Download The Structure of Collagen within Parchment

The Structure of Collagen within Parchment A Review
by CRAIG J. KENNEDY & TiM J. WESS
SKIN STRUCTURE; THE IN vivo STRUCTURE OF COLLAGEN
Structure of the skin
Skin is composed of three primary layers: the epidermis, the collagen-rich dermis layer and an
underlying layer of fat-producing cells called the subcutaneous tissue.
The epidermis is composed of four layers of cells. The Stratum Basale (basal layer), the Stratum
Spinosum (spinous or prickle cell layer), the Stratum Granulo-sum (granular layer) and the Stratum
Corneum (surface layer)1. The epidermis is the outer layer of the skin. The epidermis is rich in die
protein keratin, with the outermost part of the epidermis composed of fully keratinised, fused flat
cells that act as a barrier to external elements such as microorganisms and chemicals that may enter
the body and cause damage.
The dermis lies under the epidermis, and is separated from the epidermis by the basement
membrane. It is in this layer diat collagen is predominant2. The dermis is composed of two layers:
the papillary layer, which is closest to the epidermis, and the reticular layer. The reticular layer
consists of more dense, thicker collagen fibres compared to the papillary layer, which has thinner
fibres. The collagen within the dermal layer provides the skin with mechanical strength.
Cells within the dermis include fibroblasts, and their role is to synthesise new collagen and elastin
for the dermis. This allows a mechanism of collagen turnover, with damaged collagen being
removed and being replaced with new collagen.
The subcutaneous tissue that lies under the dermis is involved in the formation of fat. This layer is
unevenly distributed over the body.
Collagen fibres in skin
Collagen in skin has a hierarchical structure that is key to the success of collagen as a biomaterial.
At the ultrastructural level, collagen fibres exist. The fibres are
Fig. 1: Collagen fibres from the surface of parchment, as shown by Scanning Electron
Microscopy. 123x magnification.
composed of fibrils, which are made up of collagen molecules, which are in turn composed of
individual peptide chains.
Collagen fibres exist in tissues such as skin, ligaments and tendons. Fibres are approximately 50300μ in diameter, and are composed of tightly packed collagen fibrils5. The alignment of collagen
fibres is a key factor in the overall mechanical characteristic of a tissue. Fibres in tendons are
aligned in a parallel fashion along the length of the tissue, so as to resist tensile loads in the
direction of the tissue.
In skin, and subsequently in parchment, the fibres are arranged in a predominantly two-dimensional
felt-like network4 (Fig. 1). The fibres lie at random orientations, though confined to being
perpendicular to the plane of the parchment, giving the parchment tensile strength in all directions5.
The arrangement of fibres in skin allows a certain degree of flexibility, but is strongly resistant to
breaking, especially within the plane of the skin surface6. In some areas of the skin, however, the
collagen lies in a preferential orientation. Examples of this include along the spine and under the
legs of animals, where there is greater strain put on the skin in one direction. Fibre direction and
orientation are indicative of the prevalent tensile stresses that the tissue is subjected to.
Fig. 2: The hierarchical arrangement of collagen from skin, (a) Diagram of a cross-section of skin,
visible at the microscopic level, (b) Transmission Electron Microscope image of collagen. The
banding pattern is characteristic of the repeating gap/overlap function of collagen. This exists at the
mesoscopic level, (c) Representation of the quarter-staggered arrangement of collagen molecules
within a fibril at the nanoscopic level, (d) Diagram of the collagen triple helix; this exists at the
molecular level.
Collagen fibrillar structure
The fibrils that make up fibres are frequently organised into bundles or lamellae, and the size and
higher-order arrangement of fibrils gives tissue specific bio-mechanical properties. Interfibrillar
covalent crosslinks are formed during tissue maturation that give the tissue strength and play a role
in forming these structures7.
The arrangement of collagen molecules into fibrils is characteristic of the hierarchy of structure that
collagen displays (Fig. 2). Collagen fibrils are the principal, tensile strength-bearing components of
connective tissues8. There are over twenty different types of collagen molecules. Collagen
molecules from types I, II, III, V and XI are capable of self-assembling to form fibrils9. The
collagen fibrils are approximately cylindrical with diameters ranging between 10 and 500nm10, and
range from 40-100nm in skin11. This varies due to age, collagen type composition and tissue source.
Fibrils of large diameter that are aligned in a parallel fashion are generally found in highly tensile
structures such as tendon and ligaments, where they are tightly packed in thick, stiff parallel
bundles aligned with the direction of the force that is applied to it. Fibrils whose molecules run
helically have a typically uniform diameter, variable from tissue to tissue but uniform locally, are
widespread in tissues such as skin and blood vessels. The fibrils in these cases are formed in loose,
thin, wavy bundles forming three-dimensional networks12.
Fig. 3: Representation of the arrangement of collagen molecules within a fibril and the gap/ overlap
function that is characteristic of the molecular packing of collagen. A) Each arrow represents a
collagen molecule in the staggered array. B) The D-period: The five collagen triple helices are
shown; the non-helical regions represent the telopeptides at the end of the molecules. The four
molecules that do not display the gap area are approximately 234 amino acids in length; the
molecule that displays the gap is approximately 108 amino acids in length (0.46 of D). C) The
electron density profile of the gap/overlap region, displaying the characteristic 'step' function of a
collagen fibril.
In living tissue, in the axial direction of the fibril, there is a long-range order. The 300nrn long
collagen molecules are staggered relative to their neighbouring molecules by D, ~67nm in tendon or
~65.5nm in skin13-15, comprising gap and overlap regions (Fig. 3). The arrangement is staggered by
integral multiples of D. This is known as the Hodge-Petruska model16-17'
The D repeating unit is characteristic of collagen. The D stagger leaves a gap between linearly
adjacent molecules, as the molecular length is not an exact multiple of the D period, which results
in a gap region and an overlap region in each D repeat. The gap region comprises 0.54 of D, and the
overlap subsequently comprises 0.46 of D18.
The quarter-staggered array of collagen molecules allows the strength of the molecules to be
translated to the next level of the structural hierarchy of collagen. This is essential to the ability of
collagen to function within connective tissues.
X-ray diffraction has shown that in dried collagen samples including parchment, the sharp interface
between the gap and overlap regions is less apparent19' 20. This effect is presumably due to the
removal of water from the gap region during the drying process, although the structural changes are
still poorly understood. Also, the 65.5nm periodicity of the molecules in skin is reduced to ~64nm.
Fig. 4: Diagrammatic representation of the binding of proteoglycan groups to collagen fibrils. The
four open circles represent the collagen fibrils, viewed down the main fibril axis. The shaded circles
represent the protein core of the proteoglycans. The lines emanating from the protein cores are
representative of the branched glycosaminoglycan (GAG) chains of the proteoglycans.
Proteoglycan-collagen relationships
Collagen is also bound to water-soluble, carbohydrate-rich polyanions called proteoglycans (Fig. 4),
which influence nucleation and growth in developing fibres, and fibre diameter in mature fibres21.
Proteoglycans (PGs) are macromolecules composed of a protein core bound to glycosaminoglycan
(GAG) chains22. Proteodermo-chonan sulphates are the principal PG associated with mature type I
collagen23.
Proteoglycans are anionic in nature and can bind collagen at four sites per D period24. The protein
cores of the proteoglycan bind non-covalently to the collagen molecules and anionic
glycosaminoglycan (GAG) chains. Proteoglycans have a role in determining the distance between
collagen fibrils. Two protein cores based on adjacent collagen fibrils are linked by a bridge of two
or more antiparallel GAG chains. The GAG chains are bonded together through hydrogen bonds
and hydrophobic interactions.
Proteoglycans bind to collagen at regular intervals, equivalent to the D period spacing of collagen.
The ratio of proteoglycan to collagen correlates to the ratio of the circumference (2πr) to the crosssectional area of the fibril (πr2). Thus, the upper dermis of calf skin, containing 'fine' fibres has
twice the dermatan sulphate/collagen ratio of the lower dermis, containing 'coarse' fibres25.
Collagen molecular structure
At the molecular level, collagen shows a discrete structural hierarchy. Collagen molecules are made
up of three polypeptide chains, which assemble in to a triple helical structure. The positions,
orientations and side chains of the amino acids of the chains have implications with regard to the
structural hierarchy of collagen, in particular fibril formation.
Collagen molecules are approximately 300nm in length. The three collagen chains exist as lefthanded helices, and they are coiled to give a right-handed super-
Fig. 5: Inter-molecular cross-linking between neighbouring collagen molecules. Non-helical regions
represent the telopeptides at the termini of the molecules. Cross-linking can be seen between the
telopeptides of the central molecule and the helical regions of neighbouring molecules. The Nterminal telopeptides from each molecule are on the left.
helix. This gives the collagen molecule a great deal of stability and strength; a similar process is
used to make ropes. All collagens display this conformation. The main forces holding the triple
helix together are hydrogen bonds within the triple helix26, and covalent cross-links between
molecules.
Intra-molecular and inter-molecular cross-links also play a role in stabilising the collagen
molecules. In addition to ionic and hydrogen bonds present in the original molecules, covalent
bonds have been suggested to form randomly along and between the molecules resulting in an
increasing insolubility and rigidity of the fibre27.
In terms of fibril formation, the telopeptide regions are essential28 29. The strength of the collagen
fibres is dependent on the telopeptides forming covalent crosslinks with the adjacent collagen
molecules18,30-32 (Fig. 5). Type I collagen has four cross-linking sites; two in the telopeptide regions,
and two in the helical domain at residues 87 and 930. This allows for two cross-links between
molecules to be generated. The telopeptides of one collagen molecule bind to the helical domain of
its neighbouring molecule.
The character of the crosslinks in collagen change with collagen type. In type I collagen, the main
crosslinks are between the non-helical telopeptides and the helical domains of neighbouring
collagen molecules, particularly between lysine residues on the telopeptides and hydroxylysine
residues on the helix. Within type I collagen, there are differences in the telopeptides. The αl chains
have C-telopeptides 25 amino acid residues in length; α2 chains have shorter, 9 residue Ctelopeptides18.
The cross-linking sites on the helical region of type III collagen are identical to the corresponding
cross-linking sites on type I collagen. The amino acid sequences of the C telopeptide regions from
collagen I and III, which is a major cross-linking site, are markedly different indicating a different
secondary structure of the domain. The cross-link forming hydroxylysine occupies the same position on both the type I and type III C telopeptide, which allows copolymers to form between the
two types of collagen33.
Collagen molecules have the prerequisite of repeating arnino acid sequences of Gly-X-Y, where
Gly is the amino acid residue glycine and X and Y are any amino acid residues; most commonly X
and Y are proline and hydroxyproline34. Hy-droxylysine is another residue that commonly takes the
X or Y position. Proline and hydroxyproline are large, rigid, cyclic imino acids and their presence
in the backbone of the molecule prevents a movement and rotation in the molecule, and thus
contributes to the stability of the triple helix35. Glycine is the smallest of all amino acids, and its
presence between the larger residues allows for efficient twisting of the chain in to a helix.
In type I collagen, there are two distinct types of chains, α 1(1) and α2(I), with the triple helix
comprising two α1(1) chains and one α2(I) chain. Each α1(1) chain has 1014 residues present as
repeating tripeptide sequences. Type III collagen, which is present along with type I in skin consists
of only one kind of chain, the αl(III) chain, and comprises a homopolymer of three αl(III) chains.
The different types of al chains are homologous, particularly in terms of the location of charged
residues, but substitutions are present in up to 40% of non-glycine residues13. Also, collagens from
different animal types are found to contain substitutions in their α 1(1) and α2(I) chains.
Skin comprises both type I and type III collagen. Type III collagen is usually present in tissues that
display elastic qualities at the macroscopic level11' 36. However, there is no evidence for elasticity in
the type III collagen molecule itself. The fibrils in tissues with type I and type III collagen present
are typically composed of 80% type I molecules and 20% type III molecules. Type I collagen molecules are shorter than type III molecules, and the arrangement of the molecules in skin has the effect
of reducing the D periodicity of the collagen compared to a fibril comprised entirely of type I
collagen37.
The regular Gly-X-Y pattern is consistent throughout the length of the helical region of the collagen
molecule. However, the regions at the ends of the molecules, called telopeptides, do not display this
sequence regularity, and are not helical in conformation28,29,38. The telopeptide regions account for
2% of the molecule and are essential for fibril formation as they contain cross-linking sites that
connect neighbouring molecules.
THE PARCHMENT MAKING PROCESSES AND IMPACT ON COLLAGEN STRUCTURE
Preparation of the skin
From the foregoing it can be seen that the hierarchical structure of collagen is key to the production
of a functional tissue. The effect of processing the tissue to cre-
Fig. 6: Illustration of the effect of dehydration on the D periodic stagger of collagen fibrils. In the
hydrated state the collagen molecules are approximately parallel. Upon dehydration the areas of the
molecule in the gap and overlap regions undergo tilting, although during collapse the gap region
may display greater distortions; this has the effect of shortening the quarter-staggered array and
altering the length of the D period.
ate parchment and subsequent effects on each level of the hierarchical structure of collagen needs to
be known for a full understanding of parchment stability.
The methods by which a functional writing material is produced may be at odds with the overall
stability of the material as a long term effective storage media. The following will discuss
parchment preparation from skin in the context of effects that the processes may have on the
collagen.
The first step in the process is flaying the hide or skin from the animal me-'chanically. As large an
area as possible is taken from the animal, to allow a greater amount of parchment to be made from
it.
The second step is to dry the skin to avoid putrefaction. This is sometimes known as curing the
skin. This step is designed to preserve the skin temporarily so that it can either be stored until the
next step in the preparation process can begin, or allow for transportation of the skin. There are
several methods of curing the skin; they include drying the skin in air, dry-salting the skin with
sodium chloride, wet salting (brining) and lyophilisation (freeze drying). Ideally the moisture
content of the skin would be reduced by approximately 40% in this stage.
The drying of parchment has the effect of water being evacuated from the collagen molecules. This
brings about a degree of tilting in portions of the collagen chains, most likely in the centre of the
gap region where molecular shear may occur after molecular collapse on dehydration (Fig. 6). The
D-period of collagen is reduced, which indicates a molecular-level contraction of the staggered
array19.
In the next step of parchment preparation, the hide is washed in cold running water. This can be
done directly after flaying, or after curing. This step is done to remove blood, dung, dirt, protein
substances from blood such as albumins and globulins or to remove salt from the curing process39.
Proteoglycans are non-covalently linked to collagen. They are bound to the collagen molecules by
interactions between the PG protein core and the collagen
through hydrogen bonds. The parchment preparation process is likely to remove the proteoglycans,
by the addition of water that will interfere with the hydrogen bonds.
Historically the process of parchment preparation has changed40 with the use of materials such as
excrement and seawater being replaced with materials such as lime.
Liming
At this stage in the parchment making process the skin is soaked in a solution containing lime
(Ca(OH)2). The liming loosens and expands the dermal fibre network. Non-collagenous materials
including hair, the materials in between the der-mis and epidermis and lipids are removed in the
lime bath. The lime solution has a high pH, usually 10-12. This has the effect of placing most amino
acid residues above their isoelectric point, and thus making the carboxy terminus negatively
charged. The major chemical modification of collagen during liming is the hydrolysis of some of
the amido groups attached to aspartic and glutamic acid residues. The liming process lowers the
isoelectric point, the pH at which the positive and negative charges in collagen are equally
numbered. A small portion of arginine residues is also converted to ornithine and releases urea.
The increased pH may also neutralise the positively charged lysine residue that is implicated in
collagen-proteoglycan interactions. The liming brings about the progressive removal of
proteoglycans from the collagen fibres by interrupting the covalent bonds between the side chains
and protein core of the proteo-glycan41.
Liming may be enhanced by duration process, raising the temperature and pH of the liquor.
Unhairing and liming may be carried out at the same time by immersing the hides and skins
completely in lime and water mixtures and by the addition of chemical 'sharpeners' such as sodium
sulphide (Na2S), which hydro-lyse the proteins of the hair bulb and can degrade the keratinised hair
shaft in skin42. Liming process may be carried out without lime in certain cases, e.g., with greasy
skins the lime is replaced by other alkali such as caustic soda. On drying, calcium carbonate
(CaCO3) is formed from the lime in parchment reacting with carbon dioxide from the air.
Differential Scanning Calorimetry (DSC) has shown that by this stage in the parchment making
process the collagen is less stable than in fresh hides. In fresh hide, the temperature of collagen
denaturation is 63°C compared with 50.6°C after the liming process43. This indicates that the
processes thus far have reduced the overall stability of the collagen relative to the collagen from
living tissue.
Scraping
The skin is then placed over a wooden beam and the epidermal structures such as hair and fat
materials are removed using blunt knives. Fleshing and levelling the flesh side is carried out by
scraping off muscle tissue and hypodermic layers with sharp knives. The last traces of dirt, grease,
hair, pigmentation and lime from the skin surface are removed by scraping with knives.
At this stage of the skin treating process, parchment differs from leather as leather is from this stage
subjected to tanning agents which parchment is not. The tanning agents covalently cross-link with
the collagen molecules, giving the leather more chemical stability. The wet skin in parchment
manufacturing is dried under tension. This has the effect of aligning the fibrous collagen network in
skin to a configuration more parallel to the surface of the skin5.
Finishing
The final process in parchment preparation is finishing. This includes processes such as mechanical
thinning of parchment, shaving, bleaching, dying, scraping, cleaning of the surface and polishing.
This leaves the parchment ready for whatever purpose awaits it.
With the outer epidermal layers removed, the finished parchment consists almost exclusively of the
dermis layer, or corium, which is composed of approximately 95% collagen. The term flesh side of
the parchment refers to the inside layer of the skin (the side connected to the animal), and the term
grain side refers to the outer surface of the skin.
The condition of collagen in new parchment can be described as a modified form of collagen from
skin. Studies from X-ray diffraction have shown that there is a greater level of statistical disorder in
parchment compared to dry skin44. The regular repeating pattern is not as sharp as in tissue samples,
as shown by changes in the gap/overlap regions. The regularity of the long-range order of collagen,
in terms of fibrillar structure, is reduced.
THE DETERIORATION OF COLLAGEN IN PARCHMENT
The deterioration of collagen in parchment can be described as two effects; the deteriorations
brought about by the parchment making process, and deteriorations that occur after parchment has
been made.
The parchment preparation process renders the collagen within parchment in a state of disorder
relative to native collagen; the molecules have more ability to
assume different conformations and interact with other molecules in ways that were not previously
possible. From the point in time at which parchment has been created from skin, the collagen in
parchment can deteriorate via oxidation, hydrolysis and gelatinisation of the collagen molecules.
Outside factors that may accelerate degradation of the collagen have a statistically greater chance of
interacting with the molecules.
Parchment exists as a post-mortem tissue. The in vivo mechanisms of collagen turnover and repair
that existed in living skin are no longer present. With no repair mechanism in place, the collagen
present in skin is subjected to irreversible degradation.
There are several levels at which collagen deterioration can be assessed; from the molecular level to
alterations in the hierarchy of collagen structure (staggered array in fibrils, fibres, etc.). These
factors are linked; if the collagen molecules break down the structural hierarchy of collagen will be
lost45.
Factors that can bring about collagen degradation in parchment include biological sources of
degradation such as bacteria, fungi and rodents46. In humid environments at temperatures above
40°C, the prospect of microbial attack increases. Parchment has a pH of between six and eight, and
is a source of nutrition for many microorganisms.
There are three major degradation paths for collagen; molecular denaturation, hydrolysis and
oxidation39. Chemical degradation of parchment is often brought about by oxidative damage from
light and pollutants and the effects of heat. Hydrolysis of the molecules is often brought about by
the presence of acids from pollution.
Oxidation
Oxidation of the collagen molecules has an effect on the side chains of amino acids, as has been
shown by a reduction of the number of basic amino acids such as arginine, hydroxylycine and
lysine, and an increase in the number of acidic amino acids such as glutamic acid and aspartic acid.
The level of oxidation of collagen in parchment can be assessed by measuring the ratio of basic to
acidic amino acids (B/A ratio). In new collagen this ratio is 0.69, but as the collagen undergoes
oxidative change, this ratio will decrease. Historic parchments have shown B/A ratios as low as 0.5
47 49
~ .
The oxidation of the collagen molecules can occur in the main chain of the collagen molecule (Fig.
7), between the amino group of an amino acid residue and its associated Cα-atom or in the side
chains of individual amino acid residues. Oxidation caused by free radicals is capable of breaking
the N-C covalent
Fig. 7. Schematic representation of the molecular processes that brings about oxidative cleavage of
the main chain of collagen.
bonds that link neighbouring amino acid residues. The effect of this is cleavage of the main chain
the collagen molecule. Oxidative cleavage of the collagen molecules occurs preferentially at tyrosyl
residues on the collagen molecule50,51.
Cleavage of the collagen main chain purports to the molecule not contributing to the strength of the
staggered array of collagen, whereas it had previously. This reduces the stability of the collagen
hierarchy overall, and is characteristic of collagen degradation in parchment.
Oxidation can be caused by the presence of free radicals, which are formed by the interaction of, for
example, water and UV light that splits the water molecule in to two free radicals H* / *OH. The
effects of humid atmospheric air and SO2 may also bring about free radical formation7,52-54
Hydrolysis
Hydrolysis of collagen molecules is another major method of deterioration. Here the collagen
molecule is cleaved, making smaller polypeptide molecules (Fig. 8). The smaller peptides can
undergo further hydrolysis; heavily deteriorated parchments will consist of many small polypeptide
chains compared to less degraded parchments.
Hydrolysis can be brought about by acids, most commonly from atmospheric pollutants and water
combining, such as SO2 and water mixing to form sulphuric acid. Acids act in conjunction with
water to bring about a cleavage in the main chain of the collagen molecule.
With both oxidative55 and hydrolytic56 breakdown of collagen, the large 300nm collagen molecules
are broken in to smaller molecules. This has an effect on the hierarchical structure of collagen. As
the molecules are broken, they lose a great deal of integrity. This has the cumulative effect at the
hierarchical level of the loss of long-range order.
Fig. 8. Schematic representation of the molecular processes that brings about hydrolytic cleavage
of the main chain of collagen.
Fig. 9: The transition from a) the collagen triple helix to b) a collagen/gelatine intermediate to c)
gelatine.
Gelatinisation
A further mode of degradation of the collagen molecules in parchment is gelatinisa-tion57.
Gelatinisation occurs when the collagen molecules no longer have a triple helical structure, but
rather form a random coil structure58,59 (Fig. 9). This is usually brought about by the loss of internal
hydrogen bonds within the collagen molecules in the presence of water.
Water is capable of forming hydrogen bonds, and will compete with the existing hydrogen bonds
within collagen and attempt to form new bonds with the molecule. This occurs when water is
present in the system and hydrogen bonds are in a position within the molecule where they are open
to attack from the water molecules. When this occurs, the three chains of the molecule are no longer
held together and are free to form new, individual less ordered structures.
The action of heat with water also makes gelatinisation more likely to occur60. When heat is added
to collagen, there is a greater deal of energy present in the system. This has the effect of causing
molecular movement, which disrupts the hydrogen bonds that hold the triple helix in place. As the
heat is increased the hydrogen bonds move more, enhancing the chance of interaction with water.
Fig. 10: Structures of the L-and D-forms of aspartic acid.
When collagen degrades to gelatine, a partial renaturation of the triple helix is possible but native
collagen cannot be regenerated61'62.
Gelatinisation is more likely to occur in partially degraded collagen molecules compared to native
intact collagen. In intact collagen, the position and structure of the telopeptides and the position of
the hydrogen bonds internally in the triple helix makes it difficult for water to enter and interact in
this way. However, if the collagen molecule is broken, rotational freedom and entropy is increased
and hydrogen bonds are exposed at the points of breakage and as such open to the hydrogen bond
action of water.
Effects of deterioration
The breakage of collagen molecules, and increased disorder of the molecules bring about a number
of effects that cannot be described as deteriorations in themselves, but are effects of collagen
deterioration.
Extra crosslinks forming between collagen molecules is a function of the degradation of collagen in
parchment. Crosslinks are generated between adjacent collagen molecules, forming networks of
peptides. The most likely type of crosslink forms between the amino acid residues of lysine and
alanine. Crosslinking occurs naturally, or as the result of dry heating under vacuum, ultraviolet
irradiation or chemical crosslinking agents32,63,64. The presence of free radicals is also capable of
inducing cross-linking in collagen65. It has also been shown that the α2(I) chain of collagen is more
susceptible to crosslinking than the αl(I) chain66. The formation of crosslinks can help to stabilise
the collagen structure, but as the collagen degrades through gelatinisation and hydrolysis, forces
that bring stability to the collagen molecules, particularly internal hydrogen bonds, are lost. The
generation, nature and effects of cross-links of this type to collagen structure is still poorly
understood.
In living tissues, all amino acids are in the left-handed (L) conformation. This is true for the amino
acid residues present in collagen. After death, conversion of amino acid residues from the L
conformation to the right-handed (D) conforma-
tion occurs slowly until an equilibrium is reached (Fig. 10). Aspartic acid racemisa-tion is an
inevitable process during the 'natural' ageing of proteins. It can be used as a molecular indication of
protein ageing and affects long-living structural proteins such as collagen67. Conditions allowing
this include effects of the ambient temperature, pH, the amino acid sequence of the molecules and
molecular conformations. Thus, if amino acids are conformationally unrestricted, they are free to
change conformation.
As collagen turns to gelatine, and gains molecular mobility, the amino acids in the chains are
capable of changing conformation. Fragments from the Dead Sea Scrolls gave indications that over
time, as gelatinisation of collagen increases, so does the amount of D-aspartic acid57.
The change in molecular conformation from the L to D form of amino acids over time is, when
measured, an indication of the level of deterioration of collagen within parchments.
CONCLUSIONS
In skin the molecular structure and hierarchical arrangement of collagen is reasonably well
characterised. During the parchment preparation process, the regularity of this hierarchical
arrangement is in part lost and the collagen becomes more disordered and undergoes a shortening of
periodicity. The drying of the collagen plays a key role in this increased disorder.
With parchment existing as a post-mortem tissue, the in vivo mechanisms of collagen turnover and
repair are no longer present, and the collagen that is present at the time of animal slaughter is the
collagen that will make up parchment.
The deterioration of collagen is in part a co-operative process. With each deterioration event it
becomes easier for further deteriorations to occur. From the starting point in parchment, where an
element of architectural order is lost and conformational freedom is greater for the molecules,
deterioration begins and allows more deterioration and less restriction of the molecules. Eventually,
heavily deteriorated parchment will consist of collagen molecules that have been broken into many
smaller peptides, undergone oxidative change, lost their triple helical structure and lost the
hierarchical organisation that is characteristic of collagen. In this case the parchment is essentially
consisting of gelatine and a mixture of polypeptides of varying length and stability.
The detailed mechanisms of collagen degradation within parchment are relatively unclear.
Understanding the deterioration of collagen and its causes in a conservation context will in future
allow for greater care to be taken in damage assessment and preservation of parchments.
ACKNOWLEDGEMENTS
This work is supported by a grant from the National Archives for Scotland. CJK would like to
acknowledge the Conservation Workshop from the NAS for useful discussions and support. Thanks
also to Roy Sexton for assistance and guidance in operating the scanning electron microscope.
SUMMARIES
The Structure of Collagen within Parchment: A Review
For millennia parchment has been used as a writing material, commonly in the form of books,
scrolls or folded sheets. Parchment is made from animal skins, predominantly from calf, sheep or
goat. Parchment and skins have the characteristic molecular packing associated with other collagen
rich structures such as tendon, aorta, bone and cornea. For medical purposes such tissues are usually
analysed as close to the in vivo state as possible (hydrated and not degraded). This has allowed for a
wealth of information to be elucidated concerning the structure of collagen and its hierarchical
arrangements from the molecular structure to the fibrils to the organisation of fibres in a tissue.
The main considerations in understanding the structure of parchment at the molecular level is that
parchment exists in the dry state, and that it has been subjected to varying levels of deterioration
brought about by external factors.
This review centres on the structure of collagen within parchment. Discussed are the in vivo
structure of collagen in skin, alterations to the structure of collagen and alterations to the
hierarchical arrangement of collagen brought about by the parchment preparation process and
deterioration of collagen structure that is associated with parchment degradation over time.
La structure du collagene dans k parchemin : un expose sommaire
Le parchemin a ete utilise pendant des millenaires en tant que support de Pecriture, la plupart du
temps sous forme de livres, de rouleaux ou de feuilles pliees. Le parchemin est fabrique a partir de
peaux de betes, surtout de veaux, de moutons et de chevres. Le parchenmin et les peaux manifestent des structures moleculaires caracteristiques qui sont associees a d'autres structures riches en
collagene, telles que les tendons, les arteres, les os et la comee. En medecine ces substances sont
generalement analysees le plus etroitement possible dans leur etat in vivo, c.-a.-d. fortement
hydratees et non encore degradees. Ces analyses ont permis d'obtenir une foule d'informations
servant a elucider la structure du collagene et des arrangements hierarchiques allant de la structure
moleculaire en passant par les fibrilles et aboutissant a la disposition des fibres du parchemin.
La reflexion la plus importante pour comprendre la structure du parchemin a partir de son
groupement moleculaire porte sur le fait que le parchemin existe a 1'etat desseche et que lors de sa
fabrication il a ete soumis a differents processus de degradation sous Paction de facteurs extemes.
La presente analyse se concentre sur la composition et la structure du collagene contenu dans le
parchemin. On discutera de sa structure in vivo dans la peau et des alterations de la structure
de collagene ainsi que des alterations des arrangements hierarchiques du collagene apportes au
cours du processus de fabrication du parchemin et de la deterioration de la structure du collagene
associee a la degradation du parchemin qui se produit a long terme.
Die Struktur van Collagen in Pergament: ein Uberblick
Pergament wurde iiber Jahrtausende als Beschreibstoff gebraucht, meist als Buch, als Rolle oder
als gefaltetes Blatt. Pergament wird aus der Haul von Tieren gewonnen, iiberwiegend von Kal-bern,
Schafen und Ziegen. Pergament und Haul zeigen charakteristische molekulare Anordnun-gen, die
mit anderen iiberwiegend aus Collagen bestehenden Strukturen wie Sehnen, Adem, Knochen,
Homhaut etc. zusammenhangen. In der Medizin werden diese Substanzen so eng als moglich zu
ihrem Zustand im lebenden Korper analysiert, d.h. s.ark wasserhaltig und noch nicht abgebaut.
Diese Forschungen bieten eine Fiille von Informationen iiber die molekulare und iibermolekulare
Struktur von Collagen sowie den Aufbau und die Anordnung der daraus bestehenden Fibrillen in
einem Material wie Pergament.
Der wichtigste Faktor zum Verstandnis von Pergament aus seiner Molekularstruktur ist, daB es in
getrocknetem Zustand vorliegt und daB bei seiner Herstellung Abbauvorgange in unterschiedlichem AusmaB stattfanden, die von aufieren Umstanden bestimmt wurden.
Die Untersuchung gilt der Zusammensetzung des Collagens im Pergament. Es werden dessen
Struktur in vivo in der Haul und die Veranderungen in Aufbau und Anordnung sowie die Abbauvorgange diskutiert, die im Laufe der Pergamentherstellung und spater, d.h. bei langfristiger Aufbewahrung stattfinden.
REFERENCES
1. Horie, C. V.: Deterioration of skin in museum collections. Polymer Degradation and Stability 29
(1990): 109-133.
2. Burgeson, R. E.: The collagens of skin. Current Problems in Dermatology 17 (1987): 61-75
3. Woo, S. L.-Y, G.A. Johnson & B. A. Smith: Mathematical modeling of ligaments and tendons.
Journal of Biomedical Engineering 115 (1993): 468-473.
4. Kronick, P. L., & P. R. Buechler: Fiber orientation in calfskin by laser light scattering or X-ray
diffraction and quantitative relation to mechanical properties. Journal of the American Leather
Chemists Association 81 (1986): 221-231.
5. Hansen, E. F., S. N. Lee & H. Sobel: The effects of relative humidity on some physical properties
of modern vellum. Journal of the American Institute for Conservation 31:3 (1992): 325-342.
6. Purslow, P. P., T. J. Wess & D. W. L. Hukins: Collagen orientation and molecular spacing
during creep and stress-relaxation in soft connective tissues. Journal of Experimental Biology 201
(1998): 135-142.
7. Bailey, A.J.: Molecular mechanisms of ageing in connective tissues. Mechanisms of Ageing and
Development 122 (2001): 735-755.
8. Fratzl, P., K. Misof, I. Zizak, G. Rapp, H. Amenitsch & S. Bemstorff: Fibrillar structure and
mechanical properties of collagen. Journal of Structural Biology 122 (1997): 119-122.
9. Hulmes, D. J. S.: The collagen superfamily - diverse structures and assemblies. Essays in Biochemistry 27 (1992): 49-67.
10. Hulmes, D.J. S., T.J. Wess, D.J. Prockop & P. Fratzl.: Radial packing, order and disorder in
collagen fibrils. Biophysical Journal 68 (1995): 1661-1670.
11. Kielty, C. M., I. Hopkinson & M. E. Grant: Collagen, The collagen family: Structure, assembly
and organisation in the extracellular matrix. Connective Tissue and Its Heritable Disorders, ed. P.M.
Royce & B. Steinmann. New York: Wiley-Liss 1993: 103-147 .
12. Ottani, V., M. Raspanti & A. Ruggeri: Collagen structure and functional implications. Micron
32 (2001): 251-260.
13. Brodsky, B., & E. F. Eikenberry: Characterisation of fibrous forms of collagen. Methods in
Enzymology 82 (1982): 127-174
14. Stinson, R.H., & P. R, Sweeny: Skin collagen has an unusual d-spacing. Biochimica et BiophysicaActa621 (1980): 158-161.
15. Brodsky, B., E. F. Eikenberry & K. Cassidy: An unusual collagen periodicity in skin. Biochimica et Biophysica Acta 621 (1980): 162-166
16. Hodge AJ., & J.A. Petruska: Electron Microscopy vol. 1, Paper QQ.-1, ed. Breese S.S., Jr.,
Academic Press, New York (1962)
17. Hodge AJ. & J.A. Petruska: Recent studies with the electron microscope on ordered aggregates
of the tropocollagen molecule. Aspects of Protein Structure, ed. Ramachandran G.N., Academic
Press, New York (1963): 289-300
18. Orgel, J. P., T.J. Wess, & A. Miller: The in situ conformation and axial location of the intermolecular cross-linked non-helical telopeptides of type I collagen. Structure 8:2 (2000): 137-142.
19. Wess, T. J., &J. P. Orgel: Changes in collagen structure: drying, dehydrothermal treatment and
relation to long term deterioration. Thermochimica Acta 365 (2000): 119-128.
20. Wess, T J.: Analysis of collagen structure in parchment by small angle X-ray diffraction.
Handbook in the Micro Analysis of Parchments. European project SMT4-CT96-2106: 269-277.
21. Scott, J. E., C. R. Orford & E. W. Hughes: Proteoglycan-collagen arrangements in developing
rat tail tendon. Biochemical Journal 195 (1981): 573-581.
22. Kjellen, L., & U. Lindahl: Proteoglycans: structures and interactions. Annual Review of Biochemistry 60 (1991): 443-475.
23. Cribb, A.M., & J. E. Scott: Tendon response to tensile strength: an ultrastructural investigation
of collagen: proteoglycan interactions in stressed tendon. Journal of Anatomy 187 (1995): 423-428.
24. Scott, J.E., M. Ritchie, R. W. Glanville & A.D. Cronshaw: Peptide sequences in
gluteraldehyde-linked proteodermatan sulphate: collagen fragments from rat tail tendon locate the
proteoglycan binding sites. Biochemical Society Transaction 25 (1997): 663.
25. Tajima, S., & Y. Nagai: Distribution of macromolecular components in calf dermal connective
tissue. Connective Tissue Research 7:2 (1980): 65-71.
26. Beck, K., & B. Brodsky: Supercoikdprotein motifs: the collagen triple helix and the a-helical
coiled mil. Journal of Structural Biology 122 (1998): 17-29.
27. Barenberg, S.A., F.E. Filisko & P. H. Geil: Ultrastructural deformation of collagen. Connective
Tissue Research 6 (1978): 25-35.
28. Wess, T.J., A. P. Hammersley, L. Wess & A. Miller: Molecular packing of type I collagen in
tendon. Journal of Molecular Biology 275 (1998): 255-267.
29. Wess, T. J., A. P. Hammersley, L. Wess & A. Miller: A consensus model for molecular packing
of type J collagen. Journal of Structural Biology 122 (1998): 92-100.
30. Prockop, D. J., & A. Fertala: The collagen fibril: the almost crystalline structure. Journal of
Structural Biology 122 (1998): 111-118.
31. Vanderby jr, R., C. Chen & A. C. Vailas: Effect of cross-links on the quarter-staggered
molecular structure of fiber-forming collagen: a finite-element analysis Biomimetics 3:2 (1995):
91-104.
32. Henkel, W., & R.W. Glanville: Covalent crosslinking between molecules of type 1 and type III
collagen. European Journal of Biochemistry 122 (1982): 205-213.
33. Henkel, W.: Cross-link analysis of the C-telopeptide domain from type III collagen.
Biochemical Journal 318 (1996): 497-503.
34. Miller, E. J., & S. Gay: The collagens: an overview and update. Methods in Enzymology 144
(1987): 3-19.
35. Jenkins, C. L., & R. T. Raines: Insights on the conformational stability of collagen. Natural
Product Reports. 19 (2002): 49-59.
36. Miller, E. J., Epsteinjr, E. H. & K.A. Piez: Identification of three genetically distinct collagens
by cyanogen bromide cleavage in insoluble human skin and cartilage collagen. Biochemical and
Biophysical Research Communications 42 (1971): 1024-1029.
37. Cameron, G.J., I.L. Alberts,J. H. Laing & T. J. Wess: Structure of type I and type III heterotypic
collagen fibrils: an X-ray diffraction study. Journal of Structural Biology 137 (2002): 15-22.
38. Vitagliano, L., G. Nemethy, A. Zagari & H. A. Scheraga: Structure of the type I collagen
molecule based on conformational energy computations: the triple stranded helix and the Nterminal telopeptide. Journal of Molecular Biology 247 (1995): 69-80.
39. Derrick, M.: Evaluation of the state of degradation of Dead Sea Scroll samples using FT-IR
spectros-copy. American Institute for Conservation Book and Paper Group 10 (1991): 49-65.
40. Poole J. B., & R. Reed: The preparation of leather and parchment by the Dead Sea scrolls
community. Technology and Culture 3:1 (1962): 1-26.
41. Alexander, K. T. W., B. M. Haines & M. P. Walker: Influence ofproteoglycan removal on
opening-up in the beamhouse. Journal of the American Leather Chemists Association 81 (1986):
85-102.
42. Money, C.A.: Unhairing and dewooling - requirements for the quality and the environment.
Journal of the Society of Leather Technologists and Chemists. 80 (1995): 175-186.
43. Kronick P.L., & P. R. Buechler: Effects of beaming and tanning on collagen stability, studies by
differential scanning calorimetry. Journal of the American Leather Chemists Association 81 (1986):
213-220.
44. Wess, T.J., M. Drakopoulos, A. SnigirevJ. Wouters, O. Paris, P. Fratzl, M. CollinsJ. Killer & K.
Nielsen: The use of small angle X-ray diffraction studies for the analysis of structural features in
archaeological samples. Archaeomerty 43:1 (2001): 117-129.
45. Kennedy CJ.,J. Hiller, M. Odlyha, K. Nielsen, M. Drakopolous & TJ. Wess: Degradation in
historical parchments: structural, biochemical and thermal studies. PapierRestaurierung 3:4
(2002): 23-30
46. Strzelczyk, A. B., &J. Karbowska: The role of microorganisms in the decay of parchment. Acta
Microbiologica Polonica 43:2 (1994): 165-174.
47. Larsen, R., D. V. Poulsen & A. L. Jensen: Amino acid analysis of new and historic parchments.
Handbook in the Micro Analysis of Parchments. European project SMT4-CT96-2106 (1999): 199210.
48. Larsen, R., M. Vest, D.V. Poulsen, U. B. Kejser & A. L. Jensen 1997. Amino acid analysis. Deterioration and Conservation of Vegetable Tanned Leathers, ENVIRONMENT Leather Project.
European Commission DG XII Research Report No. 6 (1997): 39-53.
49. Larsen, R., V. Barkholt & K. Neilsen: Amino acid analysis of leather. Preliminary studies in deterioration, accelerated ageing and conservation of vegetable tanned leather. Das Leder 40 (1989):
153-158.
50. Larsen, R.: The possible link between collagen sequence and structure and its oxidative
deterioration pattern. STEP Leather Project European Commission DG XII Research Report No. 1
(1994): 59.
51. Deasy, C.L., & S. C. Michele sr: A study of the oxidative degradation of gelatin and collagen by
aqueous hydrogen peroxide solutions. Journal of the American Leather Chemists Association 60
(1965): 665-674.
52. Hansen, D. J., K. Nielsen & S. B. Rasmussen: Detection of radicals in collagen and parchment
produced by natural and artificial deterioration. Handbook in the Micro Analysis of Parchments.
European project SMT4-CT96-2106 (1999): 227-236.
53. Jurkiewicz, B. A., & G. R. Buettner: EPR detection of free radicals in UV-inadiated skin:
mouse versus human. Photochemistry and Photobiology 64:6 (1996): 918-922.
54. Jurkiewicz, B .A., & G. R. Buettner: Ultraviolet light-induced free radical formation in skin: an
electron paramagnetic resonance study. Photochemistry and Photobiology 59:1 (1994): 1-4.
55. Deasy, C. L.: Degradation of collagen by metal ion-hydrogen peroxide systems I. Evidence for a
free radical catalysed depolymerisation mechanism. Journal of the American Leather Chemists Association 62 (1967): 258-269.
56. Bowes, J. H., & A. S. Raistrick: The action of heat and moisture on leather part IV:
degradation of the collagen. Journal of the American Leather Chemists Association 62 (1967): 240257.
57. Weiner, S., Z. Kustanovich, E. Gil-Av & W. Traub: Dead Sea Scroll parchments: unfolding of
the collagen molecules and racemisation of aspartic acid. Nature 287 (1980): 820-823.
58. Sharma, J., & H. B. Bohidar: Gelatin-gluteraldehyde supramolecular structures studied by laser
light scattering. European Polymer Journal 36 (2000): 409-1418.
59. Koslov, P. V., & G. I. Burdygina: The structure and properties of solid gelatin and the
principles of their modification. Polymer 24 (1983): 651-665.
60. Hassel, B.: Examination of heat damaged parchment. Master thesis. Copenhagen: School of
Conservation, The Royal Danish Academy of Fine Arts 2001.
61. Pezron, I., M. Djabourov, L. Bosio &J. Leblond: X-ray diffraction of gelatin fibres in the dry
and swollen states. Journal of Polymer Science part B-Polymer Physics 28:10 (1990): 1823-1839.
62. Tanioka, A., K. Miyasaka & K. Ishikawa: Reconstitution of collagen-fold structure with
stretching of gelatin film. Biopolymers 15 (1976): 1505-1511.
63. Weadock, K .S., E. J. Miller, L. D. Bellincampi, J. P. Zawadsky & M. G. Dunn: Physical crosslinking of collagen fibres: comparison of ultraviolet irradiation and dehydrothermal treatment.
Journal of Biomedical Materials Research 29 (1995): 1373-1379.
64. Gorham, S. D., N. D. light, A. M. Diamond, MJ. Willins, A.J. Bailey, T.J. Wess & N.J. Leslie:
Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal
crosslinking. International Journal of Biological Macromolecules 14 (1992): 129-138.
65. Sobel, H., & H. Ajie: Modification in amino acids of Dead Sea Scrolls parchments. Free
Radical Biology and Medicine 13 (1992): 701-702.
66. Heidemann, E., & N. Linnert: Participation of the a2(I) chain of bovine skin collagen in the
formation of mature crosslinks. Hoppe-Seyler's Zeitschrift fur Physiologische Chemie. 365 (1984):
781-789.
67. Ritz-Timme, S., & M. J. Collins: Racemisation of aspartic acid in human proteins. Ageing Research Reviews 1 (2002): 43-59.
Craig J. Kennedy, Tim J. Wess
Centre for Extracellular Matrix Biology
University of Stirling
Stirling, Scotland, UK
Tel+44 1786467814
Fax+44 1786464994
E-mail [email protected]