The composition and chemistry of eggs and egg tempera
Alan Phenix
Introduction
In terms of technique, Italian painting of the fourteenth and
fifteenth centuries is one of the most extensively studied
periods of art history. It remains, however, that the egg tem-
pera* binding medium which is so central to painting meth-
ods of this time has received surprisingly little attention from
either the scientific or conservation points of view.
Reinkowski-Hafner has recently reviewed the history of the
use of egg tempera in depth 1 . Scientific studies have gener-
ally focused on the identification of egg in works of art
through analysis of either its proteins or lipids. 2 3, 4, 5, 6, 7
Until very recently, conservation literature relating to the
ageing/ deterioration of egg components or to the impact of
conservation treatment on egg tempera paint ftlms was vir-
tually non-existent. With regard to ageing, the primary ref-
erence for proteinaceous materials like tempera has been the
theoretical discussion by KarpowiczB, and Masschelein-
Kleiner made observations relevant to the analysis of pro-
teins in old paint ftlms9. Although still very much directed to
the analysis of egg and other proteinaceous media, impor-
tant information has been presented recently by Schilling
and co-workers on the effects of pigmentation and of ageing
on chemical constitution of egg paints. w , II, 12
The relative lack of material - at least in comparison to what
is known of drying oils and resins - concerning the proper-
ties of aged tempera paint films appreciably limits the abili-
ty of the conservator to make informed judgements as to the
likely effects of conservation treatments, particularly clean-
ing, on tempera paint ftlms: and it was this concern that ini-
tiated work on tempera at the Courtauld Institute. This
started with a student diploma project which examined
changes in surface and physical characteristics of reference
tempera films as a consequence of accelerated ageing and
exposure to organic solvents. 13 Samples of variously pig-
mented tempera paint ftlms have subsequently been further
examined for extractable components by GC and by GC-
MS, and for changes in the protein components by gel elec-
trophoresis. 14 15 I6
Although the composition, chemistry and properties of egg yolk
as a material are poorly described in the field of conse1vation, a
great deal of material on this matter exists in the literature of
other subject areas, in particular biochemistry and food science.
* It is recognised that the term egg tempera can be applied to a vari-
ety of paint vehicles based on egg, which may comprise egg white,
gums, resins or other materials in addition to egg yolk. In this pape1~
however, egg tempera is used to describe a paint medium consisting
essentially of egg yolk in the manner of Cennino Cennini.
II
The purpose of this paper is, essentially, to bring pertinent
material into the arena of conservation. The aims of this
paper, therefore, are:
- to review what is known of tl1e structure, composition and
chemistry of eggs and, especially, egg yolk;
- to identify features of the composition and chemistry of
egg yolk relevant to the hardening (curing) and ageing of
egg tempera paint films; and
- to lay the essential foundations for the papers later in these
proceedings.
The Gross Structure of Hens' Eggs
The major constituents of hens' eggs - shell, white or albu-
men, and yolk - will be familiar to all; but the egg contains
many sub-structures which are not immediately evident to
the normal user. 17. 18, 19
The arrangement of the principal parts of a hen's egg are
shown in Figure 1. The albumen (white) is not homogeneous
and consists of four layers of differing viscosity.
The yolk is suspended in the albumen and held in place by
the chalazae which are fibrous structures connected to the
inner thick white layer. The yolk itself is contained within
the vitelline membrane.
The yolk has a banded structure and contains the blastodisc.
In effect, the egg yolk is the largest single biological cell
known, in the sense that it originates from one cell division
and it contains a nucleus in the blastodisc. Cell divisions in the
blastodisc bring about embryogenesis and this is the location
of the very small amount of nucleic acid contained in the egg.
Figure 1: Longitudinal section through a hen's egg
Chalazae
Shell
Vitelline membrane
Yolk dark bands
Inner thin white
Inner thick white Latebra
Outer thin white
Outer thick white
Air cell
Egg White - the albumen yolk can be seen to contain several populations of particles
- yolk granules and yolk globules- suspended in a liquid
The albumen consists mostly of water (88-89%), the remain- p lasma phase.
der being mostly protein (10 .5 %), carbohydrate (0 .5 %) and
inorganic ions (0 .5 %). Hens' egg albumen has been exten- Figure 2 : Schematic representation qf egg)'olk at the sub-microscojJic level with
sively studied, to the point where the amino acid sequences descriptions qf the principal constituents qf each element.
of the principal constituent proteins are known. Table I lists
Insol uble yolk g lobules 0.5-5 Jlm,
the names and characteristic features of the proteins of egg contain:
white. 17, 18, 19, 20 mostl y low density lipoprotein \\~th additional
phospholipid,
water,
Table 1: Proteins qf egg white Yolk granules 0.3 - 1.6 Jlm,
constitute -25 "/o of all yolk solids,
(From Stadelman & Cotterill 18) con tam:
Phosvitin ,
Lipovitellin , }
LDLg, all are lipid / protein
Protein Amount MolWt Denaturation Mye lin figures [VLDL] complexes
of Albumen temp [0 C] water(5o%)

Plasma
Ovalbumin 54 45000 84 - 75'% of yolk solids
con tams:
soluble proteins [Li,etins, ,itamin-binding protein] - r6% of yolk solids,
Ovotransferrin I2 76000 6I Low density lipoprotein - 6o 04l of yolk solids, dissolved or dispersed as ultra-
[conalbumin] fine particles 20-30nm;
water
Ovomucoid II 28000 70
Ovomucin 35 55-83 X !06 75
Lysozyme 3-4 I4300 925 The Gross Composition of Hen's Eggs
+
G2 globulin 40 3.0-4-5 X IO
G3 globulin 40 The gross composition of hen's eggs is widely reported and
Ovo inhibitor !.5 49000 although there are slight variations between authors, typical
Ficin inhibitor o.os I2700 values are as in Table 2. Approximately 52 .5% of the fresh
Ovoglyco- yolk is solid matter. The pH of fresh egg yolk is generally
protein !.0 24400 around 6. The majority of yolk [66%] is composed of lipid;
Ovoflavo- in terms of mass, protein is the lesser component. Lipids are
protein 0.8 32000 present as triacylglycerides, phospholipids, cholesterol and
Vomacro- cholesteryl esters.Virtually all lipid is associated/ complexed
globulin o.s 7-6-9.0 x w 5 with protein through non-covalent interactions. There may
Avidin o.os 68300 be a small amount of free lipid and of covalently bound
lipid. Lipids are present in the plasma, in the yolk globules
and in the yolk granules in various associations with protein;
either as particles or as soluble complexes.
Egg Yolk The phospholipids have hydrophilic parts and lipophilic
parts and are highly surface active. The majority of lipid in
The yolk contains particles suspended in a liquid medium. fresh yolk can easily be extracted from the combination with
For many years it has been regarded, at least within the field protein by suitable organic solvents [ketones, chlorinated sol-
of conservation, somewhat erroneously as an emulsion. vents, alcohols, hydrocarbons].
Writing in their encyclopa:dia of painting materials, Gettens The dominant fatty acids in egg yolk lipids are:r6:o [palmitic
and Stout describe it as: acid], I8:o [stearic acid], I8:I [oleic acid] and 18:2 Qinoleic
an oi(y emulsion in wlziclz the oil particles are suspended in a solution acid] . Nearly so% of all fatty acid chains contain potential-
qf albumen. Lecitlzin ..... evident(y acts as an emulsijjing agent. Wizen ly reactive double bonds. Unsaturated fatty acids occur in
egg yolk is used as a painting medium, it dries to a strong film .first by both neutral lipid and phospholipid fractions.
evaporation qf the water and then by a slow hardening qf the oil wlziclz Several researchers report the presence of polyunsaturated
remains suspended in the albuminous matrix 21 fatty acids, I8:2, I8:3 etc., up to 22:6. 22 Longer chain fatty
Although it is true that egg yolk contains oily substances, water acid residues are more prevalent in the phospholipids.
and substances with emulsifying properties, it is not strictly In triacylglycerides and lecithins, saturated fatty acids pref-
speaking an emulsion. In reality, hydrophobic (lipid) and erentially occupy the I glyceryl position while unsaturated
hydrophilic (protein) components are not so separated as this fatty acids preferentially occupy the 2 glyceryl position.
model in1plies. It is hoped that the present review will clarify
the nature of fresh egg yolk and, further, elaborate on the Lipid, phospholipid and fatty acid distributions are known
processes which lead to the hardening of egg tempera films. and reported for most of the lipoprotein consitutents of egg
yolk. 17 As many as 14 distinct proteins have been identified
Egg yolk is a highly complex and heterogeneous substance. in egg yolk. The amino acid compositions of the main pro-
A complete description of its nature is perhaps best achieved tein consitutents of egg yolk [phosvitin, lipovitellins, LDL,
by considering first its gross composition, and then its sub- apovitellenins, livetins and vitamin-binding proteins] are
structure in hierarchical terms. At the microscopic level, egg also known and widely reported.2 3 2 4

I2
 Table 2: Gross composition qf hens' eggyolk components included here are not identified in every study:
[from Burley and Vadehra 17] the list of constituent phospholipids in egg yolk varies from
paper to paper, as do their relative proportions.
Component %by weight approx% of
of whole yolk dry weight The triacylclycerides predominate in abundance, followed
by phoshpolipids, especially the lecithins (phosphatidyl-
WATER 475 cholines). Phospholipids comprise a variety of different com-
LIPID 330 66 pounds of which the major species types are depicted below
PROTEIN 17-4 35 (R,R 1 = fatty acid chains) 2 5.
CARBOHYDRATE 0.2 - 0-4
[FREE] Lecithins (Phosphatidylcholines)
IN ORGANICS I. I -2.2
OTHER o.8

MACROMOLECULES
AND COMPLEXES
Low Density 30 62
Lipoprotein
Yolk granules 12 25 Cephalins (Phosphatidylethanolamines)
Livetins 8 r6
_....... o.....__
HoC c-R
R'--......__ _.......o....._,_ l II
C CH 0
II I o
Lipids in Egg Yolk: Gross Composition 0 HoC
'-.... -P-._ _c_c_NH3
/,? Ho +
0 0
/ Ho
-0
Lipids in egg yolk can be broadly divided into three classes:
triacylglycerides (neutral lipids), phospholipids and cholesterol Sphingomyelin
compounds. Proportions of each class in relation to the total
HO-......_ ~C1 3Ho7
lipid fraction are given in Table 3, which also shows the rela-
H C
tive abundance of the various phospholipids in egg yolk. The R'--......__ / N-.. . ._ 1
data in Table 3 are compiled from several sources. 17 2 5 C CH CH3
II
0
I
H C Ho .f' o
+
1
.......__o_/P_o_c_c_N-CH~
Table 3: Lipids in egg yolk. Compounds and proportions reported ftom Ho \
-0 CH3
various analyses: figures quoted are ranges qf published values
[ref. Burley & Vadehra 17 and Belitz & Grosch 2 5
Phosphatidylinositol
Lipid fraction % of total lipids As % of phospho-
lipid fraction

T riacylglycerides

Phospholipids 23 - 31.6
Lecithins
Phosphatidylcholine (PC) 6g- 77
Lysophosphatidylcholine 2.2 - 58
Cephalins Phosphatidylserine
Phosphatidylethanolamine (PE) IH - 24
Lysophosphatidylethanolamine 2.1
Sphingomyelin I- 3
Plasmalogen o.g
Phosphatidylinositol (PI) o.6
Phosphatidylserine (PS) 2-73
Diphosphatidylglycerol 3-24

Cholesterol, 39 - 6 The overall fatty acid compositions of hens' eggs have been
cholesteryl esters reported in the conservation literature3 and it is useful to
& other compounds compare with values from other sources. 17 (Table 4)
Yolk lipids, within the egg at least, appear remarkably stable
to oxidation and this is usually attributed to the presence of
Phospholipid contents are presented as ranges reported in natural antioxidants such as tocopherols, and possibly also
the literature, but it should be noted that all of phospholipid carotenoids and the protein phosvitin, though the structure
of egg lipoproteins will also contribute to this resistance to Table 5: Overall amino acid composition qf egg)iolk and egg white
oxidation of native lipids.
Amino Acid Egg Yolk, wt. % Egg H'hite, wt. % Egg Yolk, mol % Egg\ \1Ute, mol %
Table 4: Fatty acids in hens' egg lipids (%) from ~!ills & from Mills & fiom Schilling 11 from Schilling 11
ll'hite3 \Vhite3
Fatty Acid Data from Data from
Mills & White3 Burley & Vadehra 17
[ranges of 3 reports] GLY 3-5 J6 6.3 ].I
ALA 56 6.3 9-4 10.5
I4:0 trace 0.4- o.s VAL 6-4 8.3 7-4 8-7
I6:o 23-5-31.4 LEU 92 10.32 I0.4 10.3
I6:I J8-3-9 ISO [ILE) 5I 6.2 6.o 6.I
I8:o 9 8.9-I4 PRO 45 45 53 50
I8:I 44 38-4-429 PHE 3-9 52 J6 5I
I8:2 IJS 10.2-I{-2 TYR 2.8 1.4 * *
I8:3 o.s 1.0-1.4 SER 9I 58 I1.5 9I
20:4 1.3-1.6 THR s.6 3-7 8.3 53
22:5 0.4 CYS 1.9 1.9 * *
22:6 0.8 MET 2.3 1.2 1.8 2.9
ARG 55 6.8 * *
HIST 2-4 2.4 * *
LYS 5-7 8.0 6.I 6.3
Colouring Matter in Egg Yolk ASP I1.5 10-5 I2.2 II .0
GLU I5.0 I3-9 II.8 I2.7
The colour of egg yolk is strongly dependent on the hen's HPR [OHP) 0 0 0 0
diet, a fact clearly understood by Cennini:
..town hen's eggs... are whiter)iOlks than the ones which count1)1 qffann
hens produce; those [the latte1] are good, because qf their redness, for Protein is the second most plentiful substance in egg yolk,
tempe1ing flesh colours for aged or swarthy persons. and several distinct egg yolk proteins have been isolated and
The colour of egg yolks is attributed to fat-soluble pigments characterised. The principal proteins of egg yolk, their pro-
- carotenoids and xanthophylls. The types and amount of portions in relation to the total yolk solids and to total yolk
pigment, as Cennini's quote indicates, are dependent on diet proteins are given in Table 6.
and on the capacity of the hen to absorb them and to trans-
port them to the yolk. Two of the more abundant yolk pig- Table 6: Principal Proteins qf Egg Yolk
ments are: xanthophyll , also called lutein,
[3,3'- dihydroxy- a- carotene] and zeaxanthin, Component % of total Mol. Wt.
[ (3R,3R') - ~, ~ - carotene - 3,3' - diol] Solids Proteins Lipids
OH

Low density 6s 22 93
lipoprotein
fraction

Phosvitin 4 I2 0

Lipovitellin r6.o 7
UH

Live tin IO .O
a. 72-80000
Proteins in Egg ~ 42-45000
y ISOOOO
Egg white and egg yolk owe their film-forming properties 0 200000
largely to the proteinaceous components. The amino acid Others 5
distributions of the overall protein content of egg white and
egg yolk are characteristic, and form the basis of analytical Riboflavin-
identifications of egg in paint films and coatings. binding
Data for the amino acid distributions of egg white and egg protein
yolk, expressed both as weight percent from Mills and
White3 and as mol % from Schilling 11 are given in Table 5
Notable features of egg proteins are the absence of hydroxy-
proline in both white and yolk, and the abundance of serine
in yolk, which derives mostly from phosvitin.
 The Hierarchical composition of egg yolk Plasma Low Density Lipoprotein
Plasma LDL is the most abundant non-aqueous part of egg
As we have seen, the yolk is not homogeneous, but contains yolk. It is high in lipid content [> 85%), and contains a high
a variety of particles of distinct types. Some particles, espe- proportion of neutral lipids [triacylglycerides). (Table 8)
cially the larger ones - yolk spheres and yolk globules - are Two sub-fractions of low density lipoprotein have been identi-
highly unstable, whereas others such as the yolk granules are fied - LDFr and LDF2 . The gross composition, fatty acid dis-
stable and may even remain in dried tempera films. tributions and amino acid compositions for each have been
The constitution of the various sub-elements of egg yolk are given analysed and reported. The dominant fatty acids are r6:o
in Figure g, and each of these will be discussed briefly in turn. (25%), r8:r (49%) and r8:2 (ro%). LDL contains some carbo-
hydrate bound to protein.
Figure 3: Hierarchical composition qf egg yolk [Note: The number
in brackets represents the proportion of each component in Table 8: Gross composition qf plasma lipoprotein
whole egg yolk: since yolk is about so% water, the proportion (ranges qf reported values)
in dried yolk can be approximated by doubling this figure.]
Component Amount %
EGG YOLK [10o%]

WATER [48%] SOLIDS [s,%] Protein [% of total lipoprotein] !2

Lipid [% of total lipoprotein] 84- 88-7
MINOR SOUDS [''lo] LIPOPROTEINS AND PROTEINS Triacylglyceride [% lipid] 62- 6g.6
[neutral lipids, carbohydrates, ~
minerals and amino acids] Cholesterol [% lipid] 4
- -OTHER
1
- - ----------
PARTICLES,
,t - - - -- ---- Phospholipid '24-'27-'2
1
: yolk sph eres [unstable]
msoluble 1
PLASr-..fA [J8%) GRA.t'\IULES [t 2 %] Lecithin rg.g
I _ __ _ _ yolk
_ _globules
_ _ _ [stable]
___ _ I
1 Cephalin 48
I
LOW DENSI1Y FRACTION WATER SOLUBLE FRACTION
Sphingomyelin '2 .g
LDL t LDL2
cont. Bg% lipid cont. 86% lipid
LIVETINS LIPOFLAVOPROTEIN
[Ribonavinbindingprotein LDL contains a small proportion of sulfhydryl groups
I +otht rvitaminbindingproteins,
enzymes] attached to apoprotein (Cys/ 2 is present at a proportion of
0.54 mol% of total amino acids). Sulfhydryl/disulphide
interactions are at least partly responsible for the gelation of
PHOSVITIN('%] HIGH DENSITY LOW DENSITY VLDL
FRACTION [8%] FRACTION [1,%] [Myelin egg yolk LDL on heating. This process will probably be
Figures]
involved in the immobilisation of egg proteins as tempera
a LIPOVITELLIN ~ L!POVITELLIN
rComolexes with Phosvitin1 paints dry. A number of distinct apoproteins (Apovitellenin I
- Apovitellenin VI] from plasma LDL have been identified,
separated and analysed for amino acid distribution.
Yolk plasma: the aqueous phase of egg yolk
The exact state of lipoproteins in plasma (LDL) is not fully
. The yolk plasma remains when yolk granules and other established: the general concensus is that LDL is present in the
large particles (globules) have been separated off, for exam- plasma as ultra-fine particles (20- go nm in diameter) consisting
ple, by centrifugation. The plasma contains water-soluble of a neutral lipid core (triacylglycerides and cholesteryl esters)
proteins Oivetins, vitamin-binding proteins and others) and with a surface network of protein stabilised by cholesterol and
low density lipoproteins (LDL). It is effectively a water solu- phospholipids: the surface active phospholipids are oriented
tion of lipoprotein (go%) and protein (8%). The low density with their lipophilic part associated with the neutral lipid core
lipoprotein (LDL) is not present as an emulsion, but as true and their hydrophilic part with the protein/water phase.
solution comprising solubilised particulate complexes.

Soluble proteins of the plasma: the livetin fraction Stable particles in egg yolk:
Three main livetin fractions are recognised a, ~ and y. the yolk granules
Evidence for a fourth livetin fraction, D, has recently been
presented. a, ~ and y livetins correspond to known blood The yolk granules constitute about r2% of the weight of whole
serum proteins. Properties of livetins are given in Table 7. egg yolk and therefore make up about a quarter of the weight
of dry egg yolk. It seems likely that the opaque particulate
Table 7." Livetins qf egg yolk plasma structures observed in scanning electron micrographs of fresh
and, even, artificially aged egg yolk filins are yolk granules.14
Livetin fraction corresponding Mol. Wt Proportion of The general composition of yolk granules is given in Table g.
serum protein protein fraction Yolk granules have three main constituents: phosvitin, a
non-lipid containing glycoprotein, a high density lipoprotein
Serum albumin 70000 gr.6 fraction composed of a and ~ Iipovitellins, and a low densi-
<X2 glycoprotein 42000 g45 ty lipoprotein fraction comprising LDLg. In some sources
y globulin 150000 go.5 features known as Myelin Figures are reported as a (minor)
200000 r.8 component of yolk granules.2 6, 2 7

rs
Table g: Composition qf )'olk granules plexes/ cross-links can be established between phosvitin and
(From Burley &Vadehra 17) Ca(II), Mg(II) and Fe(III): the phosphate groups act as lig-
ands/ chelates. This process may contribute to the immobil-
Component % in dry granules isation of phosvitin in paint films .

Phosvitin r8 Low Density lipoprotein in yolk granules (LDLg)

a Lipovitellin 51 About 12% of the weight of yolk granules is a low density
BLipovitellin 30 lipoprotein identified as LDLg. The granules contain other
Low density lipoprotein 12 lipid-rich particles, Very Low Density Lipoprotein [VLDL]
Total lipid or Myelin figures. Granule LDL is very similar in composi-
tion to the main LDL fraction of yolk plasma (see above) .

Lipovitellins
They are the main non-aqueous constituent of egg yolk
granules. Lipovitellins are described as high density lipopro-
teins [HDL]; this characteristic is conferred by the low pro-
portion [~25%] of lipid. a and Bforms of lipovitellin differ
slightly in amino acid compositions, in amount of bound
phosphorus, in amount of bound carbohydrate and in their
apoprotein patterns (Table ro) . The amino acid and fatty
acid compositions of lipovitellins are reported. Lipovitellins
are present naturally as dimers and can complex with
phosvitin. 2 8
Lipovitellins are unlike other lipoproteins found in blood:
studies have shown that the lipovitellin complex is not the
spherical or quasi-spherical model suggested for plasma
lipoproteins (and egg LDL): lipovitellin complexes - either
as dimers or with phosvitin - are soluble and contain lipid
domains occluded within the structured globular protein. 2 9
Table w: Composition qf a and BLipovitellins qf )'Olk granules
Properties of dried tempera films
Freshly dried tempera films will be reasonbly flexible, the
mobile lipids effectively acting as plasticisers, and hygro-
scopic on account of the proteins and phospholipids present.
They will be readily swollen by water.
Lipids can be readily extracted from by a wide range of orga-
nic solvents - chlorinated solvents, hydrocarbons, alcohols
and ketones.3 This was strongly indicated by tests on fresh
and artificially aged tempera films. 14 On ageing the amount
of lipid extractable decreases considerably.
Lipid extraction leads to compaction of the paint films and
surface roughness. Some of the lipid in fresh egg yolk films
appears to be highly mobile: there is some evidence in arti-
ficially aged tempera films of efflorescence and evaporation
of volatile lipids. The refractive index of dried egg yolk films
has been measured at 1.525. The refractive index of azurite
tempera paints rose to 1.530 on ageing artiliciallyY

a lipovitellin Blipovitellin
Initial drying and hardening (curing) of
Total Lipid 20 - 24.2 20- 26.7 tempera paint films
[% dry weight]
Phospholipid rr.6 - rs rr.6- 17.0 The processes occurring during the drying and initial curing
[% dry weight] of tempera films will be discussed in more detail later in
Lecithin 10- 11.2 II - 12.4 these proceedings, supported by experimental findings.
Cephalin (PE) 2.3 2-7 Some observations, however, are relevant to an underc
Sphingomyelin o.g I. I standing of the nature of freshly dried tempera films.
+others
Triacylglycerides 6.g- 8.8 73- g.8 Lipoproteins are inherently unstable in the absence of water.
[% dry weight] The whole structure of egg yolk is stabilised by complex
Cholesterol o.g - 2.2 1.1-2-4 inter- and intra-molecular interactions in which hydro-
[% dry weight] phobic interactions play an important part. The drying and
Protein-bound 36 2.85 initial curing of yolk must involve disruption of this native
(carbohydrate) state - denaturation - due to loss of water. Significant alter-
ation of at least the tertiary and quaternary structures of
proteins is to be expected with consequent re-organisation of
Phosvitin proteins and lipids, possibly involving aggregation.
Phosvitin is a non-lipid glycoprotein, i.e it contains bound Egg yolk contains a reasonable proportion of sulphur-con-
carbohydrate (ca. 6.5%). It has a distinctive amino acid dis- taining amino acids, including the reactive cystine.
tribution, containing a high proportion [ca. s6 mol%] of Sulfhydryl [thiol] I disulphide interchange reactions to pro-
serine. About 75% of serine residues are attached to phos- duce disulphide cross-links have been implicated by many
phate. Phosvitin forms soluble complexes with Lipovitellin researchers in the formation of albumin [egg white] gels,
creating domains of occluded lipid. Metal binding is an and also in the gelation of yolk Low Density Lipoprotein
important function of Phosvitin: it binds Fe (III) strongly, as (LDL). Such crosslinks between protein chains are formed at
well as alkali metal ions. Phosvitin promotes oxidation of low energies and must surely contribute to the early harden-
Fe(II) to Fe(III) and can inhibit Fe(II) and Cu(II) catalysed ing of tempera films . The general reaction is:
oxidation of phospholipids. Inter- and intra-molecular com-

r6
 s gas cluomatography Strong evidence was fOtmd for the immobili-
p/ I + H- S - P" - H- S-P - S - S-P"
~s
There is evidence in scanning electron micrographs of dried
egg yolk films that some of the particulate structures of the
yolk, possibly the yolk granules, are retained on drying, and
that these can survive light and heat ageing. 13
Other processes which may contribute to the initial harden-
ing and immobilisation of protein components include com-
plexing of metal ions which are abundant in pigmented
films. lVIany functional groups are present in proteins/ amino
acids that may act as ligands, especially phosphate in
phosvitin. Protein complexing to or through metal ions may
serve as crosslinks.
Evidence fo.r chemical change m egg
tempera pamts
Important information on chemical changes in egg tempera
paints are presented in the paper by Boon in these proceed-
ings. Some evidence already has been reported before in the
conservation literature. Cholesterol is known to oxidise
rapidly. Principal oxidation products will include:
cholesterol 7 - hydroxycholesterol 7 - ketocholesterol
lVIills and White reported the almost complete loss of cho-
lesterol from egg tempera paints less than 20 years old.
Some preservation of cholesterol was observed for verdigris
samples, suggesting a protective effect by verdigris on the
oxidation of lipids, similar to that observed with oleate.3 2
Schilling reported several observations on the consequences
of accelerated ageing on amino acid composition of egg
tempera paints. 11 Amino acid content of egg yolk paints
deoeased substantially (up to 25% loss) after ageing, espe-
cially light ageing. This is explained by the high proportion
of unstable amino acids in egg yolk proteins. The chemical
composition of egg yolk (especially, presence of oxidising
lipids) promotes photo-oxidation and radical induced
decomposition of proteins. The most vulnerable amino
acids to oxidative degradation are histidine, tyrosine,
methionine and cysteine. Schilling's conclusion, therefore, is
that reliable identification of egg yolk tempera in paints is
most validly based on the distribution (by mol%) of stable
aminoacids: Alanine, Valine, Isoleucine, Leucine, Glycine,
Proline and Hydroxyproline.
Khandekar and PhenL-x prepared samples of egg yolk alone
and lead white, vermilion, azurite and verdigris tempera
paints for artificial ageing by light exposure (xenon-arc Fade-
a-meter, 2 durations) and thermal ageing at 70C. I+
Initial work examined lipids extracted by organic solvents using
17
sation/binding of lipids within the paint matri-x. Retention of fatty
acids in paint filius was promoted by light ageing.
Further recent studies by Khandekar on the same reference
and artificially aged tempera samples (egg yolk, lead white,
vermilion, azurite and verdigris) have included electro-
phoresis of proteins and GC/lVIS of solvent extracts. 15, 1 6
Protein bands in the range of molecular weights 38,ooo -
wo,ooo that were resolvable in 3 year old reference samples
became unresolvable in samples after light ageing, indicating
an increase in molecular weight. All samples of azurite,
including the 3 year old reference sample, did not resolve in
electrophoresis, indicating very high molecular weight com-
ponents > 250,000. This highlights the activity of Cu(II)
ions on egg protein. Vermilion showed a similar effect, but
to a lesser extent. Azurite and Vermilion appeat~ then, to
accelerate the cross-linking of egg yolk proteins.
Thermal ageing of vermilion tempera paints is suggested to
promote chain breaking with consequent change in the dis-
tribution of proteins to lower molecular weight. The protein
distribution in aged lead white tempera paints was observed
to be directly comparable with those of the unpigmented
ftlms, suggesting that this pigment does not accelerate the
cross-linking of egg yolk proteins. Verdigris, however,
behaved differently to all other pigments studied. In all
verdigris samples only low molecular weight components
were detected, suggesting severe breakdown of protein (in
some cases, this is to the extent that the ftlms have no physi-
cal strength whatsoever). Copper in an acidic environment
degrades amino acids, and acid from the pigment may pro-
mote hydrolysis. The difference in behaviour of the two cop-
per pigments on the proteins in egg tempera is noteworthy.
GC/IviS analysis of chloroform extracts (1 hour immersion)
demonstrated that the dominant fatty acid in unaged sam-
ples was 18:1 [oleic] in a proportion varying from g-34% of
total lipids extracted. After light ageing, howeve1~ the pro-
portion of oleic acid [18:1] diminishes considerably in all
samples including unpigmented egg yolk: azurite has the
most pronounced effect, verdigris has the least. Stearic acid
content of chloroform extracts was relatively unaffected by
light ageing. The ratio 18: 1!I8:o provides an indicator of loss
of oleic acid by oxidation. Reduction of oleic acid [18:1] was
predominantly a consequence of light ageing. Of the pig-
ments tested, verdigris appeared to protect oleic acid from
oxidation to the greatest extent, a finding comparable with
the findings of Mills & White and Rasti & Scott.33
Khandekar also refers to findings of Hodgins who examined
by electrophoresis changes in egg yolk proteins due to expo-
sure to light, comparing whole yolk to lipid-free yolk. The
significant role of oxidising lipids in promoting cross-linking
of proteins was confirmed: egg proteins were found to cross-
link much more slowly in the absence of lipids. High mole-
cular weight ( > wo,ooo) components formed during rela-
tively short periods of light exposure (20 hours in Fade-a-
meter) whilst smaller components dii11inished; the conclu-
sion being that a large protein matrix was formed and/ or
proteins are broken into smaller fragments unresolvable by
electrophoresis.
Degradation reactions in protein-lipid
systems such as egg yolk
Inevitably, the ageing and degradation pathways in egg yolk
paint systems will be complex, given their highly heteroge-
neous nature. Karpowicz has proposed a number of possi-
ble reactions that may play a part, to which others reported
in the biochemistry and food industry literature might use-
fully be added, as summarised as below:B
Sulfhydryl - disulphide agglomeration/ crosslinking
as described above in Initial drying and hardening (curing) of tem-
pera paintfilms.34
Lipid oxidation - by direct auto-oxidation or photosensi-
tised: the mechanism is well understood and has been exten-
sively investigated. 35-4 1 Linoleate is oxidised ro times faster
than oleate; linolenate is oxidised 20-30 times faster than
oleate. Oxidation is catalysed by metal ions e.g. Fe3+. Lipid
oxidation results in polymerisation of triglycerides and phos-
pholipids, formation of cross-linked lipid matrix, immobili-
sation of unsaturated fatty acids, formation of low molecu-
lar weight products, volatiles, carbonyls, unsaturated com-
pounds. Also, oxidation of cholesterol to oxycholesterols:
this process is accelerated by 18:2 and 18:3 fatty acid peroxy
radicals.
Reactions of Proteins with products of lipid oxi-
dation - the mechanism of hydroperoxide-protein interac-
tion is again well studied.4 2 -5 1 The general pattern includes
formation of protein radicals by transfer from lipid (perox-
ides and hydroperoxides), cross-linking of protein radicals
with lipids, polymerisation of protein-lipid products with
further protein molecules. Lipid hydroperoxides cause a
number of interesting reactions with various amino acid
residues in proteins. These various reactions help to account
for polymerisation observed in proteins in lipid/protein sys-
tems. It seems reasonable to assume that the properties
observed of aged egg tempera paints are connected with the
formation of an essentially protein cross-linked network. It is
well established that lipids or peroxides in the environment
of a protein cause significant change in the protein. Lipid
covalently bound to protein will not be extractable by neu-
tral organic solvents. Formation of insoluble complexes of
protein and oxidised lipids is widely reported.
Amino acid oxidation4 - may result in:
decomposition of amino acids, oxidised amino acid residues,
cross-linking, sulfhydryl/ disulphide interchange, formation
of dityrosine;
losses of amino acids Lysine, Tryptophan, Histidine,
Methionine, Cystine/ cysteine;
Methionine oxidised to its sulphoxide:
[R-S-CH3 __. R-SOCH3] by lipid peroxide, and possibly
also to sulphone, R-S0 2CH3,
Cysteine oxidised by lipid free radical to produce disulphide
bond or alanine,
possible lipid-protein covalent bonds, protein radicals com-
bining to form protein network i.e.
RO" +PH__. p + ROH; 2P" __. P- P
formation of lipid protein complexes,
formation of degradation products,
formation of secondary products of oxidation: carbonyl
compounds, discolouration.
Dehydroalanine reactions
In alkaline conditions, the unsaturated dehydroalanine
residue may be formed by ~ elimination, with inter- and
intra-chain cross-linking reactions occurring consequently
by addition of amine, or thiol to dehydroalanine. There are
several pathways that lead to the formation of dehydroala-
nine, including those from phosphoprotein.
Dehydroalanine is very reactive and, if formed, can crosslink
with Lysine, Histidine, Ornithine and Cysteine.
Lysinoalanine is often used as a marker for severely treated
proteins. 4,5 2
Metal catalysed reactions53
Key processes reported include:
Metal catalysed ester hydrolysis: Cu 2 + active on amino acid
esters,
Peptide and amide hydrolysis catalysed by Co3+,
Metal catalysed oxidation of organic compounds: particu-
larly Mn3+, Fe3+, Cu 2 +,
Metal catalysed polymerisationlinactivation of lysozyme
and chicken ovomucoid by Cu(I) ion,
Oxidation of amino acids activated by Cu(II), Co(II), Fe(II)
and Mn(II).
Other possible reactions:
Hydrolysis of phospholipids__. diglycerides
Condensation of proteins with carbohydrates
Formation of metal ion/protein complexes
Conclusion
Egg tempera dries and hardens to a tough durable paint fllm
whose properties are strongly influenced by both lipid and
protein components. Dried and aged egg tempera paints will
comprise an immobile network phase together with a mobile
phase of solvent- and water-extractable components.
It seems certain that both lipids and proteins are involved in
cross-linking processes leading to the formation of a poly-
meric network or immobile phase, and that metal ions from
pigments strongly influence both these curing processes and
subsequent deterioration.
References and notes
1. Reinkowski-Hafner, E., 'Tempera- zur Geschichte eines Maltechnischen Begriffs,' ,(eitsc/zriftfor Kunsttec/znologie und
Konservierung, Jahrgang 8, Heft 2 (I994), pp. 297-3I7
2. Mills,J. S., 'The gas-chromatographic examination of paint media, Part I. Fatty acid composition and identification
of dried oil films', Studies in Conservation, II (Ig66), pp. g2-Io6
3 Mills, J. S. and White, R., The 01ganic Chemistry qf Museum Objects, 2nd edition Butterworth-Heinemann, London
(I994)
4 Halpine, S. M., 'Amino acid analysis of proteinaceous media from Cosima Tura's The Annunciation with S. Francis
and S. Louis of Toulouse', Studies in Conservation, 37 (I992), pp. 22-38
5 White, R., 'The characterisation of proteinaceous binding media in art objects', National Gallery Technical Bulletin, (I984)
PP s-I4
6. Keck, S. and Peters, T. Jr., 'Identification of Protein-containing paint media by quantitative amino acid analysis',
Studies in Conservation, I4 (Ig6g), pp. 62-74
7- Masschelein-Kleiner, L., 'An improved method for the thin layer chromatography of media in tempera paintings',
Studies in Conservation, I9 (I974), pp. I94-206
8. Karpowicz, A., 'Ageing and deterioration of proteinaceous media', Studies in Conservation, 26 (Ig8I), pp. IS3-I6o
g. Masschelein-Kleiner, L., 'Contribution to the study of aged proteinaceous media', in: Conservation and Restoration qf
Pictorial Art, eds. Brommelle and Smith, (IIC/Butterworths I976), pp. I77-I83
ro. Schilling, M. R., Khanjian, H. P. and Souza, L. A., 'Gas chromatographic analysis of amino acids as ethyl chlo-
roformate derivatives, Part I. Composition of proteins associated with objects and monuments', Journal qf the American
Institute for Conservation, 35 (Igg6), pp. 45-59
II. Schilling, M. R., Khanjian, H. P. and Souza, L.A., 'Gas chromatographic analysis of amino acids as ethyl chlo-
roformate derivatives, Part 2. Effects of pigments and accelerated ageing on the identification of proteinaceous bind-
ing media', Journal qf the American Institute for Conservation, 35 (I gg6), pp. I23-44
I'2. Schilling, M. R. and Khanjian H. P., 'Gas chromatographic analysis of amino acids as ethyl chloroformate deriva-
tives, Part 3 Identification of proteinaceous binding media interpretation of amino acid composition data', Preprints to
!COM Committee for Conservation Meeting, Edinburgh (Igg6), pp.2II-'2I9
I3 Sharp, J., A study qf the mechanical and suiface characteristics qf tempera paint films; Diploma Report, Courtauld Institute of
Art, University of London, I993 (unpublished)
I4. Khandekar, N., Phenix, A. and Sharp, J., 'Pilot study into the effects of solvents on artificially aged egg tempera
paint films', The Conservator, I8 (I994), pp. 62-72
IS. Khandekar, N., The ifftcts qf four pigments on the molecular weight qf proteins in an artificial{y aged egg tempera paint film, Report
to the Samuel H Kress Foundation I995 (unpublished).
I6. Khandekar, N., The ifftcts qf selected pigments on lipids and proteins in artijicial{y aged egg tempera paint films, Report to the
Australian Research Council Igg6 (unpublished)
I7. Burley, R. W. and Vadehra, D. V., The Avian Egg: chemislly and biology, (Wiley I989)
I8. Stadelman, W.J. and Cotterill, O.J., Egg Science and Technology, 4th edition (I995)
Ig. Nisbet, A. D. et al, 'The complete amino acid sequence of hen ovalbumin', European] qf Biochemistry, vol. us (Ig8I),
pp. 335-345
20. Gilbert, A. B., 'The egg; its chemical and physical aspects', in: Bell, D. J., and Freeman, B. M. (eds.), Physiology and
Biochemislly qf Domestic Fowl, (I97I)
'2I Gettens, R. and Stout, G., Painting Materials, Dover (Ig66)
22. Holub, B.J. and Kuksis, A., 'Molecular species of phosphatidylethanolamine from egg yolk', Lipids, 4 (Ig6g), pp. 466-
472
23 Cook, W. H. and Martin, W. G., 'Function and properties of soluble lipoproteins in relation to structure', Can. J
Biochem. Plrysiol., 40 (Ig62), pp. I273-I285 and, Cook, W. H. and Martin, W. G., 'Composition and structure of solu-
ble lipoproteins', Bioc/zim. Bioplrys. Acta, 56 (Ig62), pp. 362-365
24. Byrne, B. M. et al., 'Amino acid sequence of phosvitin derived from the nucleotide sequence of the chicken vitel
logenin gene', Biochemistry, 23 (I984), pp. 4275-4279
25. Belitz, H. D. and Grosch, W., Food Chemistry, (Springer Verlag Ig86)
26 There is some ambiguity in the literature regarding the existence and nature of Myelin Figures. They are referred to
as distinct particles in several sources, notably Stadelman & Cotterill (I995), but not by Burley & Vadehra (Ig8g): a con-
sensus view might be that the Myelin figures are VLDL particles within the yolk granules.
27. Chang, C. M., Powrie, W. D. and Fennema, 0., 'Microstructure of egg yolk',] Food Sci., 42 (I977), pp. ug3-I200
28 Ross,J., Wrenn, R. F., Ohlendorf, D. H. and Banaszak, L.J., 'Lipid domains in the yolk lipoprotein complex',
in: Scanu, A. M. and Landsberger, F R. (eds.), Lipoprotein structure Annals qf NY Acad. qf Sciences, Vol348, pp. 408-4I7
29 Banaszak, LJ., Ross,J. M. and Wrenn, R. F., 'Lipovitellin and the yolk lipoprotein complex', in:Jost, PC. and
Griffith 0. H. (eds.), Lipid-protein interactions, (Wiley I982)
30 Larsen,J. E. and Froning, G.W., 'Extraction and processing of various components of egg yolk', Poult1y Science, 6o
(Ig8I), pp. I6o-I67

I9
31. Personal communication, Dr.J. Townsend, Tate Gallery, London.
32. Mills,J.S. and White, R., 'The identification of paint media from their sterol composition- a critical review', Studies
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33 Rasti, F. and Scott, G., 'Effects of some common pigments on the photo-oxidation of linseed oil based paint media', Studies
in Conservation, 25 (r98o), pp. 145-156
34 DeMan, J. M., Principles qf food chemist1y, 2nd edn. (AVI/Van Nostrand Reinhold 1990)
35 El-Tarras, M Fayez et al, 'Studies on rancidity of oils and fats Part XIII: the effect of phospholipids on the autoxi-
dation of fats in aqueous medium', Riv. It. delle Sostan;:,e Grasse, Vol LIII (1976), pp. 15-17
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Biochemist?y and Biophysics, rr 3 (r966), pp. rso- 155
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Additional reading
Stadelman, W.J., Egg Proteins in Food Colloids, ed. Graham, H. D. (Avi Publishing NY 1977), pp. 207-239
Nakai, S. and Modler, W. H., Food proteins: properties and clzaracteri;:.ation, (VCH Publishers 1996)
Coenders, A., The chemistry qf coo!.:ing, (Parthenon Publishing 1992)
Mitchell,]. R., and Ledward, D. A., Functional properties qf food macromolecules, (Elsevier 1986)
Zapsalis, C. and Anderle Beck, R., Food chemist?y and nutritional biochemist?y, (Wiley 1985)
Feeney, R. R. and Hill, R. M., 'Protein chemistry and food research', in: Advances in Food Research, Vol. w; eds. Chichester,
C. 0., Mrak, E. M . and Stewart, G. F., (Academic Press NY 1960)

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