Restaurator, 2009, Pp. 96 - 130 Printed in Germany - All Rights Reserved

Restaurator, 2009, pp.
96 – 130 Copyright © Saur 2009

Printed in Germany . All rights reserved
ISSN 0034-5806
Recombinant Proteins: A new material for the chemical stabilisation of

copper pigment corrosion on paper?
by Fabienne Meyer and Anke Neumann
Abstract: New possibilities for the chemical treatment of copper pigment-corroded paper objects are
examined. Gelatine has been suggested as a possible chemical stabiliser for the transition metal ion-
catalysed oxidative decomposition of cellulose. Gelatine appears to be able to interact with transition
metal ions, thus removing them from the reaction system with cellulose. However, according to
theoretical considerations, these copper-gelatine-complexes are relatively unstable, and thus dissolve
quickly if sufficient water is present. Possibilities for optimising the copper-binding properties of
gelatine were examined on verdigris-imprinted paper samples that had been aged artificially. For this
aim, the application of a short-chained gelatine and a gelatine rich in carboxyl groups were tested. In a
further step, the use of a recombinant protein was examined. The results suggest that the proteins are
able to bind copper ions by forming complexes. Short-chained gelatine seems to be more effective than
longer-chained modifications. The recombinant proteins tested exhibit the highest effectiveness in
inhibiting copper pigment corrosion in comparison to the gelatine modifications investigated.
Zusammenfassung/résumé at end of article
received: 02.02.2009 revised: 24.20.2009
1. Copper Pigment Corrosion
1.1. Damage Characteristics

Green copper-containing pigments have been important colouring matters in book
illumination and works of art although their dubious properties mainly concerning
changes in hue were well known historically. They are often in need of conservation
intervention today. Changes in hue and the subsequent destruction of the carrier
materials paper and parchment are frequently observed and known as copper
pigment corrosion. This type of decomposition is mostly associated with the use of
the artificially manufactured pigment verdigris (Banik 1980). The term verdigris
(probably derived from vert de Grèce, green from Greece) chemically is not well
defined. It describes a reaction product of metallic copper or copper alloys with acetic
acid, which under the conditions of manufacture form at least five different products
consisting of copper acetate precipitated with copper hydroxide and bound water.
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Fabienne Meyer and Anke Neumann
The series of these products is known in the literature as basic verdigris (Harley
1982) (Table 1).
A purer form of verdigris was manufactured in addition by dissolving basic
verdigris in acetic acid or vinegar and subsequent recrystallization of a pure copper
acetate hydrate. This product is known as neutral or distilled verdigris (Eastaugh et al
2008) (Table 1).
Table 1: Chemical formulas of basic verdigris and neutral verdigris

Basic verdigris [Cu(CH3COO)2]x [Cu(OH)2]y . z H2O,
while the possible factors for
x=1, 2; y=2, 3; z=2, 3, 5.
Neutral verdigris Cu(CH3COO)2 . H2O
(distilled verdigris)
Copper acetate in the two forms described above is one of the oldest
manufactured pigments. It is known since ancient time. Verdigris has been
identified in Egyptian papyrus dated about 1300 B.C. These samples already showed
severe deterioration resulting from the reactivity of the pigment present. Due to its
vibrant green colour, verdigris remained a very popular pigment until the 19th
century when through the rise of the chemical industry alternative and stable green
pigments were discovered and produced.
Copper pigment corrosion on paper resembles in its appearance to some extent
iron gall ink corrosion, although it follows a different chemical mechanism. In the
first state, the areas adjacent to the pigment layer start to discolour and show a
reduced absorbency for water. In a second state, the pigment penetrates through the
carrier material causing a green or brown discolouration on the verso side. This
might be due to the fact that the basic copper acetates are slightly soluble in water
depending on their composition up to 7 % maximum. It is also possible that the green
pigment itself turns into a brown-coloured degradation product. In an advanced
state, the paper carrier shows mechanical damage. This is manifested by increased
brittleness of the paper, the formation of tears and losses and lastly, the complete
degradation of the paper (Banik 1982).
1.2. Chemical mechanisms

The progression and sequence of paper decomposition caused by copper-containing
pigments are inconsistent and less predictable that caused by iron gall inks. A proved
chemical mechanism has not yet been presented and an effective treatment method,
Restaurator 30: 96 – 130 © 2009 Saur, Munich etc. 97

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Recombinant Proteins
as the calcium phytate/calcium hydrogen carbonate treatment for iron gall ink
corrosion is not developed yet. However, a closer look into the possible chemical
interactions of copper ions with cellulose are important with respect to iron gall ink
corrosion as all historic iron gall inks contain at least trace amounts of unbound
copper irons.
Copper, contained in verdigris, is one of the transition metals. As a transition
metal, copper can exist in its elementary form or as singly (Cu+) or doubly (Cu2+)
positively charged ion. The alternation between these charge states, i. e. the
oxidation or reduction of copper ions, assumes the presence of another reaction
partner that is reduced or oxidized as a consequence (Daniels 2002). In case of
copper pigment corrosion on paper, cellulose acts as the reaction partner and is thus
subjected to a parallel oxidative degradation. This creates radicals on the cellulose
which cause further redox reactions and accelerate the degradation process. In
addition, highly reactive hydroxyl radicals are generated by reaction of free copper
ions with reaction partners present in the surroundings, such as water and oxygen
(Daniels 2002). This precipitates a chain reaction, and as a result, the cellulose is
increasingly degraded until the mechanical stability of the paper is completely lost.
The hydrolytic degradation of cellulose in the presence of acidic components of
endogenous or exogenous origin could be a further damaging factor, since acidic
carboxyl groups are formed during the oxidation of cellulose. In addition, the release
of acetic acid, contained in verdigris, could stimulate acid-catalysed hydrolysis.
The property of the copper acetate to be particularly well adsorbed by cellulose
fibres is additionally of importance for the degradation process. The pigment has the
capacity to penetrate into the cell wall of the papermaking fibres and to initiate the
oxidative degradation process from there (Banik 1982). Scott (2002) describes this as
a process of mineralisation, during which a substance is converted to another
substance. The shape and dimensions of the original material remains unchanged,
but the material which makes up the chief component is replaced by another. This
way its original colour and also its physical properties, including the flexibility and
elasticity, change to those of the replacing material. In case of copper pigment
corrosion, the cellulose is the organic material, which is stepwise degraded by an
inorganic material, the copper acetate. During degradation, the cellulose fibers are
substituted by copper-cellulose degradation products. Finally, an offprint, a so-called
“pseudomorph” of the original fibre is created consisting only of the copper-cellulose
degradation products. The elasticity and flexibility of the original fibres is lost,
resulting in increasing embrittlement of the copper pigment corroded papers.
It is also striking that the damage of copper corrosion is mostly associated with
verdigris, whereas damage of malachite and azurite, both of which also contain

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copper, rarely occurs except under extreme climate conditions (Banik et al. 1984,
Hagadorn 2004). The reason for this appears to be due to the fact that copper acetate
is highly water-soluble (Daniels 2002). A series of tests (Meyer 2005) determined the
proportion of dissolved copper ions in a saturated solution of malachite, azurite or
verdigris in demineralised water. It turned out that the saturated solution with
copper acetate contained ten times as many dissociated copper ions compared to the
malachite solution and over forty times as many as the saturated azurite solution. It
appears that there is a link between the water solubility of a copper pigment and its
potential to cause damage. As long as a copper pigment is not dissociated, i. e. it exists
as a salt, it is unable to engage in any degrading interaction with other materials, such
as cellulose. In the presence of water, copper acetate is dissociated into divalent
copper ions and acetic acid. The ionised copper is then able to initiate the redox
reactions described above and to start degradation of cellulose by oxidation.
2. Application of complexing agents for the inhibition of copper pigment

corrosion
It appears that the free, chemically unstable copper ions are the main culprit of the
accelerated degradation of cellulose. One way to stop copper pigment corrosion
would therefore consist of binding or chemically changing these copper ions so that
they are not available for reactions with cellulose any more. The application of
complexing agents that can bind copper ions in form of stable complexes could be a
way to inhibit the progressive degradation. In paper conservation, several complex-
ing agents are already known as a means to treat transition metals in paper. They are
applied either in order to remove harmful transition metals from the paper (EDTA,
i. e. ethylenediaminetetraacetic acid) or to convert reactive metal ions into more
stable and thus less harmful forms (calcium phytate).
2.1. Complex compounds

A complex compound consists of a central ion, with which several molecules or ions,
i. e. the ligands, are associated (Mortimer 2001). This balances the positive charge of
the central ion with the ligands, which, containing a lone pair, are partially negatively
charged (Fig. 1). As a result, a regular molecular structure is generated, in which the
central ion is sequestered and therefore not available for other reaction partners. In
the case of copper pigment corrosion, the central ion would be represented by an
unstable, positively charged Cu(II) ion, which would be surrounded by a suitable
ligand, for example a carboxyl- or amino group. Hence, potentially damaging

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Figure 1: EDTA (ethylenediaminetetraacetic

acid) forming a complex with a divalent
copper ion via four carboxylate and two
amino groups.
reactions with the cellulose would not be possible. However, the formed complexes
can exhibit widely varying stabilities, a fact that considerably influences the
effectiveness of the applied treatment.
Figure 2: Possible way of two short stretches of collagen molecules (i. e. the source material of
gelatine) complexing a Cu2+ion. The amino acid sequences are GlyAlaProGlyAspProGlyAlaPro-
GlyArgPro and GlyAlaProGlyArgProGlyAspPro. Copper could also be complexed at the N or C
terminal end of the molecule (Schrieber and Gareis 2007). See page 104.

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2.2. Recent findings concerning the application of complexing agents in

iron gall ink and copper pigment corrosion
For the treatment of iron gall ink-corroded paper objects, the application of phytate
as an effective complexing agent was suggested by Neevel (1995). Phytate is
capable of chelating iron(II) ions. The formed complex is oxidized to a more stable
iron(III) phytate complex and thus blocks the detrimental effect of the iron(II)ions
on paper. The stabilising effect of calcium phytate on ink-corroded paper objects
was shown by Neevel (1999), Kolar and Strlič (2000) and Potthast (2008).
However, the effectiveness of this complexing agent with copper ions is
controversially discussed in the literature. Initial results of Kolar and Strlič
(2000) showed that phytate is unable to form complexes with copper ions
contained in iron gall inks. Rather than forming complexes, the presence of
calcium phytate accelerated the catalytic destructive activity of the copper ions on
paper. However, more recent research by Kolar et al. (2003) and by Potthast (2008)
confirm the stabilising potential of a phytate treatment on iron gall inks containing
copper, even with a Cu:Fe ratio up to 70:30. An additional application of the anti-
oxidant tetrabutylammonium bromide decreases the degradation rate on the
tested objects even further (Kolar et al. 2003).
Henniges, Schröter and Potthast (2005) examined the effectiveness of a treatment
of copper corroded papers with aqueous (magnesium hydrogen carbonate) and non-
aqueous magnesium compounds i. e. magnesium alcoholates. There are several
theories relating to the potential stabilising function of magnesium treatment on
copper corrosion. It is believed that magnesium shows complexing characteristics
with reactive, i. e. degrading peroxides (Samuelson 1970), or with copper ions
(Gilbert et al. 1973). Also a binding of magnesium ions to the carboxyl- and hydroxyl
groups of cellulose, and thus the displacement of the copper ions may occur (Sistach-
Anguera 1996).
Henniges, Schröter and Potthast (2005) further tested the addition of the anti-
oxidants Nipasol (propyl-p-hydroxybenzoate) and Nipagin (methyl-parahydroxy-
benzoate) to the aqueous and non-aqueous magnesium treatment. Both Nipa-
compounds, also known as anti-fungal preservatives, were applied as anti-oxidants in
order to bind radicals that have built up during the degradation process, and thus to
interrupt further chain reactions. The results of the authors show that an aqueous
magnesium treatment is not recommended for copper-corroded paper. The
degradation rate on samples treated with magnesium hydrogen carbonate dissolved
in water increases compared to the untreated controls. A non-aqueous treatment
with magnesium alcoholates e. g. propoxy-magnesium carbonate is able to slow

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down, but not completely to inhibit the decomposition of paper by copper ions. The
best results were produced by a non-aqueous magnesium treatment, combined with
application of the Nipa- compounds (Henniges et al. 2005). However, the high pH
created by the application of magnesium compounds is regarded sceptically
(Henniges et. al. 2005) since it induces the degradation of cellulose through ß-
elimination as described by Kerr (1982). Additionally, the authors suggest further
research concerning the long-term stability of Nipa-compounds.
Färber (2007) suggested a new anti-oxidant for the preventive treatment of
objects damaged by copper pigment corrosion. According to Färber, the anticorrosion
agent Benzotriazole is capable to bind free copper ions to form insoluble complexes,
thus making them harmless. In conservation, Benzotriazole is already applied as
anticorrosion agent for the treatment of outdoor bronze sculptures (Christman
1998). Benzotriazole has shown good results on gilding imitations based on copper
alloys, such as brass. However, its application on green or blue copper-containing
pigments proves to be more problematic since the colour of the pigments is
considerably changed by the complex formation. Such a change in colour makes the
use of benzotriazole unsuitable for treatment of paper objects damaged by copper
pigments at this time.
Ink corrosion research has suggested gelatine as another stabilisation agent with
promising potential against copper pigment corrosion. It is assumed that gelatine has
the ability to bind transition metals physically or in the form of complexes, thus
slowing down the catalytic, oxidative degradation of cellulose (Kolbe 2000). For this
reason, resizing with gelatine is recommended as an additional step in the treatment
of ink corrosion (Huhsmann 2000). However, more recent results (Potthast 2008)
could not analytically confirm the ability of gelatine to have an inhibiting effect on ink
corrosion.
3. Theoretical investigation on the complex formation with copper ions

Complexes can exhibit widely varying stabilities. The stability of some complexes is
comparable with covalent compounds, while others are very weak and therefore
completely substituted in the presence of water. The stability of a complex depends
on several factors. On a very generalised level, it is more stable
• the better the ligands are able to surround the central ion,
• the more interactions can be created between the central ion and the ligands (that
is, the more functional groups, i. e. groups capable of binding the central ion, are
attached to the ligands) and

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• the better the natural, three-dimensional form of the ligand allows a close
association to the central ion.
The stability of complexes is dependent on the pH since the charge of the
functional groups and thus their ability to form complexes changes with the pH.
3.1. The application of modified gelatine

According to theoretical considerations, it should be possible for gelatine to bind
transition metal ions in the form of complexes. This assumption is based on the fact
that functional groups are present within this protein, as well as on both ends of the
protein chain, which could function as coordination sites for free copper ions (Fig. 2).
Unfortunately, the low proportion of these functional groups and the long-chained
helix of the gelatine polymers do not favour the creation of copper-gelatine
complexes. Therefore, it can be assumed that relatively unstable complexes are
formed which would dissolve in the presence of water or high humidity. As
mentioned above, research by Potthast et al. (2008) did not find any analytical proof
for the inhibiting effect of gelatine on ink- or copper corrosion on paper. However, the
ability to form copper-gelatine complexes could be enhanced using specifically
modified gelatine. This could be achieved by the use of a short-chained gelatine or a
gelatine with a high proportion of functional, i. e. copper-binding, groups.
3.1.1. Short-chained gelatine

The basic structure of gelatine provides only the carboxyl groups on one end and the
amino groups on the other end of each protein chain which are able to bind copper
ions. The concentration of these functional terminal groups is proportional to the
length of the protein chains. This means that a protein solution with many short
protein polymers offers more functional carboxyl- and amino-terminal groups than a
solution of the equal volume and the equal concentration with few long-chained
polymers (Fig. 3a, 3b).
Accordingly, short-chained gelatine should be more effective at inhibiting copper
pigment corrosion. Furthermore, the shorter the gelatine polymers are, the better is
their mobility and thus their ability to coordinate copper ions. However, it can be
assumed that together with the chain length also the cohesive strength of the gelatine
decreases, while its adhesive strength seems to increase. This means that short-
chained gelatine would stick better to a material, but after drying it would exhibit an
greater brittleness than a longer chained modification. This should be considered

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Figure 3: The amount of functional endgroups which are able to bind copper ions (dots on the chain
ends) is proportional to the chain length of the gelatine polymers.
(a): Long chained gelatine
(b): Short chained gelatine
Figure 4: Cloning an expression of a recombinant protein

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when gelatine is used not only as a chemical but also as a mechanical stabiliser for
mending tears and losses on copper pigment corroded paper.
3.1.2. Gelatine rich in carboxyl groups

Gelatine consists of 18 different constituent units, the amino acids. It has been
suggested by Köhler (2004, personal communication) that the carboxyl groups on
the side-groups of the amino acids aspartic acid and glutamic acid are mainly
responsible for the creation of copper-gelatine complexes. It was therefore suggested
to compare unmodified gelatine with a modified gelatine rich in carboxyl groups
regarding their binding properties with copper ions.
3.2. The use of a genetically modified copper-binding protein

Above, we suggested the use of modified gelatine with improved copper-binding
properties. The idea was to modify a natural protein in such a way that its ability to
complex copper ions would be improved. Extension of this aim would be the
creation of a completely new protein with optimised copper-binding potential.
Recombination, i. e. the process by which a genetic material, the DNA (Deoxy-
ribonucleic acid) is broken and then joined to a different genetic DNA in order to
produce a new protein, would provide means to create such a new material
artificially.
All proteins are macromolecules composed of the 20 known amino acids. These
can be differentiated by their side groups and can be classified accordingly into polar,
nonpolar, acidic and alkaline amino acids. The class and the sequence of the amino
acids along with the length of the molecule define the specific physical and chemical
properties of a protein. The amino acid sequence determines how the protein chain
folds into its characteristic three-dimensional form and how it interacts with
neighbouring polypeptides. It is also responsible for the physical interaction with the
functional groups of other materials. As these properties – the folded form of a protein
and the amount and position of the functional, copper binding groups – are the
prerequisite for the formation of stable complexes, the amino acid sequence of a
protein is of great significance for its evaluation as a complex-binding agent.
The genetic information which determines the sequence of amino acids and thus
the protein formed is encoded in the DNA (Lehninger 1987). For the artificial
production of a protein (Fig. 4), its DNA – or a part of its DNA – is linked to the DNA
of another organism, i. e. the vector-DNA. This newly created (recombinant) DNA is
inserted into host cells.

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Propagation of these host cells also results in the propagation of the recombinant
DNA. By the addition of a specific inducer, the production of the new protein is
induced by the transcription of the DNA into the RNA (ribonucleic acid) and
subsequently the translation of the RNA to the production of a new protein. During
transcription, the genetic information encoded in the DNA is transferred to the RNA.
The sequence of the units of the newly created RNA, i. e. the nucleotides, is
complementary to the DNA. They provide the information for the sequence of amino
acids for the new protein to be formed. The process of transferring this information
from the RNA to build the new protein is called translation. After the expression of
the newly created proteins, these have to be isolated from their host cells.
3.2.1 Poly-His-Tag-Proteins, a promising material for complexing copper

ions
One method to isolate the recombinant proteins from their host cells is the
Immobilised Metal Ion Affinity Chromatography (IMAC) (Fig. 5). IMAC uses the
property of certain small proteins to form very stable complexes with transition metal
ions, such as divalent copper ions. The resulting copper(II)-protein-complexes can be
bound to e. g. magnetisable iron particles by organic acids at the same time. These
can be extracted from the cellular extract using a magnet. The coordination sites of
these proteins, which are able to bind copper ions are called Poly-His-Tags. Poly-His-
Tags are recombinant peptides, consisting of up to six histidines that can be attached
to any protein. Histidine is known to be able to build up stable complexes with
transition metal ions. Thus, Poly-His-Tag-Proteins are especially appropriate for the
isolation of recombinant proteins from their host cells by Immobilised Metal Ion
Affinity Chromatography.
Conversely, it is also possible to bind divalent copper ions to immobilize them and
to protect them from other reaction mechanisms. Considering this ability, Poly-His-
Tag-Proteins seem to be promising chemical inhibitors of copper pigment corrosion
on paper. However, the complexing of monovalent copper ions (Cu+) formed by
reduction is not possible. The recombination of Poly-His-Tag-Proteins is known as a
standard method. There are several recombinant DNA constructs already developed
that could be used for the expression of proteins with Poly-His-Tags. The expression
of Poly-His-Tag-Proteins only takes a short period of time (one to two weeks) and can
be performed with relatively low costs.

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Fig. 5: Partial structure of a recombinant protein with a 6-His-Tag, binding to a copper. The copper ion
is immobilized with iminodiacetic acid (red) bound to a chromatographic material. According to Chen
(Chen 2000) the histidines number 1, 3 and, 4 will coordinately bind to the copper. The positions
taken by the iminodiacetic acid could also be taken by water molecules or further histidines from the
His-Tag of the protein.
Fig. 6: Unaged sample, imprinted with basic verdigris, dispersed in 30 % aqueous solution of gum
Arabic (Source: Henniges and Schröter 2005).
Fig. 7a: Detail of an unaged sample. The pigment layer

shows a blue-greenish colour lying as a flat, homogeneous,
slightly shiny application on the paper surface. Single
pigment particles are not visible.
Fig. 7b: Detail of the sample after artificial pre-ageing 678,
10 % RH. A slight shift in pigment colour towards green is
observed.

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3.2.2. Natural occurring copper proteins

Further, there are different kinds of naturally occurring copper binding proteins,
distinguishable in their structure and function. All these proteins have in common
that the divalent copper ion is bound by the side chains of several amino acids. One
example is the Cox17 from yeast where the copper is bound by two cysteines
(Abajian 2004). Metallothioneins are able to bind up to 12 copper ions using several
cysteines in the active centre (Oh 1999 and Calderone 2005). Even the prion protein
which has been implicated in bovine spongiform encephalopathy (BSE) is complex-
ing one to four copper ions using the several histidines (Vieles 1999). In rusticyanin –
a blue copper protein – the copper is bound by four amino acids: two histidines, a
cysteine and a methionine (Giri 2004). Tests were carried out to assess the ability of
the materials mentioned above -short chained gelatine, gelatine rich in carboxyl
groups and Poly-His-Tag-Proteins- to prevent the degradation of paper through
copper pigment corrosion. This was achieved by artifical ageing of paper samples
printed with verdigris.
4. Experimental set-up
As carrier, hand-woven cotton paper without additives was used. A coating of cooked
wheat starch paste was applied onto the paper. This surface treatment was carried
out to imitate the characteristics of Islamic papers on which the damage of copper
pigment corrosion is often observed. A rhomb formed pattern was printed on the
coated papers with basic verdigris [[Cu (CH3COO)2]x [Cu (OH)2]y . z H2O], dispersed
in 30 % aqueous solution of gum Arabic (Fig. 6).
The printed paper samples were pre-aged at 678C and 10 % relative humidity.
After the artificial pre-ageing they were treated with one of the following test
solutions: Sol D®, Imagel®, methylcellulose, Poly-His-Tag-Proteins or water.
Sol D® is a Type B modified gelatine, which has been degraded by acid hydrolysis
and is therefore particularly short-chained. It is soluble in cold water. The formation
of a gel is not possible. Despite its extreme short chain length, Sol D® has high
adhesion power and higher swelling ability than longer-chained gelatine types but its
cohesive strength is lower, which is manifested by a greater brittleness in a dried
state. Sol D® was tested assuming that the chain length of the gelatine influences its
ability to complex copper ions.
The product Imagel® is a modified gelatine with a high proportion of carboxyl
groups. Therefore it should be especially effective in inhibiting copper pigment
corrosion. Since the carboxyl groups have to be present in ionised form in order to

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bind copper ions, the pH of the solution was adjusted to pH 8 by the addition of
aqueous calcium hydroxide.
The recombinant Poly-His-Tag-Proteins were also tested. It is a slightly cloudy,
yellowish liquid. Its viscosity, based on visual assessment, lies between the one of Sol
D® and water. However, Poly-His-Tag-Proteins do not provide any adhesive strength
and are thus not suitable for a mechanical stabilisation of copper pigment corroded
paper.
Methylcellulose (MH 400), a relatively short-chained cellulose ether of low
viscosity, was used as a non-proteinaceous-reference material. It was applied to
ensure that the effect of the other test solutions was not due to the formation of a
protecting film which prevents the influence of oxygen from the air and humidity on
the pigment layer. Oxygen and a high relative humidity are decisive factors that
promote the catalytic degradation reactions of cellulose in the presence of copper
ions.
One set of samples was used as reference. However, it was treated with water
since water was also applied on the other samples, acting as solvent for the treatment
materials tested.
The application of the treatment materials was performed with an airbrush on a
suction table. The different types of gelatine were applied in 5 % concentration at a
distance of about 20 cm. The application was repeated three times, without allowing
any drying between each application. As methylcellulose is more viscous, it was used
in 2.5 % concentration, but with six instead of three applications. For these
experiments, Poly-His-Tag-Proteins could be produced and therefore used only in a
relatively low concentration of 0.6 %. After the application of the test solutions, the
samples were subjected to dynamic accelerated ageing at 508C and a relative
humidity alternating between 50 and 80 % every 3 hours. The samples were aged as
single sheets suspended in an ageing oven. Samples were taken after 3, 5, 8 and 15
days. One set of treated and untreated samples were kept as unaged reference.
4.1. Observations
The assessment of the ageing characteristics was performed by direct vision and also
through a reflected-light stereomicroscope. Microscopy photographs were used for
documentation. To be able to evaluate objectively the colour changes that take place
on the samples during artificial aging, the treated and aged samples were measured
using a spectral photometer.
In the unaged state the pigment shows a blue-greenish colour lying as a flat,
homogeneous, slightly shiny layer on the paper surface (Fig. 7a). Single pigment

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particles are not visible even under a magnification of 32 times. During artificial pre-
ageing, a slight shift in pigment colour towards green is observed on the samples
(Fig. 7b). Apart from this, the slightly shiny pigment layer does not change. A
blackening of the pigment does not occur even at an increased temperature of 678C
and 10 % relative humidity, although verdigris has been described in the literature as
heat sensitive at higher temperatures (Kühn 1993).
After the application of the treatment materials and during subsequent dynamic
artificial ageing similar changes were observed on all samples. However, the extent of
the changes and the point of time at which they occurred were considerably different
and dependent on the respective treatment. The general observations of the ageing
characteristics which more or less occurred on all samples will be described below.
Subsequently, ageing behaviours of the differently treated samples will be discussed.
4.2. General ageing characteristics of the pigment applications

During the ageing, the outflow of acetic acid was noticed by the smell when opening
the ageing oven. The splitting of acetic acid from the copper acetate pigment can be
demonstrated by the discoloration of Acid-Detections-Strips that were placed in the
ageing oven. Acid-Detection-Strips are pH indicator papers which change their
colour in the presence of gaseous acetic acid from blue over green to yellow. The
discoloration of Acid-Detection-Strips, which were deposited on samples stored at
room climate shows that the separation of acetic acid from the pigment also takes
place during natural ageing.
Once the process of dynamic artificial ageing was started, the pigment layers
changed their appearance from a smooth, slightly shiny surface to a matte layer with
a grainy structure. At the same time, the colour shade turned from bluish green to a
brighter green with a higher content of yellow (Fig. 8a). This observation could be
confirmed by spectrometric measurements.
By the next ageing process a gradual browning of the pigment layer was observed
(Fig. 8b). However, this browning does not occur homogeneously. It starts in the
spaces between the newly formed pigment grains. Compared to the general pigment
layer, these areas exhibit a stronger glossiness. Under a magnification of 32 times,
they show an amorphous appearance that reminds of a solidified liquid. During the
browning, this amorphous material in the spaces between the pigment particles
looses its cohesion and adhesion to the paper carrier. As a result the pigment particles
become powdery.
In an advanced stage of ageing, the pigment also starts to darken gradually
(Fig. 8c). The pigment layer of the samples treated with methylcellulose and water

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turns to an almost completely black product (Fig. 8d and Fig. 9d). No adhesive
power to the paper carrier remains, and the particles start to powder at the slightest
touch with a brush.
4.3. Differences in the ageing behaviour of the samples: effectiveness of

the different treatment agents
Similar changes could be observed on all samples during the artificial ageing process
as they were described in the section above. Depending on the treatment applied,
these processes occurred at a different speed and to a different extent. The ageing of
the samples treated with methylcellulose or water proceeded considerably faster
than for those treated with gelatine. While the browning of the spaces between the
pigment particles took place in the samples treated with methylcellulose or water
after three or five respectively days (Figs. 8 and 9), this is observed in the samples
treated with the modified gelatines only after eight days of accelerated ageing, and
even then to a significant lesser degree. An overall browning of the whole pigment
layer was not observed (Figs. 10 and 11).
A treatment with Sol D® is most effective in retarding the described ageing
phenomenon. Even after 15 days most of the pigment grains are still green. A loss of
adherence of the pigment layer and an increase of powdering cannot be observed.
The ageing characteristics of the samples treated with Poly-His-Tag-Proteins are
much more inconsistent (Fig. 12 a–d). Compared to a treatment with methylcellu-
lose and water, and also with both modified types of gelatine, the ageing results are
significantly better in some parts: the pigment is not browned yet and does not
powder at all. In other parts, no difference can be observed compared to the
methylcellulose or water treatment: the browning of the pigments and of the binder
has taken place. In an advanced state, the pigment application starts to powder
severely. These comparatively good results are even more remarkable, considering
that the application of the Poly-His-Tag-Proteins was carried out at a concentration of
0.6 % while the other gelatine types were applied in a concentration of 5 % i. e. at
almost ten times higher concentration.

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Fig. 8a–d: Detail of the reference

sample (see Fig. 7b) treated with
water after 3 days (a), 5 days (b), 8
days (c) and 15 days (d) of dynamic
artificial aging. The pigment layer
gradually discolours from green
over yellowish-green and finally
turns to an almost completely
black product. No adhesive power
to the paper carrier remains, and the
particles start to powder at the
slightest touch with a brush.

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Fig. 9 a–d: Ageing characteristics of

the samples treated with methyl-
cellulose (after 3 days (a), 5 days (b),
8 days (c) and 15 days (d) of
dynamic artificial ageing. The pig-
ment layer gradually discolours
from green over yellowish-green
and finally turns to an almost
completely black product. No adhe-
sive power to the paper carrier
remains, and the particles start to
powder at the slightest touch with a
brush.

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Fig. 10 a–d: Ageing characteristics

of the samples treated with gelatine,
rich in carboxyl groups (Imagel®)
after 3 days (a), 5 days (b), 8 days (c)
and 15 days (d) of dynamic artificial
aging. Browning of the pigment
layer, starting in the amorphous,
glossy spaces between the pigment
particles, is observed only after eight
days. Thus it proceeds significantly
slower than on the samples treated
with water or Methylcellulose.

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of the samples treated with short-
chained gelatine (Sol D®) after 3
days (a), 5 days (b), 8 days (c) and 15
days (d) of dynamic artificial aging.
The treatment with Sol D® is most
effective in retarding the described
ageing phenomenons. Even after 15
days most of the pigment grains are
still green. A loss of adherency of
the pigment layer and an increase of
powdering cannot be observed.

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of the samples treated with the
recombinant proteins (Poly-His-Tag-
Proteins) in a concentration of 0.6 %
after 3 days (a), 5 days (b), 8 days (c)
and 15 days (d) of dynamic artificial
aging .The ageing characteristics of
the samples treated with Poly-His-
Tag-Proteins are heterogeneous.
Compared to a treatment with
methylcellulose and water, and
also with both modified types of
gelatine, the ageing results are sig-
nificantly better in some parts: the
pigment is not browned yet and
does not powder at all. In other
parts, no difference can be observed
compared to the methylcellulose- or
water treatment: the browning of
the pigments and of the binder has
taken place. In an advanced state,
the pigment application starts to
powder severely.

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4.4. Interpretation
4.4.1. Ageing characteristics of the samples
The separation of acetic acid is observed during artificial and natural ageing. This
seems to be the result of a chemical reaction, during which copper acetate is changed
into a new product or the copper ions are bound to surrounding materials, separating
the acetic acid.
At the beginning of the dynamic accelerated ageing process the smooth, shiny
pigment layer turns to a matte, grainy structure. Spectrometrically, a brightening and
a shift towards yellow can be measured. The brightening of the pigment colour can
be explained with the change of the surface morphology. A smooth film into which
the pigment is enclosed appears darker and glossier than a structured, grainy surface.
The reasons for this are the changes in light reflection and -refraction, caused by the
changed surface structure (Hansen 1993). However, the changes in the surface
structure do not seem to be the only reason for the changing optical appearance since
the colour of the pigment application does not only become lighter, but also turns in a
more yellow shade. This means that a chemical reaction must have taken place. The
resulting product cannot be accurately determined only by optical means. However,
the substances that are available as potential reaction partners in the environment are
known:
• Oxygen, contained in the air
• Carbon dioxide, contained in the air
• Water in gaseous state
• Carbohydrates which are present as cellulose (paper) or in the binder gum Arabic
Considering these possible reaction partners and the pale green colour of the
changed pigment imprint, one can draw the conclusion that the grainy reaction
product should be spertiniite (Cu(OH)2) or malachite (CuCO3·Cu (OH)2).
In the next ageing state the glossy amorphous material in the spaces between the
pigments started to brown. It is probable that this substance is the binding medium
gum Arabic that decayed in the presence of copper ions and turned into a brown
degradation product. Together with the proceeding browning of the amorphous
substance, the pigment layer became powdery. This confirms the suggestion that the
browned substance is gum Arabic which is subjected to a degradation process that is
demonstrated by the decrease of its adhesive power.
In an advanced stage of the ageing, a complete blackening of the whole pigment
layer was observed in the samples treated with water, methylcellulose and also to an
extent in those treated with Poly-His-Tag-Proteins. This black reaction product is

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probably tenorite (CuO). Tenorite is described as a material of a dull black appearance

(Scott 2002) that is formed when copper is heated. Thus, the creation of tenorite is an
artefact of artificial ageing at increased temperatures, which does not occur during
natural ageing.
Overall it can be assumed that the observed browning of the pigment application
can be explained by two different reaction mechanisms:
• The degradation of the binder -gum Arabic- probably by an oxidation process

which takes place in the presence of copper ions.
• The transformation of copper acetate to a black product, probably tenorite, along
with an intermediate product of a pale greenish colour which on visual
observation could be spertiniite or malachite.
The observed changes seem to take place only in the presence of a high relative
humidity, whereas only a high temperature is not sufficient. This was shown by the
fact that a clearly visible change of the pigment layer takes place only at ageing
conditions with an increased relative humidity cycling between 50 % and 80 % and a
temperature at 508C. During the pre-ageing process of the samples at 10 % relative
humidity and a temperature at 678C, this process could not be observed.
4.4.2. Degradation of paper by copper pigments during artificial ageing

The degradation of the paper carrier by the copper pigments could not be simulated
on any of the samples with the chosen ageing method. Degradation characteristics
like the browning of the paper, an increase of brittleness or the formation of tears
could not be achieved.
In addition to the described optical and haptical assessment of the samples, the
Christian Doppler Labor für Zellstoffreaktivität in Vienna kindly agreed to analyse the
treated and the non-treated, artificially aged and unaged samples chemically using
the CCOA-method. This method combines the measurement of carbonyl groups
formed by oxidative degradation of cellulose by fluorescence detection together with
the determination of the molar mass of the cellulose. Even with this highly sensitive
examination method, decomposition of the paper by copper pigment corrosion could
not be detected on any of the samples through an increase of the carbonyl groups
content or the decrease of the molar mass during the artificial ageing.
It is likely that the cycling and high relative humidity during the artificial ageing
was not sufficient to cause the migration of the copper ions into the paper-matrix.
Further, the high content of gum Arabic of 30 % in the pigment layer as well as the
wheat starch coating of the samples did inhibit the degradation of the paper: the

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pigment reacts first with the binder containing carbohydrates and possibly also with
the starch-coating before it is able to reach and thus decay the paper support. In
addition, the high degree of polymerisation of the cotton- cellulose which was used
as raw material for the sampling papers was responsible for our inability to detect
smaller changes in the molar mass of the cellulose (Potthast, 2005, personal
communication).
4.4.3. Ageing of gum Arabic

However, although no detectable damage by copper pigment corrosion was observed
in the paper samples, it is still possible to draw conclusions from the ageing results
relating to the effectiveness of the tested treatment solutions. Instead of paper, the
degradation of another carbohydrate contained in the samples could be observed:
the one of gum Arabic, which is in direct contact with the copper pigment. The
degradation of gum Arabic and of paper in the presence of copper ions causes
comparable damage characteristics: both processes start with the browning of the
initial material under creation of amorphous copper-carbohydrate products. In an
advanced stage this leads to a decrease of the mechanical stability, which is shown in
cellulose by the brittleness of the paper, in gum Arabic by the loss of its function as a
binder to adhere the pigment particles to each other and to the carrier. It was
discovered that the use of gelatine and the Poly-His-Tag-Proteins as treatment
materials could slow down the decay of gum Arabic and also significantly inhibit the
colour change of the pigment. This leads to the assumption that the proteins were
able to bind free copper ions and thus to inhibit the described degradation processes.
It seems to be possible that gelatine and Poly-His-Tag-Proteins could have the same
inhibiting effect on the degradation of paper in the presence of copper ions.
The above described experiments support the conclusion that the gelatine
hydrolysate Sol D® has greater stabilising potential compared to Imagel®. This seems
to confirm the assumption that short-chained gelatine could be especially effective in
inhibiting copper pigment corrosion. The hypothesis that gelatine rich in carboxyl
groups could also be very effective could not be confirmed, since the ageing results
are worse than those of Sol D®. However, to fully reject this hypothesis, a direct
comparison of a gelatine rich in carboxyl groups with unmodified gelatine with the
same chain lengths would be necessary.

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4.4.4. Effectiveness of Poly-His-Tag-Proteins in the inhibition of copper

pigment corrosion
The significantly heterogeneous ageing characteristics of the samples treated with
Poly-His-Tag-Proteins seem to lead to the conclusion that these proteins are generally
able to bind copper ions and therefore to inhibit the progressive copper corrosion.
The lower (almost ten times) concentration of the Poly-His-Tag-Proteins
compared to Sol D® and Imagel® was obviously not sufficient to bind all free copper
ions and to prevent them from reacting with other reaction systems. However, a
direct comparison of the effectiveness of Poly-His-Tag-Proteins with the modified
gelatine types was not possible because of the different concentration of the
treatment materials. For this reason, further examination was carried out using also
0.6 % solution of Sol D®, the most promising treatment material. Both materials, Sol
D® and the Poly-His-Tag-Proteins, were applied on the verdigris imprinted samples
and aged artificially as described above. Using both testing materials in the same
concentration, their effectiveness could be directly compared (Figs. 13 and 14).
After five days of artificial ageing, Sol D® at the low concentration also showed the
heterogeneous greenish-brown appearance which was typical in the former
investigations for Poly-His-Tag-Proteins. The partial browning of the pigment layer
is obviously produced when the treatment material is generally able to inhibit the
change of the green pigment layer to a brownish reaction product, but is not present
in a sufficient amount to completely stop the process.
It can be concluded with some precaution that the Poly-His-Tag-Proteins have a
greater potential to complex copper ions than Sol D® when used at the same
concentration. Thus, they seem to be most promising of the tested treatment
solutions in their ability to inhibit copper pigment corrosion. In addition, the above
experiments demonstrated that the effectiveness of an applied treatment material
depends on the concentration of the applied material.
5. Conclusion
The results of our investigations seem to indicate that proteins are able to complex
copper ions and thus to inhibit or at least slow down the degradation of paper by
copper ions. The recombinant Poly-His-Tag-Proteins show the best results, followed
by the short-chained type of gelatine, i. e. Sol D®. It could be shown that the
protective effect of the applied material was not due to the formation of a surface film
on the pigment layer that inhibits the penetration of oxygen and humidity, but that
complexing of the detrimental copper ions by the tested proteins must have taken

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Figure 13 a–c: Ageing character-

istics of the samples after a treat-
ment with Poly-His-Tag-Proteins in
a 0.6 % concentration 5 days (a), 8
days (b) and 15 days (c) of dynamic
artificial aging. The samples show
heterogeneous ageing characteris-
tics: In some parts, the pigment
does not discolour or become pow-
dery at all. In other parts, a severe
browning of the pigments and of the
binder takes place.
place. However, these results could not be proven in a direct way since a
decomposition of the carrier material paper by verdigris could not be simulated by
artificial ageing. Instead, the degradation and change of another carbohydrate, gum
Arabic, and of the pigment itself was evaluated. The potential effectiveness of the
tested treatment material was measured by the deceleration of the observed changes
of gum Arabic and the pigment layer.
To confirm our hypothesis on the effectiveness of the tested material, a method of
artificial ageing should be developed which causes the degradation of paper by

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Figure 14 a–c: Ageing character-

istics of the samples after a treat-
ment with Sol D® in a 0.6 %
concentration after 5 days (a), 8
(b) and 15 days (c) of artificial
ageing. Treated with Sol D® at a
low concentration, the samples
show the same, heterogeneous age-
ing characteristics as the samples
treated with 0.6 % of Poly-His-Tags.
The low concentration of both treat-
ment materials are obviously not
sufficient to bind all free copper ions
and to prevent them from reacting
with other reaction systems.
copper pigments and which better imitates the natural ageing characteristics of
copper pigment corrosion. This was not possible during the ageing tests carried out
since copper acetate turns black in combination with increased temperatures and
relative humidity. This is an artefact which does not occur during natural ageing.
The degradation of the paper carrier did not take place at the chosen experimental
parameters since the carbohydrate-based binder gum Arabic and the paper coating
containing wheat starch acted as a protective material which was degraded before

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the pigment could be decayed by the copper pigments. For further research the
application of pure copper acetate on the paper samples without binding medium or
paper coating would be suitable. The application of a saturated copper acetate
solution using a pen or a computer plotter would allow direct contact of copper
acetate with the absorbent paper fibres avoiding the use of a binding agent. The
recombinant Poly-His-Tag-Proteins showed to be the most effective of the treatment
materials tested for the inhibition of the degradation of the pigment layer and the
binder, gum arabic. This result is based on a series of experiments in which this
material could only be applied in a relatively low concentration. Further research
could use of Poly-His-Tag-Proteins at higher concentration.
Acknowledgements
We would like to express our gratitude to Ute Henniges, Christian Doppler Labor für
Zellstoffreaktivität, Universität für Bodenkultur (BOKU), Vienna, who kindly agreed
to analyse the paper samples using the CCOA method. Furthermore we would like to
thank Gerhard Banik and Ute Henniges for their helpful advice and support during
this research. Gangolf Ulbricht, Berlin, kindly supported us by producing the paper
samples.
References
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Materials
Material Specification Source
Gum Arabic Pulverised Kremer Pigmente GmbH & Co KG
Hauptstrasse 41 – 47
D-88317 Aichstetten/Allgäu
T: +49 7565 91120
Acid Detection Strips for the detection of the vinegar Mono C GmbH
Strips syndrom Königstor 14a
D-34117 Kassel
T: +49 0561 93519 0
Imagel® Gelatine rich in carboxyl groups DGF Stoess
Uferstrasse 7
D-69402 Eberbach
T: +49 6271 8401
Methylcellulose MH 400 Aqualon
Paul Thomas Strasse 58
D-40551 Düsseldorf
T: +49 211 7491 07
Paper Cotton paper, vélin, Dipl. Ing. Gangolf Ulbricht
free of alkaline filler Mariannenplatz 2
unsized D-10997 Berlin
T: +49 30 6158155
Poly-His-Tag-Pro- Copper binding protein Dr. Anke Neumann
teins Universität Karlsruhe (TH)
Institut für Bio- und Lebensmittel-
technik
Bereich II: Technische Biologie
(TeBi)
Engler-Bunte-Ring 1
D-76131 Karlsruhe
T: +49 721 608 21 25

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Materials (Continued)
Sol D® Short-chained gelatine hydrolisate DGF Stoess
Viscosity 2.19 mPas (10 %/308C) Uferstr. 7
Average molecular Weight: 5 – 7 kD D-69402 Eberbach
T: +49 6271 8401
Verdigris Basic copper acetate Kremer Pigmente GmbH & Co KG
[Cu(CH3COO)2]x [Cu(OH)2]y . z H2O Hauptstrasse 41 – 47
D-88317 Aichstetten/Allgäu
T: +49 7565 91120
Wheat starch Karl Roth GmbH
Schoemperlenstrasse 3
D-76185 Karlsruhe
T: +49 721 5606 0
Devices
Devices Source
VEGA Airbrush device Badger-Air Brush Co.
9128 W. Belmont Ave.
Franklin Park, IL 60131
USA
Climate Chamber (VC 0020) Vötsch-Hereus
Beethovenstrasse 34
D-72336 Balingen-Frommen
T.: +49 4433 3030
Drying cabinet Memmert GmbH
Postfach 1720
D- 91107 Schwabach
T.: +49 7631 795 0
Printing block Lutz Walter
Nöschenröderstr. 46
D-38855 Wernigerode
T.: +49 3943 55 39 351

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Zusammenfassung
Rekombinante Proteine: Ein neues Material zur chemischen Stabilisierung kupferfraßgeschädigter

Papiere?
Neue Ansätze zur chemischen Behandlung kupferfraßgeschädigter Papierobjekte werden untersucht.

Im Einsatz von Gelatine wird eine Möglichkeit zur chemischen Stabilisierung von Übergangsmetall-
Ionen auf Papier vermutet. Gelatine soll hierbei befähigt sein, Wechselwirkungen zu Übergangsme-
tall-Ionen aufzubauen und sie so dem Reaktionssystem mit der Cellulose zu entziehen. Nach
theoretischen Überlegungen handelt es sich bei den Wechselwirkungen zwischen Kupfer-Ionen und
Gelatine aber nur um relativ instabile Komplexverbindungen, die sich in Anwesenheit von genügend
Wasser wieder lösen. In dieser Arbeit wurden an Grünspan bedruckten, künstlich gealterten Proben
Möglichkeiten zur Verbesserung der kupferbindenden Eigenschaften von Gelatine getestet. Hierfür
wurde eine kurzkettige und eine carboxylgruppenreiche Gelatinemodifikation verwendet. In einem
weiteren Experiment wurde der Einsatz eines, durch Klonierung künstlich hergestellten Proteins
untersucht. Die Ergebnisse deuten darauf hin, dass Proteine Kupfer-Ionen in Form von Komplexen
binden können. Kurzkettige Gelatinen scheinen wirksamer zu sein als deren langkettige Modifika-
tionen. Die klonierten Proteine zeigen im Vergleich zu den Gelatinemodifikationen bezüglich der
Inhibierung von Kupferfraß die besten Ergebnisse.
Résumé
Protéines créées artificiellement par clonage: un nouveau moyen pour le traitement chimique du
papier corrodé au vert-de-gris
De nouvelles possibilités de traitement chimique s’appliquant aux objets en papier corrodés par le vert-
de-gris sont analysées. On a supposé que la gélatine pouvait éventuellement servir de stabilisateur
chimique lors de la transition des ions métal sur le papier. Il semble que la gélatine soit capable
d’interagir physiquement avec les ions métal de transition en les soustrayant ainsi du système de
réaction avec la cellulose. Cependant, selon des considérations théoriques, lors de ces interactions
entre les ions de cuivre et la gélatine il ne peut s’agir que de complexes relativement instables qui se
dissolvent à nouveau rapidement en présence d’une quantité d’eau suffisante. Dans ce contexte on a
examiné des possibilités visant à optimiser les propriétés de liaison chimique de la gélatine sur des
échantillons de papiers imprimés, corrodés au vert-de-gris et qui avaient été soumis au vieillissement
artificiel. A cette fin on a testé l’application d’une gélatine de chaîne courte et d’une gélatine riche en
groupe carboxyle. Lors d’une expérience subséquente on a analysé l’action d’une protéïne fabriquée
artificiellement par clonage. Les résultats ont démontré que les protéïnes étaient capables de se lier
avec des ions-cuivre sous forme de complexes. Les chaînes courtes de gélatine semblent être plus
efficaces que leurs modifications à chaîne longue. Les protéïnes clonées qui ont été testées présentent
le plus fort degré d’efficacité en ce qui concerne l’inhibition du pigment de cuivre.

Authenticated
Authors
Fabienne Meyer obtained her diploma in paper conservation at the State Academy of Art and Design
in Stuttgart in 2005, after an apprenticeship in book binding between 1996 – 1999. During her
studies, she worked as an intern in several collections preserving Islamic objects, developing
experience with the treatment of copper-corroded Islamic manuscripts. Between 2005 and 2008, she
was employed as a paper conservator for the collection of prints and drawings at the Saarlandmuseum
Saarbrücken. Since 2008, she is working as conservator for Islamic manuscripts at the Authority for
Culture and Heritage (ADACH) in Abu Dhabi.
Dr. Anke Neumann obtained her diploma in technical biology from the University of Stuttgart in
1993, where she studied an anaerobic dechlorinating bacterium. In 1998 she received her Ph.D.
degree from the University of Stuttgart for her biochemical and genetic studies on the tetrachloroe-
thene dehalogenase from Dehalospirillum multivorans. After 5 years of postdoctoral research and
teaching at the Friedrich-Schiller-University of Jena in the Group of Prof. Dr. G. Diekert she joined the
Section of Technical Biology of Prof. Dr. C. Syldatk at the University of Karlsruhe in 2003. Besides
lecturing on genetics her research interests focus on enzymatic conversion of glycolipids and the
fermentation of yeast and fungi.
Contact
Fabienne Mayer
Abu Dhabi Authority for Culture and Heritage
Conservation Department
P.O. Box 2380
UAE-Abu Dhabi
United Arabic Emirates
T. : +971 2 4445572
Mail: meyer_fabienne@yahoo.de
Anke Neumann
Universität Karlsruhe (TH)
Institut für Bio- und Lebensmitteltechnik
Bereich II: Technische Biologie (TeBi)
Engler-Bunte-Ring 1
D-76131 Karlsruhe
T.: +49 721 608 21 25
Mail: anke.neumann@tebi.uni-karlsruhe.de

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Restaurator, 2009, pp.

96 – 130 Copyright © Saur 2009

Recombinant Proteins: A new material for the chemical stabilisation of

by Fabienne Meyer and Anke Neumann

Zusammenfassung/résumé at end of article

received: 02.02.2009 revised: 24.20.2009

1. Copper Pigment Corrosion

1.1. Damage Characteristics

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Table 1: Chemical formulas of basic verdigris and neutral verdigris

1.2. Chemical mechanisms

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2. Application of complexing agents for the inhibition of copper pigment

2.1. Complex compounds

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Figure 1: EDTA (ethylenediaminetetraacetic

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2.2. Recent findings concerning the application of complexing agents in

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3. Theoretical investigation on the complex formation with copper ions

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3.1. The application of modified gelatine

3.1.1. Short-chained gelatine

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Figure 4: Cloning an expression of a recombinant protein

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3.1.2. Gelatine rich in carboxyl groups

3.2. The use of a genetically modified copper-binding protein

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3.2.1 Poly-His-Tag-Proteins, a promising material for complexing copper

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Fig. 7a: Detail of an unaged sample. The pigment layer

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3.2.2. Natural occurring copper proteins

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4.2. General ageing characteristics of the pigment applications

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4.3. Differences in the ageing behaviour of the samples: effectiveness of

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Fig. 8a–d: Detail of the reference

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Fig. 9 a–d: Ageing characteristics of

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Fig. 10 a–d: Ageing characteristics

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Fig. 11 a–d: Ageing characteristics

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Fig. 12 a–d: Ageing characteristics

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