study of the molecular and supramolecular organisation of elastic tissue by x-ray diffraction
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Radiation Physics and Chemistry 71 (2004) 951–952
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doi:10.1016/j.ra
Study of the molecular and supramolecular organisationof elastic tissue by X-ray diffraction
Layla Alia, E.M. Greena, R.E. Ellisa, D.A. Bradleya,J.G. Grossmannb, C.P. Winlovea,*
aBiomedical Physics Group, School of Physics, University of Exeter, Exeter EX4 4QL, UKbCCLRC, Daresbury Laboratory, Synchrotron Radiation Department, Daresbury, Warrington, WA4 4AD, UK
1. Introduction
Elastin is the principal elastic protein in mammals. It
constitutes up to 50% of the dry weight of tissues such
as lung, blood vessels and ligaments where it is
associated with a family of elastic microfibrillar glyco-
proteins to form tissue-specific networks or fibres. Little
is known about the internal organisation of these
structures. Early reports (Serfani-Fracassini and Field,
1977) of regular organisation in dried fibres using X-ray
diffraction or electron microscopy were often dismissed
as artefactual, largely because such ordered structures
were believed to be inconsistent with an entropic
mechanism of elasticity. This view is now recognised
to be erroneous and, as part of an analysis of the
molecular bases of elasticity, we have begun to re-
examine the organisation of elastin and its associated
proteins.
2. Experimental methodology
Equine nuchal ligament and porcine aorta obtained
from the abattoir were studied, either intact, after alkali
extraction (which produces pure elastin, but may cause a
small amount of hydrolysis (Soskel et al., 1987)) or after
GuHCl/collagenase extraction with or without DTE
(preserving elastin structure and, in the latter case, the
microfibrillar glycoproteins (Spina et al.,1999). Nuchal
ligament fibres of dimension B4 cm� 1mm were
ing author. Tel.: +44-1392-264140; fax: +44-
ess: [email protected] (C.P. Winlove).
ee front matter r 2004 Elsevier Ltd. All rights reserv
dphyschem.2004.05.020
dissected from the samples of ligament. Rings of aorta
were of thickness B 2mm and diameter B1 cm. Each of
these samples was mounted on a purpose-made rig on
either beamline 14.1 (wide-angle X-ray scattering,
WAXS) or 2.1 (small-angle X-ray scattering, SAXS) at
the SRS Daresbury, UK. The rig allowed the specimens
to be equilibrated with water or primary alcohols and to
be strained up to 100%.
3. Results and discussion
The wide-angle diffraction patterns of relaxed nuchal
elastin showed two broad diffraction rings correspond-
ing to spacings of 0.45 and 0.93 nm that did not orient
on stretching (Fig. 1). However at above 60% extension,
the rings were not observed. Another sharp intense ring
at spacing of 4.5 nm in which at small strains an
equatorial reflection was produced, while at higher
strain the ring was not visible. In addition, a diffuse
equatorial peak with spacing extending between 5.5 and
8.0 nm appeared in relaxed and extended specimens.
Clear meridional reflections at spacing of 9.1, 7.0, 5.8,
5.3 and 3.25 nm, became clearer with increasing strain in
collagenase extracted fibres which could be due to the
microfibrillar component in elastin. It appears that
fibrillin does not affect the packing of elastin as the main
diffraction pattern was preserved in both treatments of
ligament fibres. The X-ray diffraction pattern of
ligamentum elastin was partially reproduced in arterial
elastin in particular the two broad diffraction peaks and
the sharp intense ring appeared in identical position.
In order to investigate the reversibility of the elastic
properties of the tissue at a molecular level, samples
ed.
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Fig. 1. WAXS of unstretched elastin fibre.
Fig. 2. Background subtracted SAXS profiles of elastin fibres
obtained under different conditions.
L. Ali et al. / Radiation Physics and Chemistry 71 (2004) 951–952952
were returned to original length. Most of the features in
the pattern were preserved but the diffuse equatorial
peak and most meridionals were not immediately visible.
SAXS data showed an axial periodicity of approxi-
mately 65 nm where the third and fifth order of
diffraction were predominant and this pattern did not
change significantly as the samples stretched (Fig. 2).
The meridionals visualised in WAXS was found to be
the higher orders of the 65 nm spacing.
Replacing water by methanol or butanol, resulted in
loss of features in the diffraction pattern relating to
molecular level organisation and a significant increase of
about 0.5 nm in the spacing of the SAXS diffraction
peaks and changes of their intensities (Fig. 2).
Elastin is a highly hydrophobic protein in which
extensive b-structured hydrophobic domains are re-
peated by short, a-helical cross-linking segments. The
former are believed to be increasingly exposed to solvent
as the molecule is strained and hydrophobic effects are
thought to be important in determining elasticity. Our
results show, for the first time, the structural correlates
of this behaviour.
References
Serfani-Fracassini, A., Field, J.M., 1977. X-ray analysis of
enzymically purified elastin from bovine ligamentum
nuchae. Adv. Exp. Med. Biol. 79, 679–683.
Soskel, N.T., Wolt, T.B., Sandberg, L.B., 1987. Isolation and
characterisation of insoluble and soluble elastins. Methods
Enzymol. 144, 196–214.
Spina, M., Friso, A., Ewins, A.R., Parker, K.H., Winlove, C.P.,
1999. Physicochemical properties of arterial elastin and its
associated glycoproteins. Biopolymers 49, 255–265.