of the titration results to a single group. The linearity also suggested that electrostatic interactions were constant over the titration range, (i.e., they were dominated by the basic groups). Strong electrostatic interactions were presumed to be responsible for the pK of 3.5 ± 0.2, which is much lower than the value of 4.6 found for side-chain carboxyls in small molecules [16] and significantly lower than the value of 4.1 found for collagen [21]. In the basic region satisfactory linearity was achieved by assuming titration of phenolic groups was complete at pH 10.5 and the pK of 10 was close to the accepted value [16]. The region between 9.5 and 11.5 was also linear, but the pK ~ 11 was higher than that expected for sidechain amino groups [16] and so this portion of the curve may be affected by interference from more basic groups.
(2) Enthalpies of Ionization. Typical enthalpy time curves for step changes of the pH of the solution from 6.2 to 12.5 and back are shown in Fig. 3. The initial peak occurred within a few minutes, as expected from the rate of mixing of solution in the calorimeter. In some experiments enthalpy changes continued for many minutes. This may arise from the buffering capacity of the elastin or changes in conformation of the elastin which occur on this time scale. Figure 4 summarizes enthalpy measurements on 2 samples of alkali extracted elastin. The greatest heat changes were associated with ionization of the basic groups. The dotted line in Fig. 4 is the enthalpy change predicted from our titration data assuming that the carboxyl, imidazole, and e-amino groups make the dominant contribution and that their enthalpies of ionization are those measured for collagen [21]. Very good AUGUST 1992, Vol. 114/295
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AQ
pH
(mW/g)
/
\
pH 6.2 -> pH 12.5
10
t (min) 30
V _ _ ^ ^ ^ ' p H 12.5 -* pH6.2 11 pH
Fig. 3 Calorimeter trace showing the rate of heat release with time following a step change in the pH of the perfusing solution. Top curve: pH 6.2 - pH 12.5, Bottom curve: pH 12.5 - pH 6.2.
10
AH (J/g)
PH
10
12
PH
Fig. 4 Net heat release following step changes in the pH of the perfusing solution calculated from the area under curves like those in Fig. 3. The arrows indicate the direction of the pH change. The dotted line is the predicted net heat release using heats of ionization for charged groups obtained for collagen [21] and our estimates of the numbers of charged groups in elastin.
'(^) Fig. 2 Plots of pH versus ln(, is the number of salt ions Transactions of the ASME
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Table 1 Uptake of Ions by alkali digested elastin Otmoles/g dry weight) Na +
Ca 2 +
CI"
20±2 21 ± 2 29±2
19±1 24±2 27 ± 2
110±10 32±3 20±2
210±6 54±3 54±3
94 ±16 16±3 16±3
300 ± 3 0 330±40 350 ±30
270 ± 4 0 380 ± 5 0 370 ± 3 0
440 ±30 380±10 360±10
450 ±15 490 ±50 620 ±30
500 ±90 330 ±70 350 ±70
pH .01 M salt 2.5 7.0 10.5
so^
. r
.15 Msalt 2.5 7.0 10.5
100
c
150
(iM)
Fig. 5 The change in H,, with the addition of CaCI2, NaCI2, and Cal2. The top curves are for an initial pH of 2.5, the bottom curves are for an initial pH of 10. The data are corrected for salt effects on the measured pH of the water.
1
of charge z, "bound" to the protein [16]. The parameter A depends on the geometry of the fixed charges and because this is unknown for the elastin matrix, it is not possible to use the equation quantitatively. However, the sign of the pH change indicates that more anions than cations are bound. The smaller change produced by CaCl2 is also expected because of the divalency of the cation. In deionized water the fixed charge density at the isoionic point was approximately - 6 x 10 ~3 /xmoles/g dry weight. Changes of H6 following small additions of salt are shown in Fig. 5. At acid pH the addition of salt increased the amount of hydrogen binding. Sodium and calcium salts had similar effects when allowance was made for the difference in anion concentration. The nature of the anion seemed to be the major determinant of the degree of dissociation, chlorides being much more effective than iodides. We presume that the increase in Hfc arises because anion binding to basic groups, as has been reported for a-elastin [28], allows increased electrostatic attraction for H + to the elastin. This clearly dominates any interaction of cations with acidic groups or neutral sites which would tend to reduce ¥Lb. Under alkaline conditions addition of salt caused a release of bound hydrogen, as expected from the change in the titration curves. This, too, is presumably due to anion interaction because although Ca2+ binding to phenolic groups and incomplete ionization of Ca salts of carboxylic acids could occur [29], neither would account for the anion dependence we observed. (b) Radiotracer Uptake Experiments. Tracer uptake for different ions at different pHs and ionic strengths are given in Table 1. The measured uptake reflects tracer "interacting" with the elastin and ions "free" in the water pool. As discussed in the Methods section, these contributions cannot be distinguished either experimentally or theoretically. The distribution in the intra and extrafibrillar water spaces depends on the electrostatic field produced by the fixed charges on the elastin. This interaction was present under all the conditions we investigated, as demonstrated by the fact that the uptake of both anions and cations was always smaller than that calculated by assuming uniform distribution throughout the water space. Since the Debye length is approximately 0.8 nm in 0.15 M NaCl, this is evidence that at least some charges are present around hydrated pores of similar dimensions. Journal of Biomechanical Engineering
0 -c pH
Fig. 6(a) The change in water volume with changing pH for alkali extracted elastin. The starting point for each sample is indicated by the closed dot. .18 M
AV„/V„
NaCl
NaCl CaCl 2
CaCl 2 Nal
Nal
Fig. 6(D) The percentage change in water volume following a change in the solution from deionized water to .15 M and 1.5 M NaCl, CaCU, and Nal Fig. 6 The effect of pH and ionic composition of the solution on elastin water content
Both cations showed a slight increase in uptake with increasing pH which was not accounted for by changes in water content (see Fig. 6). This is consistent with earlier observations which were attributed to interaction with carboxyl groups [6]. The continuation of the change beyond pH 7 suggests that changes in the ionization of the basic groups are also involved [13]. The similarity in the behavior of Na+ and Ca2+ indicates that neutral site binding makes a minor contribution to uptake under these conditions [23]. Anion uptake shows the reverse dependence on pH and the ionization of the carboxyl groups AUGUST 1992, Vol. 114 / 297
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is clearly the major determinant of uptake. Sulphate uptake was smaller than that of the halides and there was a significant difference in uptake between chloride and iodide. Iodide uptake is approximately two-fold greater despite its small effect on titration curves and its inefficacy in displacing bound hydrogen. This difference in reactivity was also observed in experiments using a medium containing both ions which showed preferential uptake of iodide which was reduced only by great excesses of chloride. Uptake of cations increased approximately linearly with concentration up to 0.15 M. Anions showed more complex concentration dependence. Chloride and sulphate uptake was proportional to concentration at pH 7 and 10.5 but iodide uptake increased more slowly with concentration. At pH 2.5 uptake of all three anions was nonlinear and appeared to be saturable. The apparent fixed charge density, {Uinion - t/ calion )C bu i k) varied with ionic species and ionic strength and was not consistent with the fixed charge density determined by titration. This could reflect the peculiarities of the ionic distribution or nonelectrostatic interactions. Experiments on the washout of radioactivity from the elastin rings showed that —4.5 /xmoles of calcium/g dry weight could not be removed, but all of the sodium could be removed by the washing procedure. These observations were consistent with experiments with the elastin packed column which showed that at neutral pH calcium binding was —3.7 /imoles/g dry weight. None of the radioactivity was removed by increasing the pH of the eluate to 10.5 or by reducing it to 2.5. No sodium binding could be detected throughout the pH range. At neutral pH chloride binding was —4.1 ^moles/g dry weight and all of the radioactivity was recovered on increasing the pH to 11. At an initial pH of 2.5, chloride binding was reduced to 1.9 /xmoles/g dry weight and all of this material was removed by reducing the pH to 2.0. (4) Effects of Elastin Preparation. Four preparations of alkali-extracted elastin were made using different batches of aortas. Two of these batches of aortas were also used to prepare CNBr digests. For each type of digest the titration curves were qualitatively similar but quantitative differences were sufficient to give rise to standard errors of the means of up to 20 percent. It is not clear whether the variations arise from differences in the composition of the native elastin, the efficiency of removal of other components of the tissue or modifications occurring during preparation. For a single preparation of elastin no difference was detected between rings and ground elastin. The difference in numbers of ionizable groups between the alkali digest and CNBr preparation were no greater than the batch to batch variation in either preparation. Biochemical analysis shows that CNBr digestion produces a smaller number of rt-terminal residues and that hydrolysis leads to a loss of cross-linking amino acids [30, 31]. Our results suggest, however, that the effects are quantitatively small. There was no significant difference in pK for the basic groups, but the acid branch of the titration curve was generally rather steeper for the CNBr digest, perhaps indicating a change in conformation in the vicinity of the carboxyl groups. Radiotracer measurements showed only small differences in uptake between elastin preparations which could be explained by the differences in water content. (5) Effects of Strain. Initial experiments were conducted at 50 percent strain but the elastin tended to rupture at extreme pH and so later experiments were performed at an extension of 30 percent. The cause of rupture has not been investigated further. There was no difference in the number of titratable groups but the pK of the carboxyl groups of stretched elastin was shifted approximately 0.2 units towards the acidic in deionized water. Smaller shifts were observed in salt solutions. The change in proton binding was also reflected in a change in 2 9 8 / V o l . 114, AUGUST 1992
solution pH as the elastin was stretched. In deionized water at the isoionic point the change in pH corresponded to a change of - .036 ± .020 /xmoles H bound/g dry weight but was almost a thousand fold greater at an initial pH of 3.0. The change was slowly reversible as the elastin was allowed to relax. These results suggest that the conformation changes associated with stretching of the elastin modify charge-charge interactions principally of the carboxyl groups. Strain increased cation uptake and decreased anion uptake as we have reported previously [15]. The effect was greatest at high pH and low ionic strength, where the apparent fixed charge density reversed in sign, going from 25 to - 19 /unoles/ g dry weight. Although stretching produced a small water loss from the tissue its contribution was negligible. The titration experiments indicate that the effect does not arise from the unmasking of charged groups. Furthermore, since experiments on the displacement of protons by the addition of salt gave similar results in stretched tissue, the effects are not due to changes in competitive interactions. We presume, therefore, that they are due to larger length scale changes in the geometry of the electrostatic field. (6) Effects of pH and Solution Composition on Matrix Structure. Preliminary experiments showed that sample weight changed for up to 24 hours following a change in the composition of the surrounding medium. We have noted such long time constants in earlier experiments on the mechanochemical properties of elastin [18] and similar observations have been made on swelling collagen [32] where they were attributed to changes in the cohesion of the fibres. The effects of changing pH on the water content of several samples of elastin are summarized graphically in Fig. 6. Samples differed in initial water content. There was also some variation in the initial density of the solid elastin component, but this remained constant as pH varied. The water content of the elastin was a minimum at or slightly above the isoelectric pH. There was very little swelling at low pHs but a rapid increase in volume above pH 10, indicating that the degree of ionization of the most basic groups is an important determinant of matrix conformation. These observations are consistent with a study of the effects of pH on the mechanical properties of elastin where the decrease in unstressed length at pH 2 relative to the reference conditions at pH 7 was not significant but there was a large increase at pH 11. At both extremes of pH there was an increase in the elastic modulus [18], In the present experiments the effects were qualitatively reversible when the pH changes were reversed. There was some hysteresis due either to lack of elastic forces to aid rehydration of the tissue or to chemical modifications produced by the long exposure to extremes of pH. The data in Fig. 6(a) were obtained using deionized water. In salt solutions the changes were similar but smaller in magnitude. Changes in salt concentration at constant pH also changed the water content of the tissue as illustrated in Fig. 6(b). NaCl and CaCl 2 both caused the matrix to shrink. The effect was small in 0.15 M solution, CaCl2 having the larger effect as expected because of its higher ionic strength, but increased with salt concentrations up to about 1.5 M. This behavior is consistent with the elastin network being held apart by electrostatic interactions which are shielded by the ionic solutions. Furthermore, since the Debye length in 0.15 M NaCl is —0.8 nm, we may infer that the functional charges are distributed on this length scale. Iodide has a very different effect on matrix structure, causing swelling even at low ionic strengths. The process is reversible in the sense that replacement of iodide by chloride caused shrinkage to a similar volume to that attained without prior exposure to iodide, but further work is required to clarify the underlying mechanisms. Studies on the swelling of collagen and gelatin [32] show it to be qualitatively similar to the behavior we report. The data Transactions of the ASME
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have been fitted to a simple model in which osmotic swelling is opposed by stress within the collagen matrix. The conclusion that the "bulk modulus" of the matrix was a strong function of pH appears, however, to cast some doubt on the utility of the model. Discussion Titration and uptake measurements made on soluble proteins [e.g., 16, 17] provide a much more detailed description of charge structure than we were able to obtain with similar measurements for elastin. The major source of uncertainty for fibrous elastin is the existence of a number of water "pools" within its structure. In addition to the extrafibrillar water spaces of microscopic dimensions, a significant fraction of the water in vascular elastin is contained in intrafibrillar pores accessible only to molecules < 1000 Daltons [33]. The distribution of ions between these compartments will depend on the fixed charge structure of the elastin and its conformation, the size and charge of the ion, and the pH and ionic strength of the bathing solution. This introduces difficulties into the interpretation of both titration and ion binding experiments which cannot be adequately addressed without a detailed model of the structure and charge distribution of the elastin matrix. Though a model has been developed for the collagen fibril [34] which formed a satisfactory framework for the interpretation of experimental data on ionic interactions, the nature of the assumptions which would have to be incorporated into a model of elastin coupled with the uncertainties in our experimental data suggest that it would not represent a significant advance. One of the most striking observations in the present study was the affinity of elastin for anions. Iodide is well known to interact with proteins [27] and binding of chloride to a-elastin has been reported previously [28] and our work extends this observation to the fibrillar protein. It also shows considerable differences in the behavior of chloride and iodide which we have also observed calorimetrically [23], The reason for this difference is unclear and it could be explained by steric considerations only if the interaction involved bare rather than hydrated ions. It might be valuable to investigate the binding of phosphates and anionic macromolecules such as glycosaminoglycans and glycoproteins and degradative enzymes whose interactions with elastin are of physiological or pathological importance. Because of the wide variety of definitions of "binding" which have been employed, it is difficult to make a quantitative comparison between our data on cation binding and the earlier literature. However, over our range of experimental conditions, sodium and calcium behaved in a very similar manner suggesting that the behavior of calcium was dominated by electrostatic interactions rather than by neutral site binding [8]. The relative importance of the two types of interaction depends strongly on the conformation of the elastin. The effects of changes in conformation produced by altering the composition of the solvent have been described [9, 23], but it might be useful to repeat some of our experiments on immature or degenerated elastin to explore the effects of naturally occurring variations of conformation. Vascular remodeling in response to changing haemodynamic conditions and links between haemodynamics and atherogenesis have both been extensively documented [35]. These observations imply complex mechanochemical couplings in the vascular interstituum and our results suggest two ways in which elastin may be involved. On the one hand, changes in the composition of the surrounding medium influences the dimensions and, as we have shown in earlier experiments [18], the mechanical properties of the elastin network. On the other hand, strain influences the apparent density and pKs of the ionizable groups, perhaps influencing the interactions of elastin with other molecules or its susceptibility to proteolytic degJournal of Biomechanical Engineering
radation. Similar behavior is reported for collagen, where the unmasking of charged groups is believed to be responsible for its strain dependent reaction to Masson's trichrome stain [36]. We have found in unpublished experiments that the staining of elastin with Masson's trichrome is also strain dependent, but the colouring is the reverse of that of collagen (i.e., the relaxed tissue is stained red and the stretched tissue green). The mechanism for elastin might be more complex because it has quite recently been shown that for elastin peptides and similar molecules, changes in hydrophobic interaction rather than changes in electrostatic interaction can give rise to shifts inpK [37]. We showed that the polyelectrolyte properties of elastin are not particularly sensitive to the method of preparation. The possibility remains, however, that they are greatly modified by the other components of the extracellular matrix which are intimately associated with elastin in the intact vessel. This possibility must be examined in the future by experiments using more complex tissue preparations.
Acknowledgments We would like to thank Dr. J. Powell, Departments of Surgery and Biochemistry, Charing Cross Hospital Medical School for carrying out the amino acid analyses of our elastin samples. CPW holds a Wellcome University Award. KHP would like to thank the Clothworkers' Foundation for their generous support. References 1 Hornebeck, W., and Robert, L., "Elastin-lipid Interaction in Atherogenesis," Olsson, A. G., ed., Atherosclerosis: Biology and Clinical Science, Churchill Livingstone, Edinburgh, Chapter 16, 1987. 2 Blumenthal, H. T., Lansing, A. I., and Gray, S. H., "Interrelation of Elastic Tissue and Calcium in Genesis of Atherosclerosis," Am. J. Pathol, Vol. 26, 1950, p. 989. 3 Vander Lei, B., Wildevuur, C. R. H., and Nieuwenhuis, P., "Compliance and Biodegradation of Vascular Grafts Stimulate the Regeneration of Elastic Laminae in Neo-arterial Tissue: An Experimental Study in Rats," Surgery, Vol. 99, 1986, pp. 45-52. 4 Noma, A., Takahashi, T., and Wada, T., "Elastin Lipid Interactions in the Arterial Wall," Atherosclerosis, Vol. 38, 1981, pp. 373-382. 5 Molinari Tosatti, M. P., Gotte, L., and Moret, V., "Some Features of the Binding of Calcium Ions to Elastin," Calcif. Tiss. Res., Vol. 2, Suppl., 1968, p. 88. 6 Eisenstein, R.,.Ayer, J. P., Papajiannis, S., Haas, G. M., and Ellis, H., "Mineral Binding by Human Arterial Elastic Tissue," Lab. Invest., Vol. 13, 1964, pp. 1198-1204. 7 Seligman, M., Eilberg, R. G., and Fishmean, L., "Mineralization of Elastin Extracted From Human Aortic Tissues," Calcif. Tiss. Res., Vol. 17, 1975, pp. 229-234. 8 Urry, D. W., "Neutral Sites for Calcium Ion Binding to Elastin and Collagen: A Charge Neutralization Theory for Calcification and its Relationship to Atherosclerosis," Proc. Nat. Acad. Sci. USA, Vol. 68, 1971, pp. 810-814. 9 Rucker, R. B., Ford, K., Goettlich-Riemann, W., and Tom, K., "Additional Evidence for the Binding of Calcium Ions to Elastin at Neutral Sites," Calcif. Tiss. Res., Vol. 14, 1974, pp. 317-325. 10 Bush, K., McGarvey, K. A., Gosline, J. M., and Aaron, B. B., "Solute Effects on the Mechanical Properties of Arterial Elastin," Conn. Tiss. Res., Vol. 9, 1982, pp. 157-163. 11 Robert, L., Jacob, M. P., Frances, C , Godeau, G., and Hornebeck, W., "Interaction Between Elastin and Elastases and Its Role in the Ageing of the Arterial Wall, Skin and Other Connective Tissues. A Review," Mechanisms of Ageing and Development, Vol. 28, 1984, pp. 155-156. 12 Bendall, J. R., "The Titration Curves of Elastin and of the Derived aand /3-proteins," Biochem. J., Vol. 61, 1955, pp. 31-32. 13 Molinari Tosatti, M. P., Gotte, L., and Moret, V., "Some Features of the Binding of Calcium Ions to Elastin," Calcif. Tiss. Res., Vol. 6, 1971, pp. 329-334. 14 Bowes, J. H., and Kenten, R. H., "Some Observations on the Amino Acid Distribution of Collagen, Elastin and Reticular Tissue from Different Sources," Biochem. J., Vol. 45, 1949, pp. 281-285. 15 Winlove, C. P., and Parker, K. H., "The Influence of the Elastin Lamellae on Mass Transport in the Arterial Wall," Staub, N. C , Hogg, J. C , and Hargans, A. R., eds., Interstitial Lymphatic Liquid and Solute Movement, Karger, Basel, 1987. 16 Tanford, C , Physical Chemistry of Macromolecules, Wiley, N.Y., 1961. 17 Urry, D. W., Krivacic, J. R., and Haider, J., "Calcium Ion Effects a
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Notable Change in Elastin Conformation by Interacting at Neutral Sites," Biochem. Biophys. Res. Comm., Vol. 43, 1977, pp. 6-11. 18 Winlove, C. P., and Parker, K. H., "Influence of Solvent Composition on the Mechanical Properties of Arterial Elastin," Biopolymers, Vol. 29, 1989, pp. 729-735. 19 Jackson, D. S., and Cleary, E. G., "The Determination of Collagen and Elastin," Meth. Biochem. Anal., Vol. 15, 1967, pp. 25-76. 20 Tanford, C , "Hydrogen Ion Titration Curves of Proteins," Shedlovsky, T., ed., Electrochemistry in Biology and Medicine, Wiley, N.Y., 1955, pp. 248265. 21 Hartman, B. K., and Bakerman, S., "Titration of Salt-Extracted Human Skin Collagen," Biochem., Vol. 5, 1966, pp. 2221-2229. 22 Partridge, S. M., "Diffusion of Solutes in Elastin Fibres," Biochim. Biophys. Acta, Vol. 140, 1967, pp, 132-141. 23 Winlove, C. P., and Parker, K. H., "Microcalorimetric Investigations of the Interactions of Small Ions with Arterial Elastin," Submitted to: Biopolymers, 1992. 24 Bowes, J. H., and Kenten, R. H., "The Amino-Acid Composition and Titration Curve of Collagen," Biochem. J., Vol. 43, 1948, pp. 358-365. 25 Chon, E. J., and Edsall, J. T., Proteins, Amino-Acids and Peptides, Reinhold, N.Y., Chapter 20, 1943. 26 Smith, D. W., Brown, D. M., and Carnes, W. H., "Preparation and Properties of Salt Soluble Elastin," J. Biol. Chem., Vol. 247, 1972, pp. 24272432. 27 Scatchard, G., Coleman, J. S., and Shen, A. L., "Physical Chemistry of Protein Solutions. VII. The Binding of Some Small Anions to Serum Albumin," /. Am. Chem. Soc, Vol. 79, 1957, pp. 12-20. 28 Partridge, S. M., "The Lability of Elastin Structure and Its Probable
Form Under Physiological Conditions," Frontiers Matrix Biol., Vol. 8, 1980, pp. 3-32. 29 Greenberg, D. M., "The Interaction Between the Alkali Earth Cations, Particularly Calcium, and Proteins," Adv. in Protein Chemistry, Vol. 1, 1944, pp. 121-151. 30 Alvarez, V. G., and Sandberg, L. B., "Quantitative NH 2 Terminal Evaluation of Elastin as a Measure of Polypeptide Integrity, "Adv. Exp. Med. Biol., Vol. 79, 1977, pp. 589-595. 31 Rosenbloom, J., "Biology of Disease, Elastin: Relation of Protein and Gene Structure to Disease," Lab. Invest., Vol. 51, 1984, pp. 605-623. 32 Bowes, J. H., and Kenten, R. H., "The Swelling of Collagen in Alkaline Solutions," Biochem., Vol. 46, 1950, pp. 1-8. 33 Winlove, C. P., and Parker, K. H., "Physicochemical Properties of Vascular Elastin," D. W. L. Hukins, ed., Connective Tissue Matrix, Part 2, McMillan, London, Chapter 7, 1990. 34 Li, S. T., and Katz, E. P., "An Electrostatic Model for Collagen Fibrils. The Interaction of Reconstituted Collagen with Ca 2 + , Na + and Cl~," Biopolymers, Vol. 15, 1976, pp. 1439-1460. 35 Schettler, G., Nerem, R. M., Schmid-Schonbein, H., Mori, H., and Diehm, C , eds., Fluid Dynamics as a Localizing Factor for Atherosclerosis, SpringerVerlag, Berlin, 1983. 36 Flint, M. H., Lyons, M. F., Meaney, M. F., and Williams, D. E., "The Masson Staining of Collagen—an Explanation of a Apparent Paradox," Histochem. J., Vol. 7, 1975, pp. 529-546. 37 Urry, D. W., Chang, D. K., Zhang H. and Prasad, K. U., "pK Shift of Functional Group in Mechanochemical Coupling Due to Hydrophobic Effect: Evidence for an Apolar-Polar Repulsion Free Energy in Water," Biochem. Biophys. Res. Comm., Vol. 153, 1988, pp. 832-839.
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E
l
a
H
lastin
The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.
Introduction Elastin is widely regarded as a particularly inert protein. form of both rings and fibrils obtained by grinding dried samHowever, accumulation of lipids and calcium in the elastic ples in order to assess the effects of the method of preparation lamellae is one of the earliest manifestations of atherosclerosis on charge structure. Since radiotracer studies suggest that [1,2] and the performance of elastin used in vascular prostheses stretching elastin increases the number of sites of ion binding is frequently compromised by similar interactions [3]. There [15] experiments were also performed on rings of elastin subject have been a number of studies on the binding of lipids [4], to mechanical strain. The enthalpies of ionization of the charge ions [5-9], and macromolecules [10] including enzymes [11] to groups were determined directly by means flow microcalosoluble and fibrous elastin in vitro, but in very few cases have rimetry. the sites and mechanisms of interaction been determined. Even We investigated ionic interactions by comparing titration the most extensively investigated problem, calcium binding, is curves obtained in media of different salt composition, by still not fully resolved. It has been shown that calcium interacts measuring the displacement of hydrogen ions by salts added both electrostatically and, under appropriate conditions, at a to the bathing solution and by measuring the uptake of raneutral site [8, 9] but the relative importance of ionic and diolabelled ions by elastin. The potentiometric and radiotracer neutral site binding under physiological conditions is unclear. techniques are complementary in the ranges of ionic concenThe present study was undertaken in the belief that the trations over which they are applicable and in the information reactivity of elastin might be clarified by a better understanding they provide. The former experiments reflect largely electroof the nature and density of charged groups on fibrous elastin, static interactions [16] whilst the latter experiments also detect their reactivity towards small ions and their role in determining nonelectrostatic interactions. the structure of fibrous elastin. We also investigated the role of electrostatic interactions in Biochemical analysis shows that the elastin molecule con- determining the conformation of the matrix using a simple tains both anionic and cationic groups but the degree of ion- picnometric technique. Changing the pH or ionic composition ization of these groups and their accessibility in elastin fibers . of the bathing solution changes the conformation of soluble has been established only for elastin derived from the liga- elastin [17] and by establishing whether similar changes occur mentum nuchae [12-14]. Our potentiometric titration meas- in the fibrous protein we should obtain insight into the interurements were performed on porcine arterial elastin isolated actions occurring between elements of the elastin matrix. by alkali, formic acid, or cyanogen bromide extraction in the Methods Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENGINEERINO. Manuscript received by the Bioengineering Division June 28, 1991; revised manuscript received March 17, 1992. Associate Technical Editor: M. Friedman.
Materials. Elastin was prepared by sodium hydroxide extraction or cyanogen bromide digestion of the thoracic aortas of young pigs as detailed elsewhere [18] or by formic acid
Journal of Biomechanical Engineering
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extraction [19]. The material was returned to neutral pH with HCI or NaOH and washed very extensively with deionized water before use. The elastin was used either as rings 1-2 cm in width or after drying, grinding to a fine powder consisting of particles 200-800 fim in diameter and rehydrating. The amino acid composition of a sample of the alkali digest was determined by Dr. J. Powell, Department of Biochemistry, Charing Cross Hospital. Reagents were prepared by standard methods using deionized water and Analar grade chemicals. Titration Experiments. pH measurements were performed with a glass electrode (Orion, model 8103SC) connected to an electrometer (Keithley 614). The electrode was calibrated in standard buffer solutions (pH 2.00 and 12.00, Aldrich Chemicals, pH 4.01, 7.00, 9.19 Orion Research) before and after each series of measurements. The preparation to be titrated, consisting of a sample of elastin with a dry weight of approximately 500 mg and 20 ml of degassed solution, was placed in a glass vial in a water bath thermostated to 25.0 ± 0.20°C and stirred by a gentle stream of nitrogen bubbles. Before the experiment, the elastin was hydrated and equilibrated with several changes of solution for at least 48 h until a constant pH was attained. Titrant was added by micropipette and pH was recorded after equilibration. Equilibration was almost instantaneous at the extremes of pH but between pH 6 and 8 the voltage usually overshot initially and equilibration could take up to 25 min. We were unable to establish whether this behavior reflects differences in the accessibility of ionic groups or their rate of reaction or is due to conformation changes in the elastin. To study the effects of strain, a small rig was constructed containing two parallel glass rods 5 cm long and 3 mm in diameter whose separation could be varied by a locking screw mechanism. An elastin ring was looped over the rods and extended to the chosen strain relative to a reference length which was defined as the position at which the sides of the elastin ring first became parallel. Only the glass part of the apparatus came into contact with the solution during a measurement. In order to investigate the effects of changes in the ionic composition of the bathing solution on hydrogen ion binding by elastin, pH changes resulting from the addition of 20 ^1 aliquots of 0.01 M salt solution were measured. Calibration experiments were conducted using buffer alone. Titration curves were computed from the equation: K([acid] - [base] + [OH"] - [H + ]) *~ J*ll-[OH"]) where Hb is the number of moles of H bound per g dry weight of elastin, V is the volume of the bathing solution, W is the dry weight of elastin and [acid], and [base] are the moles of acid or base added per unit volume of the solution. [H + ] = 10" pH and [OH"] = 10" 1 4 + p H are the measured concentrations of H + and O H " ions in the solution. (Corrections were made for the activity coefficients of the hydrogen and hydroxyl ions, determined from the titration curves of water blanks under the assumption that the coefficients were unaffected by the presence of protein [20].) We have assumed that after the initial washing procedure the protein was at its isoionic point and adopted this as the reference state [21]. In deionized water, the isoionic point was pH = 6.93 ± 0.82 averaged over all alkali extracted preparations. At this pH, a variation of 0.82 pH units is equivalent to an Hft of about 0.1 /xmoles/g dry weight which represents a negligible uncertainty in the ordinate of the titration curve. The difference in isoionic point in the various salt solutions, discussed below, was also not significant in this context. Radiotracer Uptake Experiments. Rings of elastin approximately 1 cm wide were weighed in air and then suspended 294 / Vol. 114, AUGUST 1992
in 200 ml of the required medium containing the radiolabelled ion and incubated at room temperature for 4 h with constant stirring. Preliminary experiments showed that this period was sufficient to attain equilibrium. After the incubation period the tissue was either counted immediately or washed in several changes of buffer until no more radioactivity appeared in the washing solution. Samples for 7-counting were counted immediately together with aliquots of the incubation medium and then dried to constant weight. Samples for ^-counting were solubilized in Optisolv (Pharmacia) and HiSafe 3 (Pharmacia) was used as scintillant for both tissue and medium samples. Control experiments were undertaken to provide a correction factor for the tissue quenching of ^-activity. Because the quench correction was rather large, particularly for 45Ca, some experiments were repeated using only a 10 ml volume of tracer and uptake was calculated from the loss of radioactivity from the bathing medium. Both procedures gave comparable results. Solutions containing 22 NaCl, Na 36 Cl, Na 125 I, Na 2 35 S0 4 , and 45 CaCl2 at concentrations of 0.15M, 0.05M, and 0.01M and pHs (adjusted by addition of acid or alkali and unbuffered) of 2.5, 7.0, and 10.5 were employed. A series of experiments was also undertaken with rings of elastin extended to 30 percent strain in the apparatus described above. Tracer uptake, U, was defined as: counts/g dry weight counts/^mole of ion in the medium where the dry weight was determined directly for the experiments on -/-emitting isotopes and for the /3-emitters was calculated from the wet weight and mean water content of matched specimens. For unwashed tissue the measured radioactivity arose largely from tracer contained within the water spaces of the elastin. Because of electrostatic interactions the ionic composition of this fluid was not necessarily identical to that of the bathing solution and it was impossible to distinguish between ions "free" in this water space and those "bound" to the elastin fibers. It is tempting to refer to the tracer remaining after extensive washing as being "irreversibly" bound. However, this definition is also imprecise because radioactivity in the washing tended only asymptotically to zero. These difficulties confound comparisons with other data in the literature and dictate that uptake values be regarded only as comparative indicators of the behavior of different ions. In an attempt to obtain a more rigorous measure of interactions, ground elastin was also used as a substrate for affinity chromatography [32]. A suspension of elastin fibers was packed into a 12 cm by 1 cm column [Pharmacia]. The column was equilibrated with eluate at a flow rate of 8 ml/hr generated by a peristaltic pump [LKB] at the column outlet. 1-2 ml aliquots of radiolabelled solution of known concentration and specific activity were applied to the column. Eluate was collected and counted. The quantity of radioactivity retained on the column after eluate activity had fallen to background was taken to be the irreversible binding capacity of the elastin. The amounts of radioactivity removed by washing with solutions of different ionic composition or pH were also measured. At the end of a series of experiments, the elastin was quantitatively recovered from the column and its dry weight determined so that binding could be expressed as /u,moles/g dry weight. Calorimetry. Approximately 50 mg of dry, ground elastin were packed in the chamber of a flow microcalorimeter (Microscal, Model 3S) as described elsewhere [23]. Solution flowed through the chamber at a rate of 3.3 ml/h and the heat change following a step change in solution pH was recorded. In order to avoid the effects of changes in ionic composition which would accompany the use of pH buffer solutions these experiments were performed using degassed, deionized water whose pH was adjusted by small additions of NaOH or HCL. Transactions of the ASME
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The enthalpy changes resulting from changes in Na + or Cl~ concentration were negligible in comparison with the heats of ionization [23]. Enthalpies of ionization were derived from the areas under the heat-time curves. Density Measurements. A ring of elastin was attached to a clip on one arm of a lever arm balance. The sample was immersed in a beaker of deionized water and its apparent weight was measured (M„) after equilibration. The beaker of water was then removed and the specimen was carefully blotted dry and weighed in air ( M J . The elastin ring was then reimmersed in a beaker filled to the same level with the test solution, allowed to equilibrate and the weighing procedure was repeated (Mj). When the required cycle of solution changes was completed the elastin ring was immersed in deionized water to allow any solutes to diffuse out and then dried for 72 hr at 60 °C and its dry weight was measured (Me). The densities of the test solutions (pw and ps) were measured with a specific gravity bottle. All weighings in solution were corrected for the buoyancy of the clip assembly. The volume (Ve) and density (pe) of the elastin, the initial water space (V„) and the solution space after equilibration with test solution (Vw) were calculated from the following formulae: Ve=(Me-Mw)/pw Vw=(Ma-Me)/Pw pe =
pwMe/(Me-Mw)
Ms-Me + peVe V =(Pw-Ps)
Results (1) Analysis of Titration Curves. Our titration curves (Fig. 1) were similar to those obtained for ligamentum elastin [12-14]. We normally confined measurements to the range pH 2 to 11.5 where the curves were reversible. Most curves reached a plateau at pH « 2 but occasionally we found an increase in Hb at low pH due either to strongly acidic or weakly basic groups or degradation of the substrate. Though earlier work on collagen favoured the former explanation [24], we found that long exposure to very low pH does solubilize elastin. We could not detect a plateau at the basic end of the curve.
-400
Fig. 1 Titration curves of alkali extracted aortic elastin in different salt solutions. (o )-deionized water, (o )-.15 M NaC1, ( • )-.15 M CaCI2, (o )-.15 M Nal.
Journal of Biomechanical Engineering
The attribution of different portions of the titration curve to specific groups has been discussed by many authors [e.g., 20,25]. We assume that the region from pH 2 to 6.25 represents e-carboxyl groups, pH 6.25 to 8 corresponds to imidazole groups, and that from pH 8 to 11 all basic groups other than guanidyl are titrated. With these assumptions there is satisfactory agreement between titration results and biochemical analysis for alkali extracted elastin. The amino acid composition was very similar to that previously reported for this type of preparation {26]. Titration showed 120 ± 1 5 /xmoles carboxyl groups/g dry weight and the total of glutamine/glutamic acid and asparganine/aspartic acid was 200 /xmoles/g dry weight. Given the uncertainty about the forms of glutamate and aspartate this agreement seemed satisfactory and suggested that only a few of the carboxyl groups are masked in the fibrous protein and that, as for collagen, there are few a-carboxyl groups. However, compensatory intrafibrillar interactions between carboxyl and amino groups have been suggested to occur in ligament elastin [12] and we cannot discount this possibility in our preparation. Between pH 6.25 and 8 only the imidazole groups of histidine are titrated and their number, 17 ± 4 /xmoles/g dry weight is reasonably consistent with the 30 detected by amino acid analysis. The greatest problem is the interpretation of the portion of the curve beyond pH 8. The pKs of the e-amino groups of lysine, the phenolic groups of tyrosine and the a-amino and imino groups of proline and hydroxyproline lie close together and overlap the guanidyl groups of arginine [20]. The problem of attribution has been discussed for collagen [21], and our difficulties were further compounded because the number of titratable groups was very sensitive to the ionic strength of the medium. Adopting the usual upper bound of pH 11.5 for the titration of e-amino and phenolic groups gave more charges than detected biochemically but with an end point of 10.5 gave good agreement with the total tyrosine and lysine content (19 biochemically against 16 ± 4 from titration). Over the chosen range of carboxyl group titrations, a plot of log (a/1 - a ) versus pH, where a is the degree of dissociation, was linear (Fig. 2) which provides some confidence in the attribution of the titration results to a single group. The linearity also suggested that electrostatic interactions were constant over the titration range, (i.e., they were dominated by the basic groups). Strong electrostatic interactions were presumed to be responsible for the pK of 3.5 ± 0.2, which is much lower than the value of 4.6 found for side-chain carboxyls in small molecules [16] and significantly lower than the value of 4.1 found for collagen [21]. In the basic region satisfactory linearity was achieved by assuming titration of phenolic groups was complete at pH 10.5 and the pK of 10 was close to the accepted value [16]. The region between 9.5 and 11.5 was also linear, but the pK ~ 11 was higher than that expected for sidechain amino groups [16] and so this portion of the curve may be affected by interference from more basic groups.
(2) Enthalpies of Ionization. Typical enthalpy time curves for step changes of the pH of the solution from 6.2 to 12.5 and back are shown in Fig. 3. The initial peak occurred within a few minutes, as expected from the rate of mixing of solution in the calorimeter. In some experiments enthalpy changes continued for many minutes. This may arise from the buffering capacity of the elastin or changes in conformation of the elastin which occur on this time scale. Figure 4 summarizes enthalpy measurements on 2 samples of alkali extracted elastin. The greatest heat changes were associated with ionization of the basic groups. The dotted line in Fig. 4 is the enthalpy change predicted from our titration data assuming that the carboxyl, imidazole, and e-amino groups make the dominant contribution and that their enthalpies of ionization are those measured for collagen [21]. Very good AUGUST 1992, Vol. 114/295
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AQ
pH
(mW/g)
/
\
pH 6.2 -> pH 12.5
10
t (min) 30
V _ _ ^ ^ ^ ' p H 12.5 -* pH6.2 11 pH
Fig. 3 Calorimeter trace showing the rate of heat release with time following a step change in the pH of the perfusing solution. Top curve: pH 6.2 - pH 12.5, Bottom curve: pH 12.5 - pH 6.2.
10
AH (J/g)
PH
10
12
PH
Fig. 4 Net heat release following step changes in the pH of the perfusing solution calculated from the area under curves like those in Fig. 3. The arrows indicate the direction of the pH change. The dotted line is the predicted net heat release using heats of ionization for charged groups obtained for collagen [21] and our estimates of the numbers of charged groups in elastin.
'(^) Fig. 2 Plots of pH versus ln(, is the number of salt ions Transactions of the ASME
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Table 1 Uptake of Ions by alkali digested elastin Otmoles/g dry weight) Na +
Ca 2 +
CI"
20±2 21 ± 2 29±2
19±1 24±2 27 ± 2
110±10 32±3 20±2
210±6 54±3 54±3
94 ±16 16±3 16±3
300 ± 3 0 330±40 350 ±30
270 ± 4 0 380 ± 5 0 370 ± 3 0
440 ±30 380±10 360±10
450 ±15 490 ±50 620 ±30
500 ±90 330 ±70 350 ±70
pH .01 M salt 2.5 7.0 10.5
so^
. r
.15 Msalt 2.5 7.0 10.5
100
c
150
(iM)
Fig. 5 The change in H,, with the addition of CaCI2, NaCI2, and Cal2. The top curves are for an initial pH of 2.5, the bottom curves are for an initial pH of 10. The data are corrected for salt effects on the measured pH of the water.
1
of charge z, "bound" to the protein [16]. The parameter A depends on the geometry of the fixed charges and because this is unknown for the elastin matrix, it is not possible to use the equation quantitatively. However, the sign of the pH change indicates that more anions than cations are bound. The smaller change produced by CaCl2 is also expected because of the divalency of the cation. In deionized water the fixed charge density at the isoionic point was approximately - 6 x 10 ~3 /xmoles/g dry weight. Changes of H6 following small additions of salt are shown in Fig. 5. At acid pH the addition of salt increased the amount of hydrogen binding. Sodium and calcium salts had similar effects when allowance was made for the difference in anion concentration. The nature of the anion seemed to be the major determinant of the degree of dissociation, chlorides being much more effective than iodides. We presume that the increase in Hfc arises because anion binding to basic groups, as has been reported for a-elastin [28], allows increased electrostatic attraction for H + to the elastin. This clearly dominates any interaction of cations with acidic groups or neutral sites which would tend to reduce ¥Lb. Under alkaline conditions addition of salt caused a release of bound hydrogen, as expected from the change in the titration curves. This, too, is presumably due to anion interaction because although Ca2+ binding to phenolic groups and incomplete ionization of Ca salts of carboxylic acids could occur [29], neither would account for the anion dependence we observed. (b) Radiotracer Uptake Experiments. Tracer uptake for different ions at different pHs and ionic strengths are given in Table 1. The measured uptake reflects tracer "interacting" with the elastin and ions "free" in the water pool. As discussed in the Methods section, these contributions cannot be distinguished either experimentally or theoretically. The distribution in the intra and extrafibrillar water spaces depends on the electrostatic field produced by the fixed charges on the elastin. This interaction was present under all the conditions we investigated, as demonstrated by the fact that the uptake of both anions and cations was always smaller than that calculated by assuming uniform distribution throughout the water space. Since the Debye length is approximately 0.8 nm in 0.15 M NaCl, this is evidence that at least some charges are present around hydrated pores of similar dimensions. Journal of Biomechanical Engineering
0 -c pH
Fig. 6(a) The change in water volume with changing pH for alkali extracted elastin. The starting point for each sample is indicated by the closed dot. .18 M
AV„/V„
NaCl
NaCl CaCl 2
CaCl 2 Nal
Nal
Fig. 6(D) The percentage change in water volume following a change in the solution from deionized water to .15 M and 1.5 M NaCl, CaCU, and Nal Fig. 6 The effect of pH and ionic composition of the solution on elastin water content
Both cations showed a slight increase in uptake with increasing pH which was not accounted for by changes in water content (see Fig. 6). This is consistent with earlier observations which were attributed to interaction with carboxyl groups [6]. The continuation of the change beyond pH 7 suggests that changes in the ionization of the basic groups are also involved [13]. The similarity in the behavior of Na+ and Ca2+ indicates that neutral site binding makes a minor contribution to uptake under these conditions [23]. Anion uptake shows the reverse dependence on pH and the ionization of the carboxyl groups AUGUST 1992, Vol. 114 / 297
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is clearly the major determinant of uptake. Sulphate uptake was smaller than that of the halides and there was a significant difference in uptake between chloride and iodide. Iodide uptake is approximately two-fold greater despite its small effect on titration curves and its inefficacy in displacing bound hydrogen. This difference in reactivity was also observed in experiments using a medium containing both ions which showed preferential uptake of iodide which was reduced only by great excesses of chloride. Uptake of cations increased approximately linearly with concentration up to 0.15 M. Anions showed more complex concentration dependence. Chloride and sulphate uptake was proportional to concentration at pH 7 and 10.5 but iodide uptake increased more slowly with concentration. At pH 2.5 uptake of all three anions was nonlinear and appeared to be saturable. The apparent fixed charge density, {Uinion - t/ calion )C bu i k) varied with ionic species and ionic strength and was not consistent with the fixed charge density determined by titration. This could reflect the peculiarities of the ionic distribution or nonelectrostatic interactions. Experiments on the washout of radioactivity from the elastin rings showed that —4.5 /xmoles of calcium/g dry weight could not be removed, but all of the sodium could be removed by the washing procedure. These observations were consistent with experiments with the elastin packed column which showed that at neutral pH calcium binding was —3.7 /imoles/g dry weight. None of the radioactivity was removed by increasing the pH of the eluate to 10.5 or by reducing it to 2.5. No sodium binding could be detected throughout the pH range. At neutral pH chloride binding was —4.1 ^moles/g dry weight and all of the radioactivity was recovered on increasing the pH to 11. At an initial pH of 2.5, chloride binding was reduced to 1.9 /xmoles/g dry weight and all of this material was removed by reducing the pH to 2.0. (4) Effects of Elastin Preparation. Four preparations of alkali-extracted elastin were made using different batches of aortas. Two of these batches of aortas were also used to prepare CNBr digests. For each type of digest the titration curves were qualitatively similar but quantitative differences were sufficient to give rise to standard errors of the means of up to 20 percent. It is not clear whether the variations arise from differences in the composition of the native elastin, the efficiency of removal of other components of the tissue or modifications occurring during preparation. For a single preparation of elastin no difference was detected between rings and ground elastin. The difference in numbers of ionizable groups between the alkali digest and CNBr preparation were no greater than the batch to batch variation in either preparation. Biochemical analysis shows that CNBr digestion produces a smaller number of rt-terminal residues and that hydrolysis leads to a loss of cross-linking amino acids [30, 31]. Our results suggest, however, that the effects are quantitatively small. There was no significant difference in pK for the basic groups, but the acid branch of the titration curve was generally rather steeper for the CNBr digest, perhaps indicating a change in conformation in the vicinity of the carboxyl groups. Radiotracer measurements showed only small differences in uptake between elastin preparations which could be explained by the differences in water content. (5) Effects of Strain. Initial experiments were conducted at 50 percent strain but the elastin tended to rupture at extreme pH and so later experiments were performed at an extension of 30 percent. The cause of rupture has not been investigated further. There was no difference in the number of titratable groups but the pK of the carboxyl groups of stretched elastin was shifted approximately 0.2 units towards the acidic in deionized water. Smaller shifts were observed in salt solutions. The change in proton binding was also reflected in a change in 2 9 8 / V o l . 114, AUGUST 1992
solution pH as the elastin was stretched. In deionized water at the isoionic point the change in pH corresponded to a change of - .036 ± .020 /xmoles H bound/g dry weight but was almost a thousand fold greater at an initial pH of 3.0. The change was slowly reversible as the elastin was allowed to relax. These results suggest that the conformation changes associated with stretching of the elastin modify charge-charge interactions principally of the carboxyl groups. Strain increased cation uptake and decreased anion uptake as we have reported previously [15]. The effect was greatest at high pH and low ionic strength, where the apparent fixed charge density reversed in sign, going from 25 to - 19 /unoles/ g dry weight. Although stretching produced a small water loss from the tissue its contribution was negligible. The titration experiments indicate that the effect does not arise from the unmasking of charged groups. Furthermore, since experiments on the displacement of protons by the addition of salt gave similar results in stretched tissue, the effects are not due to changes in competitive interactions. We presume, therefore, that they are due to larger length scale changes in the geometry of the electrostatic field. (6) Effects of pH and Solution Composition on Matrix Structure. Preliminary experiments showed that sample weight changed for up to 24 hours following a change in the composition of the surrounding medium. We have noted such long time constants in earlier experiments on the mechanochemical properties of elastin [18] and similar observations have been made on swelling collagen [32] where they were attributed to changes in the cohesion of the fibres. The effects of changing pH on the water content of several samples of elastin are summarized graphically in Fig. 6. Samples differed in initial water content. There was also some variation in the initial density of the solid elastin component, but this remained constant as pH varied. The water content of the elastin was a minimum at or slightly above the isoelectric pH. There was very little swelling at low pHs but a rapid increase in volume above pH 10, indicating that the degree of ionization of the most basic groups is an important determinant of matrix conformation. These observations are consistent with a study of the effects of pH on the mechanical properties of elastin where the decrease in unstressed length at pH 2 relative to the reference conditions at pH 7 was not significant but there was a large increase at pH 11. At both extremes of pH there was an increase in the elastic modulus [18], In the present experiments the effects were qualitatively reversible when the pH changes were reversed. There was some hysteresis due either to lack of elastic forces to aid rehydration of the tissue or to chemical modifications produced by the long exposure to extremes of pH. The data in Fig. 6(a) were obtained using deionized water. In salt solutions the changes were similar but smaller in magnitude. Changes in salt concentration at constant pH also changed the water content of the tissue as illustrated in Fig. 6(b). NaCl and CaCl 2 both caused the matrix to shrink. The effect was small in 0.15 M solution, CaCl2 having the larger effect as expected because of its higher ionic strength, but increased with salt concentrations up to about 1.5 M. This behavior is consistent with the elastin network being held apart by electrostatic interactions which are shielded by the ionic solutions. Furthermore, since the Debye length in 0.15 M NaCl is —0.8 nm, we may infer that the functional charges are distributed on this length scale. Iodide has a very different effect on matrix structure, causing swelling even at low ionic strengths. The process is reversible in the sense that replacement of iodide by chloride caused shrinkage to a similar volume to that attained without prior exposure to iodide, but further work is required to clarify the underlying mechanisms. Studies on the swelling of collagen and gelatin [32] show it to be qualitatively similar to the behavior we report. The data Transactions of the ASME
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have been fitted to a simple model in which osmotic swelling is opposed by stress within the collagen matrix. The conclusion that the "bulk modulus" of the matrix was a strong function of pH appears, however, to cast some doubt on the utility of the model. Discussion Titration and uptake measurements made on soluble proteins [e.g., 16, 17] provide a much more detailed description of charge structure than we were able to obtain with similar measurements for elastin. The major source of uncertainty for fibrous elastin is the existence of a number of water "pools" within its structure. In addition to the extrafibrillar water spaces of microscopic dimensions, a significant fraction of the water in vascular elastin is contained in intrafibrillar pores accessible only to molecules < 1000 Daltons [33]. The distribution of ions between these compartments will depend on the fixed charge structure of the elastin and its conformation, the size and charge of the ion, and the pH and ionic strength of the bathing solution. This introduces difficulties into the interpretation of both titration and ion binding experiments which cannot be adequately addressed without a detailed model of the structure and charge distribution of the elastin matrix. Though a model has been developed for the collagen fibril [34] which formed a satisfactory framework for the interpretation of experimental data on ionic interactions, the nature of the assumptions which would have to be incorporated into a model of elastin coupled with the uncertainties in our experimental data suggest that it would not represent a significant advance. One of the most striking observations in the present study was the affinity of elastin for anions. Iodide is well known to interact with proteins [27] and binding of chloride to a-elastin has been reported previously [28] and our work extends this observation to the fibrillar protein. It also shows considerable differences in the behavior of chloride and iodide which we have also observed calorimetrically [23], The reason for this difference is unclear and it could be explained by steric considerations only if the interaction involved bare rather than hydrated ions. It might be valuable to investigate the binding of phosphates and anionic macromolecules such as glycosaminoglycans and glycoproteins and degradative enzymes whose interactions with elastin are of physiological or pathological importance. Because of the wide variety of definitions of "binding" which have been employed, it is difficult to make a quantitative comparison between our data on cation binding and the earlier literature. However, over our range of experimental conditions, sodium and calcium behaved in a very similar manner suggesting that the behavior of calcium was dominated by electrostatic interactions rather than by neutral site binding [8]. The relative importance of the two types of interaction depends strongly on the conformation of the elastin. The effects of changes in conformation produced by altering the composition of the solvent have been described [9, 23], but it might be useful to repeat some of our experiments on immature or degenerated elastin to explore the effects of naturally occurring variations of conformation. Vascular remodeling in response to changing haemodynamic conditions and links between haemodynamics and atherogenesis have both been extensively documented [35]. These observations imply complex mechanochemical couplings in the vascular interstituum and our results suggest two ways in which elastin may be involved. On the one hand, changes in the composition of the surrounding medium influences the dimensions and, as we have shown in earlier experiments [18], the mechanical properties of the elastin network. On the other hand, strain influences the apparent density and pKs of the ionizable groups, perhaps influencing the interactions of elastin with other molecules or its susceptibility to proteolytic degJournal of Biomechanical Engineering
radation. Similar behavior is reported for collagen, where the unmasking of charged groups is believed to be responsible for its strain dependent reaction to Masson's trichrome stain [36]. We have found in unpublished experiments that the staining of elastin with Masson's trichrome is also strain dependent, but the colouring is the reverse of that of collagen (i.e., the relaxed tissue is stained red and the stretched tissue green). The mechanism for elastin might be more complex because it has quite recently been shown that for elastin peptides and similar molecules, changes in hydrophobic interaction rather than changes in electrostatic interaction can give rise to shifts inpK [37]. We showed that the polyelectrolyte properties of elastin are not particularly sensitive to the method of preparation. The possibility remains, however, that they are greatly modified by the other components of the extracellular matrix which are intimately associated with elastin in the intact vessel. This possibility must be examined in the future by experiments using more complex tissue preparations.
Acknowledgments We would like to thank Dr. J. Powell, Departments of Surgery and Biochemistry, Charing Cross Hospital Medical School for carrying out the amino acid analyses of our elastin samples. CPW holds a Wellcome University Award. KHP would like to thank the Clothworkers' Foundation for their generous support. References 1 Hornebeck, W., and Robert, L., "Elastin-lipid Interaction in Atherogenesis," Olsson, A. G., ed., Atherosclerosis: Biology and Clinical Science, Churchill Livingstone, Edinburgh, Chapter 16, 1987. 2 Blumenthal, H. T., Lansing, A. I., and Gray, S. H., "Interrelation of Elastic Tissue and Calcium in Genesis of Atherosclerosis," Am. J. Pathol, Vol. 26, 1950, p. 989. 3 Vander Lei, B., Wildevuur, C. R. H., and Nieuwenhuis, P., "Compliance and Biodegradation of Vascular Grafts Stimulate the Regeneration of Elastic Laminae in Neo-arterial Tissue: An Experimental Study in Rats," Surgery, Vol. 99, 1986, pp. 45-52. 4 Noma, A., Takahashi, T., and Wada, T., "Elastin Lipid Interactions in the Arterial Wall," Atherosclerosis, Vol. 38, 1981, pp. 373-382. 5 Molinari Tosatti, M. P., Gotte, L., and Moret, V., "Some Features of the Binding of Calcium Ions to Elastin," Calcif. Tiss. Res., Vol. 2, Suppl., 1968, p. 88. 6 Eisenstein, R.,.Ayer, J. P., Papajiannis, S., Haas, G. M., and Ellis, H., "Mineral Binding by Human Arterial Elastic Tissue," Lab. Invest., Vol. 13, 1964, pp. 1198-1204. 7 Seligman, M., Eilberg, R. G., and Fishmean, L., "Mineralization of Elastin Extracted From Human Aortic Tissues," Calcif. Tiss. Res., Vol. 17, 1975, pp. 229-234. 8 Urry, D. W., "Neutral Sites for Calcium Ion Binding to Elastin and Collagen: A Charge Neutralization Theory for Calcification and its Relationship to Atherosclerosis," Proc. Nat. Acad. Sci. USA, Vol. 68, 1971, pp. 810-814. 9 Rucker, R. B., Ford, K., Goettlich-Riemann, W., and Tom, K., "Additional Evidence for the Binding of Calcium Ions to Elastin at Neutral Sites," Calcif. Tiss. Res., Vol. 14, 1974, pp. 317-325. 10 Bush, K., McGarvey, K. A., Gosline, J. M., and Aaron, B. B., "Solute Effects on the Mechanical Properties of Arterial Elastin," Conn. Tiss. Res., Vol. 9, 1982, pp. 157-163. 11 Robert, L., Jacob, M. P., Frances, C , Godeau, G., and Hornebeck, W., "Interaction Between Elastin and Elastases and Its Role in the Ageing of the Arterial Wall, Skin and Other Connective Tissues. A Review," Mechanisms of Ageing and Development, Vol. 28, 1984, pp. 155-156. 12 Bendall, J. R., "The Titration Curves of Elastin and of the Derived aand /3-proteins," Biochem. J., Vol. 61, 1955, pp. 31-32. 13 Molinari Tosatti, M. P., Gotte, L., and Moret, V., "Some Features of the Binding of Calcium Ions to Elastin," Calcif. Tiss. Res., Vol. 6, 1971, pp. 329-334. 14 Bowes, J. H., and Kenten, R. H., "Some Observations on the Amino Acid Distribution of Collagen, Elastin and Reticular Tissue from Different Sources," Biochem. J., Vol. 45, 1949, pp. 281-285. 15 Winlove, C. P., and Parker, K. H., "The Influence of the Elastin Lamellae on Mass Transport in the Arterial Wall," Staub, N. C , Hogg, J. C , and Hargans, A. R., eds., Interstitial Lymphatic Liquid and Solute Movement, Karger, Basel, 1987. 16 Tanford, C , Physical Chemistry of Macromolecules, Wiley, N.Y., 1961. 17 Urry, D. W., Krivacic, J. R., and Haider, J., "Calcium Ion Effects a
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Notable Change in Elastin Conformation by Interacting at Neutral Sites," Biochem. Biophys. Res. Comm., Vol. 43, 1977, pp. 6-11. 18 Winlove, C. P., and Parker, K. H., "Influence of Solvent Composition on the Mechanical Properties of Arterial Elastin," Biopolymers, Vol. 29, 1989, pp. 729-735. 19 Jackson, D. S., and Cleary, E. G., "The Determination of Collagen and Elastin," Meth. Biochem. Anal., Vol. 15, 1967, pp. 25-76. 20 Tanford, C , "Hydrogen Ion Titration Curves of Proteins," Shedlovsky, T., ed., Electrochemistry in Biology and Medicine, Wiley, N.Y., 1955, pp. 248265. 21 Hartman, B. K., and Bakerman, S., "Titration of Salt-Extracted Human Skin Collagen," Biochem., Vol. 5, 1966, pp. 2221-2229. 22 Partridge, S. M., "Diffusion of Solutes in Elastin Fibres," Biochim. Biophys. Acta, Vol. 140, 1967, pp, 132-141. 23 Winlove, C. P., and Parker, K. H., "Microcalorimetric Investigations of the Interactions of Small Ions with Arterial Elastin," Submitted to: Biopolymers, 1992. 24 Bowes, J. H., and Kenten, R. H., "The Amino-Acid Composition and Titration Curve of Collagen," Biochem. J., Vol. 43, 1948, pp. 358-365. 25 Chon, E. J., and Edsall, J. T., Proteins, Amino-Acids and Peptides, Reinhold, N.Y., Chapter 20, 1943. 26 Smith, D. W., Brown, D. M., and Carnes, W. H., "Preparation and Properties of Salt Soluble Elastin," J. Biol. Chem., Vol. 247, 1972, pp. 24272432. 27 Scatchard, G., Coleman, J. S., and Shen, A. L., "Physical Chemistry of Protein Solutions. VII. The Binding of Some Small Anions to Serum Albumin," /. Am. Chem. Soc, Vol. 79, 1957, pp. 12-20. 28 Partridge, S. M., "The Lability of Elastin Structure and Its Probable
Form Under Physiological Conditions," Frontiers Matrix Biol., Vol. 8, 1980, pp. 3-32. 29 Greenberg, D. M., "The Interaction Between the Alkali Earth Cations, Particularly Calcium, and Proteins," Adv. in Protein Chemistry, Vol. 1, 1944, pp. 121-151. 30 Alvarez, V. G., and Sandberg, L. B., "Quantitative NH 2 Terminal Evaluation of Elastin as a Measure of Polypeptide Integrity, "Adv. Exp. Med. Biol., Vol. 79, 1977, pp. 589-595. 31 Rosenbloom, J., "Biology of Disease, Elastin: Relation of Protein and Gene Structure to Disease," Lab. Invest., Vol. 51, 1984, pp. 605-623. 32 Bowes, J. H., and Kenten, R. H., "The Swelling of Collagen in Alkaline Solutions," Biochem., Vol. 46, 1950, pp. 1-8. 33 Winlove, C. P., and Parker, K. H., "Physicochemical Properties of Vascular Elastin," D. W. L. Hukins, ed., Connective Tissue Matrix, Part 2, McMillan, London, Chapter 7, 1990. 34 Li, S. T., and Katz, E. P., "An Electrostatic Model for Collagen Fibrils. The Interaction of Reconstituted Collagen with Ca 2 + , Na + and Cl~," Biopolymers, Vol. 15, 1976, pp. 1439-1460. 35 Schettler, G., Nerem, R. M., Schmid-Schonbein, H., Mori, H., and Diehm, C , eds., Fluid Dynamics as a Localizing Factor for Atherosclerosis, SpringerVerlag, Berlin, 1983. 36 Flint, M. H., Lyons, M. F., Meaney, M. F., and Williams, D. E., "The Masson Staining of Collagen—an Explanation of a Apparent Paradox," Histochem. J., Vol. 7, 1975, pp. 529-546. 37 Urry, D. W., Chang, D. K., Zhang H. and Prasad, K. U., "pK Shift of Functional Group in Mechanochemical Coupling Due to Hydrophobic Effect: Evidence for an Apolar-Polar Repulsion Free Energy in Water," Biochem. Biophys. Res. Comm., Vol. 153, 1988, pp. 832-839.
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