VOL. 15, 813-832 (1976)
BIOPOLYMERS
Nuclear Spin-Relaxation Studies of Hydrated Elastin G. E. ELLIS and K. J. PACKER,* School of Chemical Sciences, University of East Anglia, Norwich N R 4 7TJ, England
Synopsis The nuclear magnetic spin-lattice and transverse relaxation processes for the ‘H and 2D nuclei in purified elastin (ligamentum nuchae), exchanged and hydrated with excess DzO, have been studied in the temperature range 276O-34O0K. The 2D relaxation results clearly show the presence of DzO 1) external to the bulk elastin sample, 2) in spaces within the bulk elastin, and 3) as an integral part of the protein on a molecular level. It is shown from these measurements that the water content of the protein itself changes from -0.8 g D20/g dry elastin a t -280°K to -0.2 g DzO/g dry elastin at -335OK, a decrease of 400%. T h e D20 content of the interfiber spaces decreases by less than 20% over the same temperature range. This fact throws considerable doubt on the validity of the values of p, the thermal expansion coefficient of elastin, used by other workers in discussion of the elastic mechanism in elastin. The elastin proton transverse relaxation shows the presence of three regions in elastin having different degrees of molecular mobility. These are assigned to protons associated with the crosslinks, a fairly mobile, hydrophobic, and lowwater-content region, and a more mobile higher water-content region. The temperature variation of the relative proportions of these three regions is explained in terms of a hypothetical temperature-composition phase diagram in which the two mobile regions are represented as two partially miscible pPases with different negative temperature coefficients of “solubility” in water. The implications of these observations for current views of the nature of elastin are assessed. I t is concluded that the spin-relaxation results are consistent with a multiphase structural model for elastin. An approximate sorption isotherm for the water/elastin system is reported and shows the relatively weak nature of the water/elastin interaction.
INTRODUCTION The protein elastin is an important structural component of animal connective tissue. With collagen it plays a load-bearing role in tissues such as ligaments and arterial wa1ls.l Elastin is characterized by its rubberlike properties when hydrated and the relatively high proportion of its constituent amino acids, which are nonpolar. The dehydrated protein is fibrous, brittle, and nonelastic and shows a strong tendency to cornify. Hydrated elastin has been investigated by several groups of workers with differing conclusions being drawn as to the nature and * To whom correspondence should be addressed. 813 0 1976 by John Wiley & Sons, Inc.
814
ELLIS AND PACKER
mechanism of its elasticity. Hoeve and Flory, in agreement with earlier workers, suggested that elastin is a random crosslinked network with an elastic behavior similar to rubber.2 Further studies led Volpin and cow o r k e r ~to~the ~ ~ conclusion that, although its elasticity is not due entirely to configurational entropy changes, elastin behaves very much like a conventional rubber. More recently, Weis-Fogh and Andersen5 concluded, from calorimetric measurements, that the elastic behavior of elastin differed fundamentally from that of a conventional rubber in that large changes in internal energy accompanied the stretching process. These workers postulated that elastin is a series of crosslinked globular “molecules,” each having a molecular weight in the region of 67,000. Stretching of elastin, it was suggested, increases the surface area of these globules producing additional waterlnonpolar peptide interactions, these being the main source of the changes in internal energy. This model was essentially based on the structural model of Partridge, which in turn was based on electron microscopy, elastin biochemistry, and gel filtration studies using elastin fibers as the stationary phase.6-8 The crosslinks in elastin have been identified as arising from lysine residues in the soluble elastin precursor and their chemical nature has been e l u ~ i d a t e d . ~ The suggestion that elastin is not a random crosslinked polymer network, but possesses a globular two-phase character, has been rejected by Hoeve and Flory in a recent paper.1° They maintain that the calorimetric measurements are consistent with and indicative of a rubberlike random network in which configurational entropy changes are the driving force in the elastic process. They also rejected the “oiled-coil” model of Gray et al.ll on the grounds that, like the globular, two-phase structure, it has intrinsically insufficient elastic response. In this paper we report the results of a study of the nuclear magnetic relaxation behavior of both the protein protons and the deuterons in the water (DzO) hydrating elastin.
EXPERIMENTAL Sample Preparation The elastin used in this paper was the ligamentum nuchae of mature beef. The fresh ligaments were scraped free of extraneous fatty tissue and cut into strips, 5-10 cm in length and approximately 1 cm2 in cross section. The strips were washed with 1% saline solution and then with distilled water. Finally, they were boiled in successive quantities of distilled water until the supernatant liquid gave a negative biuret test for the presence of protein. The purified elastin strips were then dehydrated by successive treatments with quantities of ethanol, ethanolldiethyl ether (l:l),and finally diethyl ether. The final solvent was re-
HYDRATED ELASTIN
815
moved under vacuum and the dried elastin stored under vacuum over silica gel. For the nuclear spin-relaxation measurements the elastin was soaked in excess D20 (99.9 atom %, equivalent to approximately five times the volume of the dry elastin) for several weeks with three changes of D20 during that period. This procedure replaces all exchangeable protons, i.e., those in -COOH, -NH2, -OH groups, by deuterons. The measuremeuts of the temperature dependence of the relaxation processes, reported below, were carried out on a single sample consisting of 200 mg of dry elastin and 790 mg D20 in a closed sample tube (10 mm diameter).
Sorption Isotherm The sorption isotherm of water and elastin was approximately determined by equilibrating weighed strips of dry elastin over aqueous salt solutions, of known relative humidities, in airtight containers. The elastin strips were weighed a t intervals over a period of several weeks until they achieved constant weight.
Nuclear Spin-RelaxationMeasurements Nuclear spin-relaxation times were measured using a modified Bruker B-KR 3228 pulse spectrometer. Proton resonance was observed a t 40 MHz; deuterium resonance a t 10 MHz. The magnetic fields were provided by an AE1 M12 electromagnet. Spin-lattice relaxation was studied using a 180"-~-90"pulse sequence for both *H and 2D nuclei. For the elastin protons the transverse relaxation behavior was determined directly from the free induction decay signal following a single 90" pulse. This was possible since the effects of magnetic field inhomogeneities were negligible on the time scale of these decays. The general procedure adopted was to adjust the spectrometer system to resonance using the free induction decay from a sample of glycerol and then to record the elastin 1H free induction decay, which, a t all temperatures studied, decayed much faster than that from glycerol. 2D transverse relaxation behavior was determined using Carr-Purcell/Gill-Meiboom (CPMG) pulse sequences [go,( - 7(18OYr - 27 - 180,t - T ) ~ ] . All signals were recorded using phase-sensitive detection and signal amplitudes were determined using a 512-channel digital signal averager, designed and built in this laboratory specifically for use with the spectrometer. Every lH free induction decay was recorded on two time scales, 1 and 10 p s between measurement points, with the last points measured on the longer of these time scales providing the baseline measurement. In general, between 32 and 128 scans were averaged for each measurement and each measurement was repeated a t least twice and, in many cases, more times. Spin-lattice relaxation and CPMG Tz mea-
816
ELLIS AND PACKER t IPS
t /PS
Fig. 1. The 'H free induction decay signal S ( t ) from DZO-exchanged and hydrated elastin at 277.9'K: ( O ) , lower time scale showing long-time behavior; (-), upper time scale showing short-time behavior. The continuous line is the long-time limiting slope; the broken line is the same line drawn on the expanded (upper) time scale.
surements were accumulated in the signal averager using the special modes of the averager designed to accommodate the pulse sequences used. Again between 32 and 128 measurements were averaged for each set of parameter values used in the pulse sequences. The pulse separations used in the spin-lattice relaxation measurements were sufficient to follow the relaxation through at least two decades of recovery. The 7 values used in the CPMG sequences were in the range 0.5-10 ms. All experimental data were output onto punched paper tape for off-line processing by computer or calculator or graphically by hand. In all measurements the sample temperature was controlled to approximately f0.2OK by a conventional gas-flow cryostat. Temperatures were measured using a thermocouple contained in a normal sample tube, this being substituted for the sample for temperature measurement.
RESULTS Proton Relaxation
Figure 1shows a typical lH free induction decay obtained from DzOexchanged and hydrated elastin (All the relaxation measurements re-
HYDRATED ELASTIN
817
Fig. 2. The temperature dependence of the fractional populations, p a , pb, and p e , of the two exponential and one Gaussian ‘H transverse relaxation components in DnO-exchanged and hydrated elastin. The individual symbols represent experimental points; the continuous lines give an indication of the overall trends in the values.
ported were made on one sample comprising 200 mg dry elastin and 790 mg D20). It can be seen that the decay is not monotonic. “Solid echo” e x p e r i m e n t ~ lindicated ~ , ~ ~ that only the very short-time ( t < 15 ps) part of the free induction decay was-refocused to any extent, thus leading to the conclusion that the longer time behavior arises from motionally narrowed magnetic dipolar interactions between protons.14 It was found that these ‘H free induction decay’s could be well represented by a superposition of three decays, the two slower decays being exponential, the fastest being Gaussian. Thus the lH free induction decay S ( t ) can be written as:
s(t)= sob=exp - (t/T2,)
4- P b exp - (t/T2b) 4- p c exp
- (t2/T2,2)]
where the pi and T2, are the fractional populations and transverse relaxation times of the ith component and where T2, > T2b > T2,. The ‘H free induction decays were recorded at different temperatures in the range 277O-34O0K. Figures 2 and 3 show the variation with temperature of the fractional populations and transverse relaxation times of
818
ELLIS AND PACKER
+ 100 -
I
0
I
I
I
I
I
I
I
10
20
30
LO
50
60
70
T/'C
Fig. 3. The temperature dependence of the' three 'H transverse relaxation times: (X) T2,, (+) T2*,(0) T2,;in DnO-exchanged and hydrated elastin. The continuous lines emphasize trends in the data.
T/"C
Fig. 4. The temperature dependence of the 'H spin-lattice relaxation time in DnO-exchanged and hydrated elastin. The broken lines emphasize the trends in the T Ivalues.
three free induction decay components. In every case the values of the pi and Tgi were obtained by graphical resolution of S(t).15 Spin-lattice relaxation of the nonexchangeable elastin protons was found to be virtually a single exponential recovery a t all temperatures in the range 2770-340°K and, in addition, was independent of where on the free induction decay, following the 90° pulse in the 18Oo-~-9O0 sequence, the measurement point was situated. Figure 4 shows the variation with temperature of this 'H spin-lattice relaxation time T I .
HYDRATED ELASTIN
819
t/ms
Fig. 5. 2D spin-lattice relaxation in DzO-exchanged and hydrated elastin a t 277.7'K; (X) experimental points. The resolution into two components is shown, the continuous line being the longer relaxation time component A and the broken line being the shorter relaxation time component B.
Deuterium Relaxation Figure 5 shows a typical 2D spin-lattice relaxation recovery curve obtained from the D2O-hydrated elastin sample. These curves could be resolved into two components at all temperatures, i.e., the relaxation process can be represented by: [l- R ( t ) ]= 2 b A exp - (t/T1,)
+ P B exp - (t/T~,)l
where R ( t ) = (M,(t)/Mo)and T I , > TI,. Figure 6 shows the observed , and TzBwith temperature. variation of P A , p ~TI,, Figure 7 shows a typical 2D transverse relaxation decay for the D20hydrated elastin sample. It was found that, a t all but the highest temperatures, these decays could be resolved into three exponential components, i.e., the decay can be written: Mx,(t)= Mx,(0)[Pa exp
- (t/7'2,) + PO exp - WT2J
+ P r exp - (t/T2JI Figures 8 and 9 show the variation with temperature of the populations and transverse relaxation times of the various components. As before, these were obtained by graphical resolution of the decay curves. As can be seen, the experimental scatter is fairly large but this is not surprising considering the nature of the measurements and the problems of resolving multiple exponential decays. The trends in the populations and time constants are clearly visible however.
ELLIS AND PACKER
820 0.7
0.6 Froctional poplhtion
0.5 0-4
0.3
Ib
7
20
30
LO
50
60
70
T/"C
Fig. 6. The variation with temperature of the fractional populations, pa and p ~ and , spin-lattice relaxation times, TI, and TI,, for 2D nuclei in DzO-exchanged and hydrated elastin.
f
Long time limting dope fnxn data exterdq to t = 850rns
I
200
100 t
300
/rnS
Fig. 7. 2D transverse relaxation in DzO-exchanged and hydrated elastin at 276.7OK showing the resolution into three exponential decays: (O), experimental points; (X) and (a),data derived by successive subtraction of longer time constant components.
821
HYDRATED ELASTZN 0.6
Pd
0.5 Fractonal
o . ~poplation 0.3 0.2
O
7
0.1
0
population
xx
0.34
I
O
o;
io
3b
Lo o;
$0
o;
T/OC
Fig. 8. The variation with temperature of the fractional populations, polPB. and Pr, of the three 2D transverse relaxation components in DzO-exchanged and hydrated elastin: (0) p m ,(X) PO, ( 0 )pr. The broken lines indicate the trends in the data. loo01
Fig. 9. The temperature dependence of the three ‘D transverse relaxation components: (0) Tz,,(X) Tz8,( 0 )7 ’ ~ The ~ . broken lines indicate the trends in the relaxation times.
ELLIS AND PACKER
822 0.8 -
0 0.706Weqht of
(9 H20 /g
H20
sorbed cty eklstin )
0.5-
04-
0.30.2-
0.1-
O+
100
Figure 10 shows the sorption isotherm of water (HzO) on elastin at -293‘K.
DISCUSSION In the following we first discuss the interpretation of the results presented above, independent of any models for the nature of elastin and its elasticity. Having done so we then look‘at the implications of the results for such models.
Sorption Isotherm The isotherm shown in Figure 10 illustrates the rather weak interaction of water with elastin, consistent with the rather small percentage (
BIOPOLYMERS
Nuclear Spin-Relaxation Studies of Hydrated Elastin G. E. ELLIS and K. J. PACKER,* School of Chemical Sciences, University of East Anglia, Norwich N R 4 7TJ, England
Synopsis The nuclear magnetic spin-lattice and transverse relaxation processes for the ‘H and 2D nuclei in purified elastin (ligamentum nuchae), exchanged and hydrated with excess DzO, have been studied in the temperature range 276O-34O0K. The 2D relaxation results clearly show the presence of DzO 1) external to the bulk elastin sample, 2) in spaces within the bulk elastin, and 3) as an integral part of the protein on a molecular level. It is shown from these measurements that the water content of the protein itself changes from -0.8 g D20/g dry elastin a t -280°K to -0.2 g DzO/g dry elastin at -335OK, a decrease of 400%. T h e D20 content of the interfiber spaces decreases by less than 20% over the same temperature range. This fact throws considerable doubt on the validity of the values of p, the thermal expansion coefficient of elastin, used by other workers in discussion of the elastic mechanism in elastin. The elastin proton transverse relaxation shows the presence of three regions in elastin having different degrees of molecular mobility. These are assigned to protons associated with the crosslinks, a fairly mobile, hydrophobic, and lowwater-content region, and a more mobile higher water-content region. The temperature variation of the relative proportions of these three regions is explained in terms of a hypothetical temperature-composition phase diagram in which the two mobile regions are represented as two partially miscible pPases with different negative temperature coefficients of “solubility” in water. The implications of these observations for current views of the nature of elastin are assessed. I t is concluded that the spin-relaxation results are consistent with a multiphase structural model for elastin. An approximate sorption isotherm for the water/elastin system is reported and shows the relatively weak nature of the water/elastin interaction.
INTRODUCTION The protein elastin is an important structural component of animal connective tissue. With collagen it plays a load-bearing role in tissues such as ligaments and arterial wa1ls.l Elastin is characterized by its rubberlike properties when hydrated and the relatively high proportion of its constituent amino acids, which are nonpolar. The dehydrated protein is fibrous, brittle, and nonelastic and shows a strong tendency to cornify. Hydrated elastin has been investigated by several groups of workers with differing conclusions being drawn as to the nature and * To whom correspondence should be addressed. 813 0 1976 by John Wiley & Sons, Inc.
814
ELLIS AND PACKER
mechanism of its elasticity. Hoeve and Flory, in agreement with earlier workers, suggested that elastin is a random crosslinked network with an elastic behavior similar to rubber.2 Further studies led Volpin and cow o r k e r ~to~the ~ ~ conclusion that, although its elasticity is not due entirely to configurational entropy changes, elastin behaves very much like a conventional rubber. More recently, Weis-Fogh and Andersen5 concluded, from calorimetric measurements, that the elastic behavior of elastin differed fundamentally from that of a conventional rubber in that large changes in internal energy accompanied the stretching process. These workers postulated that elastin is a series of crosslinked globular “molecules,” each having a molecular weight in the region of 67,000. Stretching of elastin, it was suggested, increases the surface area of these globules producing additional waterlnonpolar peptide interactions, these being the main source of the changes in internal energy. This model was essentially based on the structural model of Partridge, which in turn was based on electron microscopy, elastin biochemistry, and gel filtration studies using elastin fibers as the stationary phase.6-8 The crosslinks in elastin have been identified as arising from lysine residues in the soluble elastin precursor and their chemical nature has been e l u ~ i d a t e d . ~ The suggestion that elastin is not a random crosslinked polymer network, but possesses a globular two-phase character, has been rejected by Hoeve and Flory in a recent paper.1° They maintain that the calorimetric measurements are consistent with and indicative of a rubberlike random network in which configurational entropy changes are the driving force in the elastic process. They also rejected the “oiled-coil” model of Gray et al.ll on the grounds that, like the globular, two-phase structure, it has intrinsically insufficient elastic response. In this paper we report the results of a study of the nuclear magnetic relaxation behavior of both the protein protons and the deuterons in the water (DzO) hydrating elastin.
EXPERIMENTAL Sample Preparation The elastin used in this paper was the ligamentum nuchae of mature beef. The fresh ligaments were scraped free of extraneous fatty tissue and cut into strips, 5-10 cm in length and approximately 1 cm2 in cross section. The strips were washed with 1% saline solution and then with distilled water. Finally, they were boiled in successive quantities of distilled water until the supernatant liquid gave a negative biuret test for the presence of protein. The purified elastin strips were then dehydrated by successive treatments with quantities of ethanol, ethanolldiethyl ether (l:l),and finally diethyl ether. The final solvent was re-
HYDRATED ELASTIN
815
moved under vacuum and the dried elastin stored under vacuum over silica gel. For the nuclear spin-relaxation measurements the elastin was soaked in excess D20 (99.9 atom %, equivalent to approximately five times the volume of the dry elastin) for several weeks with three changes of D20 during that period. This procedure replaces all exchangeable protons, i.e., those in -COOH, -NH2, -OH groups, by deuterons. The measuremeuts of the temperature dependence of the relaxation processes, reported below, were carried out on a single sample consisting of 200 mg of dry elastin and 790 mg D20 in a closed sample tube (10 mm diameter).
Sorption Isotherm The sorption isotherm of water and elastin was approximately determined by equilibrating weighed strips of dry elastin over aqueous salt solutions, of known relative humidities, in airtight containers. The elastin strips were weighed a t intervals over a period of several weeks until they achieved constant weight.
Nuclear Spin-RelaxationMeasurements Nuclear spin-relaxation times were measured using a modified Bruker B-KR 3228 pulse spectrometer. Proton resonance was observed a t 40 MHz; deuterium resonance a t 10 MHz. The magnetic fields were provided by an AE1 M12 electromagnet. Spin-lattice relaxation was studied using a 180"-~-90"pulse sequence for both *H and 2D nuclei. For the elastin protons the transverse relaxation behavior was determined directly from the free induction decay signal following a single 90" pulse. This was possible since the effects of magnetic field inhomogeneities were negligible on the time scale of these decays. The general procedure adopted was to adjust the spectrometer system to resonance using the free induction decay from a sample of glycerol and then to record the elastin 1H free induction decay, which, a t all temperatures studied, decayed much faster than that from glycerol. 2D transverse relaxation behavior was determined using Carr-Purcell/Gill-Meiboom (CPMG) pulse sequences [go,( - 7(18OYr - 27 - 180,t - T ) ~ ] . All signals were recorded using phase-sensitive detection and signal amplitudes were determined using a 512-channel digital signal averager, designed and built in this laboratory specifically for use with the spectrometer. Every lH free induction decay was recorded on two time scales, 1 and 10 p s between measurement points, with the last points measured on the longer of these time scales providing the baseline measurement. In general, between 32 and 128 scans were averaged for each measurement and each measurement was repeated a t least twice and, in many cases, more times. Spin-lattice relaxation and CPMG Tz mea-
816
ELLIS AND PACKER t IPS
t /PS
Fig. 1. The 'H free induction decay signal S ( t ) from DZO-exchanged and hydrated elastin at 277.9'K: ( O ) , lower time scale showing long-time behavior; (-), upper time scale showing short-time behavior. The continuous line is the long-time limiting slope; the broken line is the same line drawn on the expanded (upper) time scale.
surements were accumulated in the signal averager using the special modes of the averager designed to accommodate the pulse sequences used. Again between 32 and 128 measurements were averaged for each set of parameter values used in the pulse sequences. The pulse separations used in the spin-lattice relaxation measurements were sufficient to follow the relaxation through at least two decades of recovery. The 7 values used in the CPMG sequences were in the range 0.5-10 ms. All experimental data were output onto punched paper tape for off-line processing by computer or calculator or graphically by hand. In all measurements the sample temperature was controlled to approximately f0.2OK by a conventional gas-flow cryostat. Temperatures were measured using a thermocouple contained in a normal sample tube, this being substituted for the sample for temperature measurement.
RESULTS Proton Relaxation
Figure 1shows a typical lH free induction decay obtained from DzOexchanged and hydrated elastin (All the relaxation measurements re-
HYDRATED ELASTIN
817
Fig. 2. The temperature dependence of the fractional populations, p a , pb, and p e , of the two exponential and one Gaussian ‘H transverse relaxation components in DnO-exchanged and hydrated elastin. The individual symbols represent experimental points; the continuous lines give an indication of the overall trends in the values.
ported were made on one sample comprising 200 mg dry elastin and 790 mg D20). It can be seen that the decay is not monotonic. “Solid echo” e x p e r i m e n t ~ lindicated ~ , ~ ~ that only the very short-time ( t < 15 ps) part of the free induction decay was-refocused to any extent, thus leading to the conclusion that the longer time behavior arises from motionally narrowed magnetic dipolar interactions between protons.14 It was found that these ‘H free induction decay’s could be well represented by a superposition of three decays, the two slower decays being exponential, the fastest being Gaussian. Thus the lH free induction decay S ( t ) can be written as:
s(t)= sob=exp - (t/T2,)
4- P b exp - (t/T2b) 4- p c exp
- (t2/T2,2)]
where the pi and T2, are the fractional populations and transverse relaxation times of the ith component and where T2, > T2b > T2,. The ‘H free induction decays were recorded at different temperatures in the range 277O-34O0K. Figures 2 and 3 show the variation with temperature of the fractional populations and transverse relaxation times of
818
ELLIS AND PACKER
+ 100 -
I
0
I
I
I
I
I
I
I
10
20
30
LO
50
60
70
T/'C
Fig. 3. The temperature dependence of the' three 'H transverse relaxation times: (X) T2,, (+) T2*,(0) T2,;in DnO-exchanged and hydrated elastin. The continuous lines emphasize trends in the data.
T/"C
Fig. 4. The temperature dependence of the 'H spin-lattice relaxation time in DnO-exchanged and hydrated elastin. The broken lines emphasize the trends in the T Ivalues.
three free induction decay components. In every case the values of the pi and Tgi were obtained by graphical resolution of S(t).15 Spin-lattice relaxation of the nonexchangeable elastin protons was found to be virtually a single exponential recovery a t all temperatures in the range 2770-340°K and, in addition, was independent of where on the free induction decay, following the 90° pulse in the 18Oo-~-9O0 sequence, the measurement point was situated. Figure 4 shows the variation with temperature of this 'H spin-lattice relaxation time T I .
HYDRATED ELASTIN
819
t/ms
Fig. 5. 2D spin-lattice relaxation in DzO-exchanged and hydrated elastin a t 277.7'K; (X) experimental points. The resolution into two components is shown, the continuous line being the longer relaxation time component A and the broken line being the shorter relaxation time component B.
Deuterium Relaxation Figure 5 shows a typical 2D spin-lattice relaxation recovery curve obtained from the D2O-hydrated elastin sample. These curves could be resolved into two components at all temperatures, i.e., the relaxation process can be represented by: [l- R ( t ) ]= 2 b A exp - (t/T1,)
+ P B exp - (t/T~,)l
where R ( t ) = (M,(t)/Mo)and T I , > TI,. Figure 6 shows the observed , and TzBwith temperature. variation of P A , p ~TI,, Figure 7 shows a typical 2D transverse relaxation decay for the D20hydrated elastin sample. It was found that, a t all but the highest temperatures, these decays could be resolved into three exponential components, i.e., the decay can be written: Mx,(t)= Mx,(0)[Pa exp
- (t/7'2,) + PO exp - WT2J
+ P r exp - (t/T2JI Figures 8 and 9 show the variation with temperature of the populations and transverse relaxation times of the various components. As before, these were obtained by graphical resolution of the decay curves. As can be seen, the experimental scatter is fairly large but this is not surprising considering the nature of the measurements and the problems of resolving multiple exponential decays. The trends in the populations and time constants are clearly visible however.
ELLIS AND PACKER
820 0.7
0.6 Froctional poplhtion
0.5 0-4
0.3
Ib
7
20
30
LO
50
60
70
T/"C
Fig. 6. The variation with temperature of the fractional populations, pa and p ~ and , spin-lattice relaxation times, TI, and TI,, for 2D nuclei in DzO-exchanged and hydrated elastin.
f
Long time limting dope fnxn data exterdq to t = 850rns
I
200
100 t
300
/rnS
Fig. 7. 2D transverse relaxation in DzO-exchanged and hydrated elastin at 276.7OK showing the resolution into three exponential decays: (O), experimental points; (X) and (a),data derived by successive subtraction of longer time constant components.
821
HYDRATED ELASTZN 0.6
Pd
0.5 Fractonal
o . ~poplation 0.3 0.2
O
7
0.1
0
population
xx
0.34
I
O
o;
io
3b
Lo o;
$0
o;
T/OC
Fig. 8. The variation with temperature of the fractional populations, polPB. and Pr, of the three 2D transverse relaxation components in DzO-exchanged and hydrated elastin: (0) p m ,(X) PO, ( 0 )pr. The broken lines indicate the trends in the data. loo01
Fig. 9. The temperature dependence of the three ‘D transverse relaxation components: (0) Tz,,(X) Tz8,( 0 )7 ’ ~ The ~ . broken lines indicate the trends in the relaxation times.
ELLIS AND PACKER
822 0.8 -
0 0.706Weqht of
(9 H20 /g
H20
sorbed cty eklstin )
0.5-
04-
0.30.2-
0.1-
O+
100
Figure 10 shows the sorption isotherm of water (HzO) on elastin at -293‘K.
DISCUSSION In the following we first discuss the interpretation of the results presented above, independent of any models for the nature of elastin and its elasticity. Having done so we then look‘at the implications of the results for such models.
Sorption Isotherm The isotherm shown in Figure 10 illustrates the rather weak interaction of water with elastin, consistent with the rather small percentage (
