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First published online December 14, 2006
Journal of Experimental Biology 210, 12-26 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02613
Reversibly labile, sclerotization-induced elastic properties in a keratin analog from marine snails: whelk egg capsule biopolymer (WECB)
,*
Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA 92093, USA
* Author for correspondence (e-mail: rapoports{at}email.chop.edu)
Accepted 18 October 2006
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Key words: whelk egg capsule biopolymer, elastomer, mechanical properties, marine snail
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) and
hierarchical layers of ordered fibrous constituents
(Rawlings, 1999), suggestive
of a structure analogous to an intermediate filament (IF)-based material such
as that of hard alpha-keratin (Whitely and
Kaplin, 1977).
Despite apparent structural similarities at the IF level, there are
important structural and biogenic differences between WECB and hard
alpha-keratin. For example, WECB is composed of a plywood-type arrangement of
discrete fibrous sheets (foils) arranged at various angles such that isotropic
mechanical behavior is observed in the plane formed by the transverse and
longitudinal axes of the capsule (H.S.R. and R.E.S., unpublished data). In
contrast, hard alpha-keratin is a complex cylindrically arranged composite
with concentric hierarchical outer layers around a major axis of fibrillar
orientation (Hearle,
2000).
Whelk egg capsule biopolymer (WECB) possesses unusual tensile properties
(Rapoport and Shadwick, 2002).
Quasi-static mechanical tests revealed the following stress-strain features:
an initial region of low strain (>3-5%) distinguished by relatively high
modulus of elasticity and low hysteresis, followed by an apparent yield to a
plateau-like region with relatively low modulus and high hysteresis that
persists up to strains of at least 50% (see
Fig. 1). Although this
transition has the appearance of tensile failure, it is transient only, and
complete recovery occurs during unloading, as evidenced by stable
stress-strain curves from repeated cycles to high strains
(Rapoport and Shadwick, 2002).
For simplicity in referring to these material properties, the initial stiff
portion of the stress-strain curve is termed the `Hookean' region, due to its
linear elastic response. Following the Hookean region is the plateau-like, or
`yield' region, so called due to the zone of apparent yield that precedes it,
a convention used previously (Feughelman,
1964) to describe the similar response to strain found in hard
alpha-keratin (Fig. 2).
Although similar responses to strain are generated in both materials,
significant differences in mechanical parameters exist between WECB and hard
alpha-keratin. For example, Denny (Denny,
1988) reported that keratin possesses an approximate tensile
strength, breaking strain, toughness and maximum elastic modulus of 150 MPa,
50%, 20 MJ m-3 and 4000 MPa, respectively. We have found WECB to
posses a similar order of magnitude breaking strain of 95%, but all other
parameters are greatly reduced: 5 MPa tensile strength, 3 MJ m-3
toughness, and a 100 MPa maximum elastic modulus
(Rapoport and Shadwick,
2002).
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Since hard alpha-keratin is based on hierarchical arrangements of IF
structures, and WECB is putatively similarly organized, it is reasonable to
consider differences in higher order structure, accessory proteins, matrix and
stabilization as causative factors in resultant mechanical properties. Some
insight into the mechanics of hard alphakeratin has been obtained in the
studies of hagfish slime threads, which are matrix-free IF analogues that are
free from complicating modifications listed above
(Fudge and Gosline, 2004).
These IF analogues demonstrate mechanical properties that are different from
those of either WECB or hard alphakeratin. For example, hagfish slime threads
in saline possess an approximate tensile strength, breaking strain, toughness
and maximum elastic modulus of 180 MPa, 220%, 130 MJ m-3 and 220
MPa, respectively (Fudge et al.,
2003).
It is clear in the deviation of IF-based materials from simple IF fiber
mechanics that successive levels of processing in hard alpha-keratin and WECB
are responsible for the development of end-stage mechanical properties, and
that the respective formation of these materials is very different. Hard
alphakeratin is derived from epithelial cells and possesses cellular remnants
from this complex biogenic process (Van
Steensel et al., 2000), whereas WECB precursor is stored within
glandular vesicles and formed into a functional material in the following
two-step process. The raw capsule containing embryos is first formed in the
nidamental (capsule) gland (Fig.
3), passed outside the body through the reproductive tract onto
the substratum, and then secondarily covered by the ventral pedal gland (VPG)
located on the ventral surface of the foot
(Fig. 4). Treatment over the
course of many minutes to an hour in the VPG renders the newly formed capsule
rigid and insoluble (Ankel,
1929). Because a clear formation process exists that can be
interrupted at finite stages, we are able to form the hypothesis that
treatment in the VPG involves a chemical process that operates on IFs already
present to provide the critical end-stage mechanical properties in WECB.
Moreover, we hypothesize that harsh treatments involving desiccation, thermal
treatment and change in pH, will show that the chemical treatment in the VPG
involves the addition of covalent cross-linking to WECB.
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). In this study we focus on the WECB of two species,
Busycon canaliculatum (Linnaeus 1758) and Kelletia kelletii
(Forbes 1852). Because significant similarities in fundamental mechanical
properties of WECB exist among whelk species, in particular the shape of the
stress-strain curve, we feel the results of the current study are
representative of WECB across the order Caenogastropoda.
Capsule collection/preparation
Fresh egg capsules from Busycon canaliculatum were obtained in
seawater from The Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA
02543, USA. Capsules were subsequently washed with deionized water and frozen
at -20°C until ready for use. Defrosted capsules were washed exhaustively
in deionized water. The capsule was then dissected along the medial ridge
connecting both sides of the capsules, and the inner contents discarded. Whole
thickness strips for testing were formed by using pairs of standard razor
blades at a fixed width of 6 mm. Strips were on average 10-12 mm in length, by
repeatedly selecting the same cut orientation. Thickness of egg capsule strips
varied between 100 µm and 500 µm.
Collection of immature capsules
Capsule properties change as they are manufactured and processed by the
snail in a sequence of stages. Two designations are used in describing
capsules based on processing that occurs in the ventral pedal gland. Mature
capsules are those that have been fully completed by the snail and released
from contact with the animal. Immature capsules are those that have been
intercepted before the last stage of manipulation occurs in the ventral pedal
gland. In order to obtain immature capsules one must wait for the period of
reproduction and intercept capsules as they pass out of the nidamental gland
en route to the ventral pedal gland. Another, more controllable
technique for isolating immature egg capsules has been developed
(Ram, 1977). In this
technique, a neural extract is created by homogenizing the esophageal ganglion
of either male or female snails in 0.2 µm-filtered seawater on ice and then
centrifuging at >10 000 g for 30 s. The supernatant is
subsequently injected into the sinus cavity of the fecund female snail.
Extract injection reliably induced the production of single immature egg
capsules from gravid B. canaliculatum specimens maintained in aquaria
at The Marine Biological Laboratory, Woods Hole, MD, USA. J. Ram (Wayne State
University) generously donated an immature capsule and a mature capsule from
C. giganteus for comparison. In all cases, capsules were stored and
prepared for testing as previously described.
Scanning electron microscopy
Specimens of B. canaliculatum egg capsule material were air-dried
and sputter-coated with gold prior to viewing at 20 kV and a magnification of
up to x15000 on a Cambridge S-360 scanning electron microscope.
Collection and testing of egg capsules from K. kellettii
Kelletia kellettii is a species endemic to the coast of Southern
California and can be readily collected from the subtidal zone. Specimens of
K. kellettii were maintained in an indoor aquarium with ambient
flow-through seawater. This laboratory population was observed to continue
normal reproductive behavior in synchrony with free-living cohorts in the
ocean. During a reproductive cycle following mating, a female will begin the
process of egg capsule deposition on available substrate, which in this case
was an aquarium glass wall (see Fig.
4). It is possible to watch capsule formation in progress and
intercept capsules en route to the ventral pedal gland
(Fig. 5). In this fashion an
experiment was conducted in which snail capsules (N=3) were
intercepted at different stages in processing and tested by quasi-static
methodology (Rapoport and Shadwick,
2002). Briefly, tensile tests on capsule specimens were performed
on an MTS material testing apparatus; model 858 Mini Bionix, MTS Systems
Corporation, Eden Prairie, MN, USA.
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Measurements of dynamic mechanical properties
Most biological materials possess viscoelastic properties
(Vogel, 1988). Since these
materials present time-dependent as well as temperature-dependent properties,
a standardized method for characterizing viscoelastic behavior was utilized
(i.e. forced-oscillation). Specifically, we wanted to make separate tests of
the dynamic response of the two distinct parts of the stress-strain curve,
i.e. the Hookean and the yield regions (see Figs
1 and
2). This technique might
present a method for teasing apart contributors to bulk mechanical behavior in
WECB and also addressing observations from stress-relaxation experiments made
previously (Rapoport and Shadwick,
2002). These experiments showed that when WECB was stretched into
the yield region and held at constant length, the force relaxed, and the high
stiffness (Hookean) behavior reappeared when stretch was reapplied.
Egg capsule strips 6 mm wide were clamped into a dynamic testing device.
Specimen thickness was measured with a micrometer. The initial length of the
specimen, Lo, was measured using a Vernier caliper. The
testing rig consisted of an electromagnetic vibrator (model 400, Ling Dynamic
Systems, Don Mills, Ontario, Canada) mounted vertically on the adjustable
crosshead of a test frame, which allowed different levels of pre-stretch to be
applied. Sinusoidal oscillations were imposed by a function generator (BK
Precision 3011B, Chicago, IL, USA) driving the vibrator. Specimens were tested
in two modes relating to the shape of the quasi-static stress-strain curve:
Hookean (N=5) and yield (N=6), and a third mode, vibe-ramp
(N=5). For the Hookean test mode, specimens were extended to a mean
strain of 2% by adjusting the position of the crosshead to place the
material's behavior well within the Hookean region (e.g. <
5% strain).
Since dynamic testing is done at small strains (on the order of ±0.05
mm), the material remained in that region during the extent of the dynamic
testing. In the yield test mode, pre-strain was applied as described above.
Finally, vibe-ramp denotes a test conducted in the yield region where, during
the dynamic test, a constant extension rate was applied to the material
(0.03 mm s-1) through motion of the crosshead. This had the
effect of overlaying a ramp displacement onto a sinusoidal displacement.
Displacement was measured at the vibrator through a fabricated cantilever
transducer comprising strain gauges in a Wheatstone bridge. Force was measured
through another fabricated cantilever transducer also comprised a similar
strain gauge-based Wheatstone bridge. The resonant frequency of the force
transducer was 200 Hz, sufficiently higher than the maximal testing
frequencies that approached 15 Hz. Force and displacement signals were
conditioned by matched amplifiers before being recorded digitally using a TL-2
Interface board (Axon Instruments, Burlingame, CA, USA) and Axotape 1.3.0.09
software on a 486-PC.
Formic acid treatment of B. canaliculatum specimens
In addition to dynamic and quasi-static mechanical tests of native B.
canaliculatum egg capsule strips, tests were also conducted following
treatment of specimens with formic acid. Formic acid is a common organic acid
used in a variety of protein applications. Formic acid irreversibly
destabilizes protein secondary structure when its aldehydic functions form
esters with the aminohydroxy group of the protein backbone
(Josefsson, 1962;
Josefsson, 1966). In this
fashion, we would expect dissolution of the labile or noncovalent associations
within WECB (e.g. hydrogen bonds) due to the low pH formic acid environment.
As normal protein conformation is disrupted, progressive attachment of
aldehydic functions will proceed. In destabilizing the protein, it might be
possible to study the purely covalent interactions present in the material
that are consistent with the different stages of processing.
Specimens were first measured using a micrometer to determine thickness, and then mounted between the clamps of a MTS material testing apparatus (model 858 Mini Bionix, MTS Systems Corporation, Eden Prairie, MN, USA). All strips used were pre-tested before treatment to ensure normal behavior. Formic acid was employed as follows: strips of WECB were allowed to equilibrate for 30 min in the following concentrations of formic acid prior to testing: 0% (control; N=4), 11% (N=4), 22% (N=4), 44% (N=3) and 88% (N=4). We noticed that for incubation times approaching 1 h, there was a prominent alteration to mechanics that was only partially reversible. We mitigated any damage-history effect that might also be present at 30 min incubation times by using multiple samples per concentration, and no cross-concentration treatment of samples. All concentrations of formic acid used in this study possess a pH <2.0.
Specimen starting length, L0, was measured using
Vernier calipers. To maintain specimen hydration with formic acid, the strip
was covered with moist tissues and a sheet of cellophane. Force-length
information pertaining to extension loops was recorded at an extension rate of
0.025 mm s-1 and subsequently converted to stress-strain diagrams,
where stress is the force/cross-sectional area, and strain is the
extension/L0. The following parameters were calculated
from the stress-strain diagrams: elastic moduli, yield stress and resilience.
Elastic moduli of the Hookean and yield regions were determined by taking the
slopes of linear regressions fitted to those regions. The intersection of
those two regression lines denotes the yield stress, the transition from
Hookean to yield. Resilience (R), the ratio of energy recovered to
energy imparted to the material, is calculated from the following equation:
 | (1) |
In this study, H is the hysteresis of the stress-strain loop
derived from quasi-static mechanical testing. Hysteresis is calculated
numerically in spreadsheet form by subtracting the area under the return curve
from the area under the extension curve and dividing the result by the area
under the extension curve. Resilience can be thought of as the elastic
efficiency of the material in question. Synthetic rubbers, for example,
approach a resilience of 96-97% (Gosline,
1980).
Finally, an additional strip that was allowed to equilibrate for 1 h in 88% (pH <2) formic acid was tested dynamically for comparison to the native state using the same protocol mentioned for the native specimens in the preceding section. The longer incubation time as mentioned earlier was found to irreversibly affect the mechanics of WECB.
Analysis of dynamic data
As previously detailed for viscoelastic materials (e.g.
Gosline, 1971;
Szulgit and Shadwick, 2000),
sinusoidal displacement signals are found to lag their corresponding
sinusoidal force signal by an angular displacement described as the phase lag,
. The phase lag is related to the storage modulus (E')
and to the loss modulus (E'') in the following expression:
 | (2) |
Axotape 1.3.0.09 software used in collecting the dynamic data also provides
an interface for data analysis. Spectra attained for the force and
displacement signals are used to calculate the phase lag . The complex
modulus can be determined in a similar fashion allowing calculation of storage
and loss moduli through the following trigonometric relationships:
 | (3) |
 | (4) |
where |E| is the complex modulus.
The storage and loss moduli are indexes of elastic and viscous
contributions to stiffness, respectively. A common convention in studying
viscoelastic materials is to plot both Tan and E' versus
the logarithm of the excitation frequency generated from dynamic behavior at
various temperatures (e.g. Vincent,
1990). These resultant master curves are useful for understanding
the range of viscoelastic behavior of a material and facilitate inter-material
comparisons.
Citrate/phosphate buffer pH series treatment of B. canaliculatum specimens
To further explore the effects that a wide pH range has on WECB protein
mechanics, strips (N=5) were in turn equilibrated for 30 min in
citrate/phosphate buffer (Stoll and
Blanchard, 1990) at the following pH values: 2, 5, 7 and 10. To
eliminate a possible effect of history by proceeding sequentially through the
pH range, the following order was chosen to create maximum shift in pH during
the course of the experiment: 10, 2, 7 and 5. Following equilibration, B.
canaliculatum egg capsule strips were tested at 19°C as described
previously for the formic acid-treated specimens, with the exception that
hydration was now maintained through the use of an environmental chamber. Use
of the chamber allowed samples to be equilibrated at various pH values without
removing them from the testing clamps- a potential source of variation in the
mechanical response of the material. Elastic modulus of the Hookean and the
yield regions, yield stress and resilience were measured as described
above.
Thermal series treatment of B. canaliculatum WECB
Thermal studies present an alternative to acidification that similarly
affects stabilization in proteins, primarily through the disruption of
hydrophobic interactions, electrostatic interactions and hydrogen bonding
(von Hippel and Wong,
1965).
For this study strips were placed in water in a temperature-controlled environmental chamber. Specimens tested in this fashion were clamped into the tensile testing device throughout the experiment while being subjected to temperatures ranging from 5°C to 92°C. Ten stress-strain cycles were performed after the specimen reached the target temperatures of 7°C (N=2), 20°C (N=2), 40°C (N=2), 65°C (N=2), 78°C (N=2) and 92°C (N=5). Elastic modulus of the Hookean and the yield regions, yield stress and resilience were measured as described previously. Following testing, strips were allowed to equilibrate for 24 h at 19°C in water prior to exploratory testing to determine the extent of recovery from thermal perturbation in the specimen.
Determining toughness of dehydrated WECB
Chemical treatments (formic acid, buffers) have been used to alter the
behavior of WECB. Hydration is also a related concern in testing IF materials
(Fudge and Gosline, 2004).
Therefore, the quasi-static properties of WECB as determined through toughness
calculations were determined by air-drying five strips of the material. These
strips were subsequently tested as described previously with the exception
that instead of repeated cycling, these strips were pulled to failure. The
area under the curve to failure is defined as a materials' toughness - the
ability to absorb energy - and a useful index for comparing properties among
materials (Rapoport and Shadwick,
2002). Initial modulus was calculated as the initial slope of the
stress-strain curve. Yield strain, yield stress and breaking strain were
obtained directly from the stress-strain curves.
Statistical analyses of dynamic and quasi-static data
For the dynamic testing data set, statistical differences among treatments
were investigated. Statistical regression analyses, ANOVA comparisons and
post-analysis Tukey HSD or Holm-Sidak tests were conducted using both
STATGRAPHICS Version 5.1 and MINITAB version 13.32 software (Statistical
Graphics Corp., Herndon, VA, USA and Minitab Inc., State College, PA, USA,
respectively). For dynamic data, regression lines were fit to pooled storage
modulus/frequency and Tan/frequency plots for each test mode (Hookean,
yield and vibe-ramp) and each parameter, Tan and storage modulus.
Pair-wise comparisons of regression coefficients were made. For quasi-static
data, one-way ANOVAs were carried out for the following parameters calculated
for each test mode: Hookean modulus, yield region modulus, yield stress and
resilience. Sample size is reported elsewhere. Statistical significance was
calculated using a 95% confidence level.
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Immature capsule collection and SEM
Immature capsules roughly approximate the shape of the fully processed
mature capsule as illustrated for three species in
Fig. 5. Additionally the
immature capsules can be readily solubilized whereas mature capsules are
resistant to degradation (H.S.R. and R.E.S., unpublished). Attempts to
mechanically test immature capsules either quasi-statically or dynamically
were unsuccessful due to a lack of material cohesiveness when subjected to
even low-level deformation. SEM studies of immature and mature B.
canaliculatum (Fig. 6)
revealed structured lamina with distinct fibrous presence (foils) in differing
orientations. Capsules isolated from K. kelletii during various
stages of processing and subsequently quasi-statically tested revealed that
the characteristic bimodal stress-strain behavior was not evident until some
time had been spent in the ventral pedal gland
(Fig. 7). Specifically, these
capsules possess long-range elasticity consistent with the yield region of the
bimodal stress-strain diagram for mature capsule material, but lack the
high-stiffness Hookean region found at lower strains. The Hookean region
develops only after longer exposure times in the ventral pedal gland.
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Mechanical properties: formic acid concentration series The formic acid treatments and the buffered pH treatment elicited seemingly similar results in that there was an apparent change in the shape of the stress-strain curve (Figs 8, 9). Specifically, the Hookean region is diminished with increasing concentrations of acid while the yield region seemed unaffected. We found that by increasing the formic acid incubation time from 30 min to 1 h at higher acid concentrations, the effect became pronounced enough to completely eliminate the Hookean region. Since this behavior was partly irreversible (Fig. 10), and probably represented damage in the material through excessive aldehydic attachment, we only analyzed the data from the 30-min incubations. In contrast, with shorter time intervals, changes to stress-strain behavior were apparently fully reversible upon returning the specimen to native conditions. In sum, the formic acid effect acts on a labile structure that provides the high-stiffness (Hookean) behavior.
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1.7 MPa. In
experiments with formic acid that persisted for longer incubation times
(1 h), the yield region was removed completely. Lastly, the elastic
modulus of the yield region was not altered significantly with formic acid
treatment (P=0.136; Fig.
13). However, the Hookean region modulus did vary significantly
with formic acid (P=0.002) concentrations above 11%, all varying with
the 11% concentration and ranging from 30 MPa to 10 MPa. The overall
formic acid effect (especially at longer incubation times) appears to be
consistent with the preliminary heating results previously discussed
(Rapoport and Shadwick, 2002)
as well as the heating results from this study that follow.
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Citrate/phosphate buffer pH series treatment
Replicates were cycled ten times each at pH of 2, 5, 7 and 10. The tenth
cycle from each sample was used for comparison in order to ensure stability of
the stress-strain response. Visual inspection of the results of each piece of
WECB in buffer of differing pH suggests that only pH 2 has any noticeable
effect (Fig. 9). Statistically,
resilience and elastic modulus of the yield region did not vary
(P=0.319) and (P=0.986), respectively, across pH (Figs
11,
12 and
13). Yield stress did vary
significantly across treatments (P=0.003) with a Tukey HSD test (95%
confidence limit) showing that pH=10 and pH=7 were both significantly
different from the pH=2 treatment. The pH=5 treatment was not significantly
different from the pH=2 treatment although qualitatively this appeared to be
the case (Fig. 12). The
Hookean region modulus did not vary significantly across treatments based on a
Tukey HSD test (95% confidence limit), but differences were suggested through
an ANOVA (P=0.046) (Fig.
13).
Thermal treatment series
Fig. 14 shows the extension
results from a representative specimen showing the transition that occurs as
the yield stress decreases with increasing temperature. At 92°C the
Hookean behavior disappears completely. Not shown is the recovery curve
following the thermal treatment that illustrates, as in the other treatments,
the ability of WECB to apparently return to its initial properties following
perturbation with harsh treatments that normally cause protein denaturation or
solubilization. There are qualitative behavioral similarities between the
92°C treatment, and the 1-h incubation time in 88% formic acid, both of
which appear similar in mechanical behavior to WECB retrieved from the VPG
before processing is complete (Fig.
7II). The Hookean region modulus decreases steadily with
increasing temperature, but due to high variability these changes are not
statistically significant (P=0.702;
Fig. 13). Since there was no
Hookean region in the 92°C treatment, it was not considered in this
comparison. Similarly, there was no yield stress for statistical comparison
from the 92°C treatment. For the remaining temperature regimes, yield
stress decreased steadily but, like the Hookean modulus, these changes were
not statistically significant (P=0.702;
Fig. 12). Elastic modulus in
the yield region was unaffected statistically by the temperature treatments
(P=0.695; Fig. 13).
Lastly, resilience values did show a significant difference
(P=0.043), but only in a comparison between resilience values from
the 92°C and 7°C treatments. Qualitatively, differences could be seen,
but variation in the experiment failed to reveal strong statistical
differences.
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Dynamic mechanical tests
Results from dynamic testing of native B. canaliculatum specimens
are expressed in two graphs consisting of frequency as the independent axis
and two respective dynamic mechanical parameters as the dependent axes.
Tan, an index of phase shift between the force generated in a specimen
and its excitation displacement (viscous damping) is one parameter. Tan
provides information about mechanical hysteresis as well as resilience in a
material. The relationship between Tan and hysteresis is directly
proportional, while the relationship of Tan with resilience is
inversely proportional. The other parameter that can be realized from dynamic
testing is the storage modulus (E'), an index of elastic energy storage.
Results from the three test modes (Hookean, yield and vibe-ramp) were plotted
together to assess differences. Fig.
15 shows Tan and storage modulus (E') results from
the aforementioned experiments plotted against frequency. In all cases, a weak
regression coefficient is indicated with r2 values
<0.5, and in several cases, r2 is <0.1: (Tan
results: Hookean, N=5, r2=0.0149; yield,
N=6, r2=0.0625; vibe-ramp, N=5,
r2=0.4077; E' results: Hookean, N=4,
r2=0.30; yield, N=6, r2=
0.27; vibe-ramp, N=5, r2=0.535). Prior to
cross-test mode statistical comparisons, within-test mode statistical
comparisons were made and from these it was determined that within-test mode
replicates were not significantly different from each other at a 95%
confidence level (Tan results: Hookean, N=5, P=0.593;
yield, N=6, P=0.448; vibe-ramp, N=5,
P=0.815; E' results: Hookean, N=4, P=0.584;
yield, N=6, P=0.367; vibe-ramp, N=5,
P=0.464). The storage modulus (E'), of the Hookean test mode
was significantly greater than E' both for the yield (P=0.001)
and vibe-ramp test modes (P=0.20), which were not themselves
statistically different from each other (P=0.116). Tan results
show that the Hookean test mode is significantly different from the vibe-ramp
test mode (P=0.0). The Hookean test mode, however, is not
statistically different from the yield test mode (P=0.743).
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results indicated that the
formic acid treatment is significantly different from the other tests
(w/Hookean, P=0.0015; w/yield, P=0.0; w/vibe-ramp,
P=0.0). Similarly, the storage modulus is significantly lower than
Hookean and vibe-ramp tests (P=0.0129; and P=0.0008,
respectively), but not the yield mode (P=0.0610).
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To summarize these results, storage modulus and Tan results for the
three test modes all possessed weak regressions as a function of frequency,
with Hookean and yield Tan possessing extremely weak regressions
indicative of virtually no frequency dependence. Within-test mode statistics
failed to show differences, thus indicating fidelity among the replicates.
Variability among the test modes is summarized as follows. The Hookean storage
modulus is different from the storage modulus of yield and vibe-ramp, which
are not different from each other. The Tan for the Hookean test mode is
different from vibe-ramp, but not yield. Lastly, the formic acid Tan is
different from all other experiments, and the storage modulus from formic acid
treatment is different from Hookean and vibe-ramp, but not yield.
Qualitatively, the storage modulus for the formic acid-treated specimen was
lower than either yield or vibe-ramp, as expected, but these differences
failed to materialize in the statistics.
Toughness of dehydrated specimens
Fig. 17 illustrates the
results from five dehydrated specimens extended to failure. Failure ranged
from approximately 40% to 140% strain. There is an order of magnitude increase
in yield stress [mean value (± s.e.m.) = 37.0±5.56 MPa,
N=5], and little to no change in yield strain (mean yield strain was
3.5±1.0%, N=5), when compared with hydrated tested specimens.
Initial modulus was also an order of magnitude greater than hydrated specimens
(mean value = 900.96±58.91 MPa, N=5). Mean toughness of these
specimens was 72.57±16.67 GJ m-3 (N=5). Mean
breaking strain was 84.9±19.5% (N=5).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Chapman, 1969;
Feughelman, 1979;
Wortmann and Zahn, 1994;
Hearle, 2000). One important
question is how matrix and IFs each contribute to the mechanical properties.
These models were revisited (Fudge and
Gosline, 2004) and the compelling argument, based on examination
of a matrix-free IF analog (hagfish slime threads), was presented that the
matrix limits hydration of IFs, thus regulating water's role as a plasticizer;
this in turn leads directly to the observed stress-strain responses.
Furthermore, this `matrix squeeze' hypothesis
(Fudge and Gosline, 2004) is
based on the presence of covalent disulfide crosslinks that appear to function
in IF-IF, matrix-IF and inter-IF roles. It is thought that crosslinks, in
binding matrix together with IFs, restrict the hydration of IFs, which in turn
directly impacts mechanical performance through alterations in elastic modulus
(i.e. stiffness). WECB is hydrated in its native state, and therefore matrix-squeeze modeling is not entirely appropriate for this material. However, there are aspects to the model that apply to WECB. Observations on how IFs can or should behave with various modes of covalent crosslinking and differing hydration levels may be relevant to WECB. In particular, simple crosslinking between adjacent coiled-coils and possibly combined with crosslinking between non-coiled-coil terminal regions in IFs should lead to high initial modulus (stiffness) and relatively low yield stress, as seen in WECB. The lack of these crosslinks and matrix in hagfish slime threads means that there should be low initial modulus and low yield stress, which is indeed the case. However, by dehydrating hagfish slime threads, modulus and yield stress can be substantially increased because the lack of a plasticizer allows H-bonding to dominate. In hard alpha-keratin, a very stiff and brittle material, the addition of water acts as a plasticizer allowing a decrease in stiffness, but not much due to the `matrix squeeze model'. WECB fits somewhere in between these two materials. It is normally hydrated and thus possesses lower stiffness and strength, but covalently crosslinked, and when dry, exhibits marked increases in stiffness.
WECB's mechanical behavior is interesting because it can be tied to a discrete manufacturing process terminating in the snail's ventral pedal gland. Based on SEM comparisons of immature capsules with mature capsules, we now know that the mechanical integrity of WECB does not arise from any gross structural changes resulting from manipulations occurring in the pedal gland. Rather, the capsule structure is completed in the nidamental gland and is both molded and somehow stabilized to a mechanical cohesiveness by activity of the ventral pedal gland. We conclude that since the hierarchical fibrous structure is already present in the immature capsules, it is highly likely that treatment by the ventral pedal gland induces formation of crosslinks and possibly adds matrix, ultimately leading to the creation of an insoluble protein polymer. Furthermore, qualitative observations of the mechanics of these capsules as they are processed prior to complete stabilization suggest that the complex bimodal mechanical behavior develops sequentially. The preliminary mechanical properties seem likely to be related to the long-range elasticity seen in the yield region of the native egg capsules, or alternatively, a much simpler rubber-like behavior existing in the nascent capsule prior to its final crosslinking. Irrespective of the nature of the initial properties at the early stages, the Hookean region appears to develop later in processing and heralds the completion of the capsule maturation process.
Model
Based on our observations, and observations for alpha-keratin and hagfish
slime threads, we advance the following model on the structural stability and
mechanics of WECB (Fig. 18).
(A) In general WECB consists of sheets (foils) of 1 µm diameter
macrofibrils consisting of hierarchical assemblages of intermediate filament
(IF)-type structures arranged in parallel (cylindrical structures). SEM images
(Fig. 6) suggest that the
macrofibrils may not be discrete structures, and that there may be connections
among macrofibrils. There is presumably matrix associated with the IFs, but it
is not well characterized at this time. Successive layers are arranged in
varying orientations throughout the thickness of the material and are
presumably responsible for bulk orthotropic mechanical behavior in the plane
formed by foils. As the inset illustrates, each macrofibril comprises the
staggered head to tail arrangement of coiled-coil molecules at its smallest
level of organization (Gathercole,
1969; Flower et al.,
1969). Alignments of coiled-coils favored by charge-based
self-assembly are responsible for repeat striation patterns seen in EM
sections. For simplicity, IF structures, the next hierarchical level of
organization, are not shown in the model. (B) Tensile loading results in
shearing and sliding of successive layers, leading to material failure.
Immediately following formation in the nidamental gland (NG), WECB is white in
color and still soluble, indicating lack of sclerotization. Bulk mechanical
measurements at this stage are hampered by a lack of cohesiveness in the
material, but noncovalent interactions (further supported by the ease at which
material at this stage can be rendered soluble; H.S.R., unpublished
observation) are probably responsible for maintaining its structure and
rudimentary cohesiveness. Here we conjecture that the application of tensile
force allows the foils to slide apart fairly easily. Since the individual
foils appear to be discontinuous and interdigitated with other layers (foils
appeared to be laid down in a fashion analogous to disordered strokes of a
broad painting brush; thus, the foils do not necessarily span the entire
length of the egg capsule), the loose material associations resulting from
self-assembly are not sufficient to hold the material together and difficult
to measure at the bulk material scale. (C) Shearing of successive sheets with
restoring force provided by sporadic linkages, primarily among macrofibrils
(idealized crosslinks, illustrated as black lines in inset). During treatment
in the ventral pedal gland (VPG) crosslinks begin to stabilize WECB by linking
successive foils as the muscular action of the VPG brings them closer
together. These crosslinks are hierarchically ubiquitous, stabilizing IFs,
macrofibrils and matrix. At early stages of crosslinking, the density of the
crosslinking is minor (see inset as well), and the material behaves much like
a pliant rubber with a restoring force provided by bulk deformations of
matrix. Mechanics are dominated by features on the bulk material scale (i.e.
changes in foil position and perhaps some warping of foils). (D) Tightly
crosslinked material with stresses transferred down to level of coiled-coils.
Following processing in the VPG, the crosslink density is now sufficient to
transfer mechanical stress down to the smallest hierarchical level of the
material, thus adding a level of complexity to the witnessed mechanical
response. Here, we see the development of the Hookean region that results from
strain directed to a network of stiff coils. Unraveling of coiled-coils into
random coiled configurations (see inset) begins at the transition between the
Hookean and yield regions. In the yield region, extensive unraveling of
coiled-coils occurs. Under physiological conditions this is not a stable
conformation, so recovery of the initial state occurs when the strain is
removed; the restoring force is provided by a combination of matrix
contributions and entropic mechanisms, but not necessarily due to the IFs
themselves (Kreplak et al.,
2005). The organization that allows the stiffer Hookean region to
occur at initial strain application is an in-parallel loading of both
coiled-coils and matrix.
|
Deconstructing a material's mechanical properties to gain insights into its formation
We previously found that the Hookean region of the stress-strain behaviour
could be reversibly eliminated from the material behavior with a thermal
treatment pointing to the fact that the Hookean region is heat labile and the
yield region is not. The present study determined that high concentrations of
formic acid as well as buffers with low pH could also mimic aspects of the
thermal effect. In sum, the results of these treatments suggest a crosslinked
polymer with the ability to maintain long-range elasticity in conditions that
might otherwise be destructive to proteins.
There is utility in exploring the conditions necessary for diminishment of
the Hookean region. A synthesis of the salient features of alpha-keratin
mechanical models suggest that the Hookean region represents coiled-coil
structural motifs, and that by applying strain to the material, sufficient
energy is applied to begin unraveling these domains at the transition between
the Hookean and yield regions. Additional unraveling events can accommodate
further extension within the yield region
(Kreplak et al., 2004).
How does the behavior of WECB resemble this? First, support exists in the
observation that there is a recovery feature to the mechanics of WECB at
extensions past the Hookean region that allow Hookean-like stiffness to
reappear in the yield region. For example, consider a stress relaxation
experiment conducted earlier (Rapoport and
Shadwick, 2002), during which the WECB specimen was stretched into
the yield region and subsequently held at constant strain. Stresses dropped
almost immediately. When a strain was applied to the specimen, stresses rose
in a fashion consistent with the Hookean region (high slope=high modulus=high
stiffness), instead of being consistent with the yield region where the
specimen was originally resting at constant strain. This suggests that during
the stress-relaxation experiment, holding the specimen at constant strain
allowed some sort of molecular reorganization as stresses fell that imparted
behavior upon the material commensurate with the Hookean region. This
molecular reorganization is probably due to the reconstitution of
coiled-coils. Second, with thermal/chemical treatments we are probably seeing
a decrease in the strain energy required to unravel these coiled-coils,
particularly in the case of nonreversible formic acid treatment in which
aldehydic functionalities are indiscriminately attaching to WECB in a fashion
that impedes the reformation of higher order structure. The general mechanism
of action of acid/heat treatments is the dissolution of non-covalent
stabilization, probably hydrogen bonds. Thus, the yield point can be shifted
to lower energy levels.
The data also support this sequence of events. There is a clear decrease in yield stress and elastic modulus of the Hookean region with all three denaturing treatments (Figs 10, 11). Changes in resilience (Fig. 8) are not as clear, except in the case of temperature treatment. In this particular case, high temperature was the only treatment that completely removed the Hookean region. Later explorations with longer incubation times at higher concentrations of formic acid also completely removed the Hookean region, and should be expected to exhibit the same resilience increase as ordered structure is replaced with random coils. Complete elimination of the Hookean region leads to changes in the overall shape of the stress-strain diagram and concomitant changes in resilience.
Dynamic testing afforded a better opportunity to look at the time course of coiled-coil reconstitution following perturbation. The `vibe-ramp' test mode consisted of extending the specimen into the yield region, but instead of holding at constant strain (and allowing Hookean behavior to re-develop) a small-value constant strain rate was maintained, which we believed would be sufficient to prevent molecular reorganizations from developing during the dynamic testing.
The storage modulus resulting from the Hookean test mode (85±2.3 MPa, N=48) was similar to the elastic modulus measured in quasi-static tests, but higher than the storage modulus resulting from either the yield (26±0.83 MPa, N=72) or vibe-ramp (22±0.83 MPa, N=67) test modes. This is supporting evidence that WECB possesses two distinct mechanical behaviors derived from coiled coil domains and matrix, with very different (yet not necessarily independent) properties. Curiously, results suggest that the vibe-ramp test mode did not have the desired effect of eliminating any possible Hookean contribution. That is, there are no significant differences between the behaviors of WECB under the vibe-ramp or yield test modes. Indeed, storage moduli in both these tests were higher than expected based on the elastic moduli from the quasi-static results (which range from approximately 2 to 10 MPa). This implies that the coiled-coil motifs consistent with the Hookean region (and, the likely hydrogen bonding that both stabilizes within and among these structures) are reforming or engaging even under continuous extension. This agrees with keratin models suggesting that in the yield region there are still coiled-coil motifs unraveling, and that the witnessed strain-relaxation effect during quasi-static testing must only be a partial reforming of the coiled-coil regions.
Tan (an indicator of viscous loss, related to both hysteresis and
resilience) results suggest that there is no real difference in resilience
among the three test modes when data spread is considered. This is consistent
with the idea that strain energy alone cannot remove coiled-coil structure
like the harsher of the acid/thermal treatments is capable of accomplishing.
Additionally, in dynamic testing where small oscillatory strains are applied
and material recovery is demonstrated, it suggests that not passing through a
yield zone allows a convergence of hysteresis/resilience values. This is a
departure from expected results. The Hookean region should have a larger
resilience (i.e. lower Tan) than either the yield or the vibe-ramp test
modes because of its high stiffness and low hysteresis. If we now consider the
dynamic mechanical results of the longer, 1 h incubation time of the 88%
formic acid treated specimen (which is effective at eliminating the Hookean
region behavior from the material), the mean storage modulus (2.4±0.09
MPa, N=49) is much less than for any of the other dynamic tests, but
similar to the elastic modulus of the yield region based on quasi-static
tests. The mean Tan for the formic acid treatment (0.25±0.01,
N=41) is in the same range as for the other tests (Hookean,
0.28±0.01, N=61; yield, 0.28±0.006, N=72;
vibe-ramp, 0.31±0.01, N=54), but slightly lower, which is
consistent with the disruption of the non-covalent interactions and
coiled-coil structures that generates the Hookean region.
It is the interplay of hierarchical levels of organization that allows WECB
to present interesting mechanical properties. As an extracellular keratin-like
material from the marine environment, WECB still displays characteristic
behavior that can be defined by keratin models and elucidated by matrix-free
modeling of hagfish slime threads (Fudge
et al., 2003). We have shown that WECB possesses lower stiffness
(100 MPa) when compared to hydrated alpha-keratin (2-6 GPa). This is
likely explained by the matrix-squeeze model
(Fudge and Gosline, 2004),
where WECB's native hydrated state requires less covalent crosslinking for its
function when compared to stiffer keratins such as horn, hair and hoof.
Additionally, we see that we can generate a stiffer, stronger material by
dehydrating WECB, at which point both its yield stress and initial modulus
rise by an order of magnitude (37.0±5.56 MPa, N=5) and
(900.9±58.91 MPa, N=5), respectively. This is analogous to the
changes seen between wet and dry alpha-keratin, but differing in magnitude.
Yield strain in dehydrated WECB remains essentially unchanged from the
hydrated form and breaking strain decreases from 95% to 85%, although the
dehydrated breaking strain had a degree of variation, probably due to
incomplete drying among samples. In comparison with hydrated keratin, it is
interesting to note that breaking strains for these two materials are similar:
50% (hydrated alpha-keratin) to 85±19.5% for WECB. Drying WECB appears
to make it more similar to hydrated alpha-keratin, but because crosslink
and/or matrix density is comparatively low, WECB never truly attains a modulus
on par with keratin. Again, this is probably due to the environment in which
these materials are expected to operate. A further consideration is that WECB
functionally acts as a protective, yet diffusive barrier for developing
embryos. Covalent crosslinking of the same magnitude seen in keratin (based on
comparison of detectable cys residues as a proxy for disulfide interactions)
might impede this function by creating a much more compacted and water
resistant material.
Lastly, harsh denaturants appear to cause WECB to behave in a fashion analogous to wet hagfish slime threads, although the utility in comparing these two materials in this fashion is minor because WECB presumably has matrix present irrespective of treatment, whereas slime threads do not. Based on this model, we predict that in the case of large strains, sufficient change to coiled-coil conformation and resultant fiber banding periodicity should be observable as a change in birefringence (or through the use of X-ray diffraction). Relaxation of the capsule that occurs during a constant strain experiment in the yield region will probably also present a change in conformation detectable by birefringence microscopy or X-ray diffraction as coiled-coils reform.
Future directions
We have already almost completely identified a gene for WECB. With complete
gene information, we hope to undertake expression experiments in which a more
detailed understanding of structural relationships to mechanical properties
[see other work (Keeley et al.,
2002)] can be determined. Moreover, complete gene information
should provide additional corroboration of coiled-coil structure and enhance
WECB's characterization as a keratin analog. Finally, we intend to obtain
protein sequence information from the primary protein(s) present in the
ventral pedal gland during the egglaying portion of the reproductive season.
It is our hope that the isolation of temporally expressed gene products in the
ventral pedal gland will lead to an understanding of the biochemistry
responsible for the sclerotization of the capsules.
|
Acknowledgments |
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|
Footnotes |
|---|
Present address: Division of Cardiology, Children's Hospital of
Philadelphia, 3615 Civic Center Blvd., Abramson Research Building,
Philadelphia, PA 19104, USA 
Present address: Department of Zoology, University of British Columbia,
Vancouver, BC, V6T 2A9, Canada
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ankel, W. E. (1929). Über die Bildung der Eikapsel bei Nassa-arten. Zool. Anz. Suppl. 4, 219-230.
Chapman, B. M. (1969). A mechanical model for wool and other keratin fibers. Text. Res. J. 39,1102 -1109.
Denny, M. W. (1988). Biology and the Mechanics of the Wave-swept Environment. Princeton: Princeton University Press.
Feughelman, M. (1959). A two-phase structure for keratin fibers. Text. Res. J. 29,223 -228.[CrossRef]
Feughelman, M. (1964). The post-yield region and the structure of keratin. Text. Res. J. 34,539 -545.[CrossRef]
Feughelman, M. (1979). A note on the role of
microfibrils in the mechanical properties of 
-keratins. J.
Macromol. Sci. Phys. B16,155
-162.[CrossRef]
Feughelman, M. (1997). Mechanical
Properties and Structure of -keratin Fibres: Wool, Human Hair, and
Related Fibres. Sydney: UNSW Press.
Flower, N. E., Geddes, A. J. and Rudall, K. M. (1969). Ultrastructure of the fibrous protein from the egg capsules of the whelk Buccinum undatum. J. Ultrastruct. Res. 26,262 -273.[CrossRef][Medline]
Fretter, V. and Graham, A. (1994). British Prosobranch Molluscs. Dorchester: Dorset Press.
Fudge, D. S. and Gosline, J. M. (2004). Molecular design of the alpha-keratin composite: insights from a matrix-free model, hagfish slime threads. Proc. R. Soc. Lond. B Biol. Sci. 271,291 -299.[Medline]
Fudge, D. S., Gardner, K. H., Forsyth, V. T., Riekel, C. and Gosline, J. M. (2003). The mechanical properties of hydrated intermediate filaments: insights from hagfish slime threads. Biophys. J. 85,2015 -2027.
Gathercole, L. (1969). Studies on the protein of the egg capsules of whelks. PhD thesis, University of Leeds, UK.
Gosline, J. M. (1971). Connective tissue
mechanics of metridium senile. II. Visco-elastic properties and macromolecular
model. J. Exp. Biol. 55,775
-795.
Gosline, J. M. (1980). The elastic properties of rubber-like proteins and highly extensible tissues. In The Mechanical Properties of Biological Materials, 34th Symposium of the Society for Experimental Biology (ed. J. F. V. Vincent and J. D. Currey), pp. 331-357. Cambridge: Cambridge University Press.
Hearle, J. W. S. (2000). A critical review of the structural mechanics of wool and hair fibres. Int. J. Biol. Macromol. 27,123 -138.[CrossRef][Medline]
Josefsson, L. (1962). Changes in the physico-chemical properties of proteins during the formic acid-induced transformation. Biochim. Biophys. Acta. 59,128 -136.[Medline]
Josefsson, L. (1966). Studies with model substances on the mechanism of the formic acid-induced reversible inactivation of protein enzymes. Biochim. Biophys. Acta 115,148 -159.[Medline]
Keeley, F. W., Bellingham, C. M. and Woodhouse, K. A.
(2002). Elastin as a self-organizing biomaterial: use of
recombinantly expressed human elastin polypeptides as a model for
investigations of structure and self-assembly of elastin. Philos.
Trans. R. Soc. Lond. B 357,185
-189.
Kreplak, L., Doucet, J., Dumas, P. and Briki, F. (2004). New aspects of the alpha-coil to beta-sheet transition in stretched hard alpha-keratin fibers. Biophys. J. 87,640 -647.
Kreplak, L., Bar, H., Leterrier, J. F., Herrmann, H. and Aebi, U. (2005). Exploring the mechanical behavior of single intermediate filaments. J. Mol. Biol. 354,569 -577.[Medline]
Ram, J. L. (1977). Hormonal control of
reproduction in Busycon: laying of egg capsules caused by nervous
system extracts. Biol. Bull.
152,221
-232.
Rapoport, H. S. (2003). Biomechanics, biochemistry, and molecular biology of a molluscan scleroprotein elastomer: whelk egg capsules. PhD thesis, University of California, San Diego, USA.
Rapoport, H. S. and Shadwick, R. E. (2002). Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.). Biomacromolecules 3,42 -50.[CrossRef][Medline]
Rawlings, T. A. (1999). Adaptations to physical stresses in the intertidal zone: the egg capsules of neogastropod mollusks. Am. Zool. 39,230 -243.
Stoll, V. S. and Blanchard, J. S. (1990). Buffers: principles and practice. In Methods in Enzymology. Vol. 182 (ed. M. P. Deutscher), pp. 24-38. San Diego: Academic Press.[Medline]
Szulgit, G. K. and Shadwick, R. E. (2000). Dynamic mechanical characterization of a mutable collagenous tissue: response of sea cucumber dermis cell lysis and dermal extracts. J. Exp. Biol. 203,1539 -1550.[Abstract]
Van Steensel, M. A. M., Happle, R. and Steijlen, P. M.
(2000). Molecular genetics of the hair follicle: the state of the
art. Exp. Biol. Med.
223, 1-7.
Vincent, J. (1990). Structural Biomaterials (revised edn). Princeton: Princeton University Press.
Vogel, S. (1988). Life's Devices. Princeton: Princeton University Press.
von Hippel, P. H. and Wong, K.-Y. (1965). On
the conformational stability of globular proteins. J. Biol.
Chem. 240,3909
-3923.
Whitely, K. J. and Kaplin, I. J. (1977). Letter to the editor, the comparative arrangement of microfibrils in orth-, meso-, and paracortical cells of merinowool fibres. J. Textiles Inst. 68,384 -386.[CrossRef]
Wortmann, F.-J. and Zahn, H. (1994). The
stress-strain curve of -keratin fibers and the structure of the
intermediate filament. Text. Res. J.
64,737
-743.[CrossRef]
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  L. Blackburn WHELK WONDERMATERIAL J. Exp. Biol., January 1, 2007; 210(1): i - i. [Full Text] [PDF]
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