Globin phylogeny, evolution and function, the newest update.

Our globin census update allows us to refine our vision of globin origin, evolution, and structure to function relationship in the context of the currently accepted tree of life. The modern globin domain originates as a single domain, three-over-three α-helical folded structure before the diversification of the kingdoms of life (Bacteria, Archaea, Eukarya). Together with the diversification of prokaryotes, three monophyletic globin families (M, S, and T) emerged, most likely in Proteobacteria and Actinobacteria, displaying specific sequence and structural features, and spread by vertical and horizontal gene transfer, most probably already present in the last universal common ancestor (LUCA). Non-globin domains were added, and eventually lost again, creating multi-domain structures in key branches of M- (FHb and Adgb) and the vast majority of S globins, which with their coevolved multi-domain architectures, have predominantly "sensor" functions. Single domain T-family globins diverged into four major groups and most likely display functions related to reactive nitrogen and oxygen species (RNOS) chemistry, as well as oxygen storage/transport which drives the evolution of its major branches with their characteristic key distal residues (B10, E11, E7, and G8). M-family evolution also lead to distinctive major types (FHb and Fgb, Ngb, Adgb, GbX vertebrate Gbs), and shows the shift from high oxygen affinity controlled by TyrB10-Gln/AsnE11 likely related to RNOS chemistry in microorganisms, to a moderate oxygen affinity storage/transport function controlled by hydrophobic B10/E11-HisE7 in multicellular animals.

Ontogeny of hemoglobin­oxygen binding and multiplicity in the obligate air-breathing fish Arapaima gigas.

The evolutionary and ontogenetic changes from water- to air-breathing result in major changes in the cardiorespiratory systems. However, the potential changes in hemoglobin's (Hb) oxygen binding properties during ontogenetic transitions to air-breathing remain poorly understood. Here we investigated Hb multiplicity and O2 binding in hemolysates and Hb components from juveniles and adults of the obligate air-breathing pirarucu (Arapaima gigas) that starts life as water-breathing hatchlings. Contrasting with previous electrophoresis studies that report one or two isoHbs in adults, isoelectric focusing (IEF) resolved the hemolysates from both stages into four major bands, which exhibited identical O2 binding properties (i.e. O2 affinities, cooperativity coefficients, and sensitivities to pH and the major organic phosphate effectors), also as compared to the cofactor-free hemolysates. Of note, the multiplicity pattern recurred upon reanalyses of the most-abundant fractions isolated from the juvenile and the adult stages, suggesting possible stabilization of different quaternary states with different isoelectric points during the purification procedure. The study demonstrates unchanged Hb-O2 binding properties during development, despite the pronounced differences in O2 availability between the two media, which harmonizes with findings based on a broader spectrum of interspecific comparisons. Taken together, these results disclose that obligate air-breathing in Arapaima is not contingent upon changes in Hb multiplicity and O2 binding characteristics.

Development of a Fiber-Optics Microspatially Offset Raman Spectroscopy Sensor for Probing Layered Materials.

Microspatially offset Raman spectroscopy (micro-SORS) has been proposed as a valuable approach to sample molecular information from layers that are covered by a turbid (nontransparent) layer. However, when large magnifications are involved, the approach is not straightforward, as spatial constraints exist to position the laser beam and the objective lens with the external beam delivery or, with internal beam delivery, the maximum spatial offset achievable is restricted. To overcome these limitations, we propose here a prototype of a new micro-SORS sensor, which uses bare glass fibers to transfer the laser radiation to the sample and to collect the Raman signal from a spatially offset zone to the Raman spectrometer. The concept also renders itself amenable to remote delivery and to the miniaturization of the probe head which could be beneficial for special applications, e.g., where access to sample areas is restricted. The basic applicability of this approach was demonstrated by studying several layered structure systems. Apart from proving the feasibility of the technique, also, practical aspects of the use of the prototype sensor are discussed.

A globin domain in a neuronal transmembrane receptor of Caenorhabditis elegans and Ascaris suum: molecular modeling and functional properties.

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.

Kinetic properties and heme pocket structure of two domains of the polymeric hemoglobin of Artemia in comparison with the native molecule.

In this project, we studied some physicochemical properties of two different globin domains of the polymeric hemoglobin of the brine shrimp Artemia salina and compared them with those of the native molecule. Two domains (AsHbC1D1 and AsHbC1D5) were cloned and expressed in BL21(DE3)pLysS strain of Escherichia coli. The recombinant proteins as well as the native hemoglobin (AfHb) were purified from bacteria and frozen Artemia, respectively by standard chromatographic methods and assessed by SDS-PAGE. The heme environment of these proteins was studied by optical spectroscopy and ligand-binding kinetics (e.g. CO association and O2 binding affinity) were measured for the two recombinant proteins and the native hemoglobin. This indicates that the CO association rate for AsHbC1D1 is higher than that of AsHbC1D5 and AfHb, while the calculated P50 value for AsHbC1D1 is lower than that of AsHbC1D5 and AfHb. The geminate and bimolecular rebinding parameters indicate a significant difference between both domains. Moreover, EPR results showed that the heme pocket in AfHb is in a more closed conformation than the heme pocket in myoglobin. Finally, the reduction potential of -0.13V versus the standard hydrogen electrode was determined for AfHb by direct electrochemical measurements. It is about 0.06V higher than the potential of the single domain AsHbC1D5. This work shows that each domain in the hemoglobin of Artemia has different characteristics of ligand binding.

A behavioural study of neuroglobin-overexpressing mice under normoxic and hypoxic conditions.

Neuroglobin (Ngb), a neuron-specific heme-binding protein that binds O2, CO and NO reversibly, and promotes in vivo and in vitro cell survival after hypoxic and ischaemic insult. Although the mechanisms of this neuroprotection remain unknown, Ngb might play an important role in counteracting the adverse effects of ischaemic stroke and cerebral hypoxia. Several Ngb overexpressing mouse models have confirmed this hypothesis; however, these models were not yet exposed to in-depth behavioural characterisations. To investigate the potential changes in behaviour due to Ngb overexpression, heterozygous mice and wild type (WT) littermates were subjected to a series of cognitive and behavioural tests (i.e., the SHIRPA primary screening, the hidden-platform Morris water maze, passive avoidance learning, 47h cage activity, open field exploration, a dark-light transition box, an accelerating rotarod, a stationary beam, a wire suspension task and a gait test) under normoxic and hypoxic conditions. No significant behavioural differences were found between WT and Ngb-overexpressing mice at three months old. However, one-year-old Ngb-overexpressing mice travelled more distance on the stationary beam compared with WT littermates. This result shows that the constitutive overexpression of Ngb might counteract the endogenous decrease of Ngb in crucial brain regions such as the cerebellum, thereby counteracting age-induced neuromotor dysfunction. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Is the heme pocket region modulated by disulfide-bridge formation in fish and amphibian neuroglobins as in humans?

Neuroglobin, a globin characterized by a bis-histidine ligation of the heme iron, has been identified in mammalian and non-mammalian vertebrates, including fish, amphibians and reptiles. In human neuroglobin, the presence of an internal disulfide bond in the CD loop (CD7-D5) is found to modulate the ligand binding through a change in the heme pocket structure. Although the neuroglobin sequences mostly display conserved Cys at positions CD7, D5 and G18/19, a number of exceptions are known. In this study, neuroglobins from amphibian (Xenopus tropicalis) and fish (Chaenocephalus aceratus, Dissostichus mawsoni and Danio rerio) are investigated using electron paramagnetic resonance and optical absorption spectroscopy. All these neuroglobins differ from human neuroglobin in their Cys-positions. It is demonstrated that if disulfide bonds are formed in fish and amphibian neuroglobins, the reduction of these bonds does not result in alteration of the heme pocket in these globins. Furthermore, it is shown that mutagenesis of the Cys residues of X. tropicalis neuroglobin influences the protein structure. The amphibian neuroglobin is also found to be more resistant to H2O2-induced denaturation than the other neuroglobins under study, although all show an overall large stability in high concentrations of this oxidant. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin.

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Determination of ligand pathways in globins: apolar tunnels versus polar gates.

Although molecular dynamics simulations suggest multiple interior pathways for O(2) entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.

Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes.

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima.

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where 'nerve hemoglobins' (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb 'classic' globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.

The effect of pH and nitrite on the haem pocket of GLB-33, a globin-coupled neuronal transmembrane receptor of Caenorhabditis elegans.

Out of the 34 globins in Caenorhabditis elegans, GLB-33 is a putative globin-coupled transmembrane receptor with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of electronic circular dichroism, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin.

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.

Marked difference in the electronic structure of cyanide-ligated ferric protoglobins and myoglobin due to heme ruffling.

Electron paramagnetic resonance experiments reveal a significant difference between the principal g values (and hence ligand-field parameters) of the ferric cyanide-ligated form of different variants of the protoglobin of Methanosarcina acetivorans (MaPgb) and of horse heart myoglobin (hhMb). The largest principal g value of the ferric cyanide-ligated MaPgb variants is found to be significantly lower than for any of the other globins reported so far. This is at least partially caused by the strong heme distortions as proven by the determination of the hyperfine interaction of the heme nitrogens and mesoprotons. Furthermore, the experiments confirm recent theoretical predictions [Forti, F.; Boechi, L., Bikiel, D., Martí, M.A.; Nardini, M.; Bolognesi, M.; Viappiani, C.; Estrin, D.; Luque, F. J. J. Phys. Chem. B 2011, 115, 13771-13780] that Phe(G8)145 plays a crucial role in the ligand modulation in MaPgb. Finally, the influence of the N-terminal 20 amino-acid chain on the heme pocket in these protoglobins is also proven.

Ligand migration through the internal hydrophobic cavities in human neuroglobin.

Neuroglobin (Ngb), a member of the globin superfamily, was found in the brain of vertebrates and is suggested to play a neuroprotective function under hypoxic conditions by scavenging nitrogen monoxide (NO) through a dioxygenase activity. In order for such a reaction to efficiently take place and to minimize the release of reactive intermediates in the cytosol, the cosubstrates O(2) and NO and other unstable reaction intermediates should bind sequentially to docking sites in the protein matrix. We have characterized the accessibility of these sites by analyzing the geminate CO rebinding kinetics to the heme moiety observed upon nanosecond flash photolysis of the Ngb-CO complex encapsulated in silica gels. The geminate rebinding phase showed a remarkable complexity, revealing the presence of a system of secondary docking sites where ligands are stored for hundreds of microseconds. Most kinetics steps display little temperature dependence, demonstrating that ligands can easily migrate through the cavities, except for the slowest reaction intermediate, possibly reflecting a structural conformational change reshaping the system of cavities. This conformational change is unrelated with distal His E7 binding to the heme, as it persists for the HE7L mutant. Overall, data are consistent with the presence of a discrete system of docking sites, possibly acting as reservoirs for the putative cosubstrates and for other reactive species involved in the physiologically relevant reaction.

High resolution crystal structures of the Cerebratulus lacteus mini-Hb in the unligated and carbomonoxy states.

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O(2) dissociation and ultra-high O(2) affinity. However, CerHb affinity for O(2) is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O(2) ligand, the overall binding schemes for CO and O(2) are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.

Structural heterogeneity and ligand gating in ferric Methanosarcina acetivorans protoglobin mutants.

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb-specific loops and a N-terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme-Fe-atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side-chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme-bound ligand.

Globins in Caenorhabditis elegans.

Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB-1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions.

Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA).

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.