Search

To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH.Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis.2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins.Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.évaluer l’amélioration de l’extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d’expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l’abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d’un gel 2DE JRH ont été clivés par enzyme pour l’analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l’analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d’abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d’un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées.l’extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu’il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

A three-component analytical model for single fibre curvature is developed in terms of the axial contractions, elastic moduli and geometrical properties of wool fibre cells. The elastic moduli of wool cells and single fibre curvature of selected Romney wools are measured. Using axial contraction data from the literature, the mean single fibre curvature is computed and compared with the measured mean value. The simulation is about the same as the measured value when the para- to orthocontraction ratio is 1.7:1.

Exposure of reactive chemical functional groups through controlled surface lipid removal provides a means for covalent attachment of novel molecular entities to the wool fiber surface. Cleavage of the dominant lipid in the epicuticle, 18-methyleicosanoic acid (18-MEA), from the underlying proteinaceous layer was employed, prior to further surface modification, by means of aqueous hydroxylamine treatment. Treatments utilizing covalent bonding to the surface through thiol, carboxyl, amine, or other reactive chemical groups offer improved durability to textile processing, such as dyeing and laundering. This surface modification investigation compared and assessed the attachment and durability of various wool fiber surface modification technologies and provided proof of principle for the manipulation of surface properties via covalent attachment of treatment compounds, providing enhanced fiber performance. Following removal of the surface lipid layer, wool fibers were modified using fluorescent and hydrophobic compounds and micro and nanoparticles. The extent of modification was assessed using scanning electron, light, and fluorescence microscopy, wettability testing and X-ray photoelectron spectroscopy (XPS). Surface treatments were evaluated with regard to effectiveness and durability to dyeing and laundering. Surface modification via attachment to thiol and other nucleophilic functionalities was observed to be greatly improved after hydroxylamine treatment, demonstrating the advantage of a customized exposed and accessible surface before secondary treatment. Increased durability and effective microparticle attachment was best accomplished employing zero or short-range crosslinking mechanisms, with zero-length attachment enhanced with the use of the crosslinking promoters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Fluorescent and hydrophobic surface modifications exhibited high durability to laundering and demonstrated the viability of customizing surface hydrophobicity.

This investigation provided a comparative assessment of strategies for the removal of 18-methyleicosanoic acid (18-MEA), and other surface bound lipids enveloping the wool fiber surface, by chemical and physical cleavage of the thioester bond. The removal of this lipid layer reveals an underlying proteinaceous layer, exposing functional chemical groups available for covalent attachment of new molecular or nanoparticulate entities by chosen treatments. Lipid removal treatments employing methanolic potassium hydroxide, t -butoxide in t-butanol, and aqueous hydroxylamine and a physical atmospheric pressure glow discharge (APGD) plasma treatment were compared. Treated wool fabrics were subsequently characterized by analysis of the exposed groups on the fiber surface by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), with the bulk surface properties of the fabrics assessed employing wettability testing techniques. An evaluation of chemical and plasma methods for removal of the surface bound lipid layer resulted in the selection of aqueous hydroxylamine/ non-ionic surfactant as the optimal treatment method for subsequent covalent attachment of novel entities by surface treatments. Optimized aqueous hydroxylamine treatment was found to remove up to 77% of the surface bound 18-MEA, providing a marked increase in surface wettability, without significantly affecting the handle of the treated fabric. Surface characterization demonstrated that the hydroxylamine treatment produces an increase in surface friction, with uniform and controlled removal of the surface lipid layer, and minimal surface oxidation of the surface thiols. Minimizing surface oxidation of thiols was a critical target of surface lipid removal in this study, as it maximizes the potential for subsequent surface modification via covalent attachment. The use of aqueous conditions, short reaction times, and moderate temperatures with hydroxylamine treatment are advantageous in comparison with treatments employing non-aqueous solvents such as methanol and anhydrous t-butanol. The economic and environmental advantages of an aqueous, effective, and non-damaging approach to surface lipid removal highlight this approach as a potential avenue for future textile application of novel wool surface chemistries.

Three different cell types have been identified in the cortex of wool: orthocortex, mesocortex and paracortex. Fine wool fibres, particularly Merino sheep, are noted for their bilateral distribution of orthocortical and paracortical cells, with the latter following the concave side of the crimp wave. Furthermore, studies have indicated that the paracortex has a higher concentration of cysteine than the orthocortex. This has been supported by in situ hybridization studies in the follicle that have shown that sulphur-rich proteins are initially expressed on the paracortical side of the fibre, with some becoming more uniformly spread, laterally, over the entire fibre as the keratinization process progresses. In contrast, proteins high in glycine and tyrosine tend to be expressed initially on the orthocortical side of the follicle. While these in vitro studies have pointed to where specific proteins are located in the follicle, elucidating the situation for the mature fibre has been less easy. A range of approaches have been used to separate orthocortical and paracortical cells and these have only been able to provide evidence for a higher level of cysteine in the latter. Electrophoretic studies have found a number of differences in protein expression between the two sides but have not specifically identified which proteins. Thus, there appears to be good evidence for the paracortex containing a higher proportion of proteins in the ultra-high sulphur class but there is some uncertainty regarding the exact distribution of proteins high in glycine and tyrosine.

The colour of scoured wool is a critical determinant of its value and quality, with the presence of significant non-scourable discoloration severely limiting its use. In order to develop effective protocols for the removal or prevention of non-scourable fleece wool discoloration, it is imperative that all significant contributing chromophores are characterised and their origin established. We describe the location, extraction and characterisation of chromophores from non-scourable yellow fleece wool. Yellow discoloration was found to be located predominantly in the cuticular region of the wool fibre. Chromophoric compounds were extracted, isolated and characterised by tandem mass spectrometry, with five phenazine derivatives identified: phenazine, 1-hydroxyphenazine, phenazine-1-carboxylic acid, pyocyanine and 1-methoxyphenazine. Phenazines are brightly coloured pigments that are characteristic secondary metabolites of the bacterial genus Pseudomonas, a known ubiquitous component of the wool fleece microflora. The results of this research represent significant progress in our knowledge of wool discoloration, providing insight into both the chemical and microbial origin of non-scourable wool yellowing.

This paper describes the identification of the yellow chromophore-containing residue kynurenine within photoyellowed wool. Photoyellowed Merino wool fabric was tryptically digested and the resultant peptides were separated by high-performance liquid chromatography. Peaks with absorbance at 400 nm were collected and analyzed by tandem quadrupole time-of-flight mass spec-trometry. Kynurenine was identified as a yellow chromophore in two peptide sequences characteristic of Type 1 intermediate filament and high glycine-tyrosine wool proteins. To our knowledge this is the first conclusive evidence confirming the identity of photoyellowed products and their location within the primary structure of wool proteins. A number of non-yellow modifications were identified in the course of this study and taken together with the identification of kynurenine modifications, these results support the current mechanistic theory of the radical oxidative process occurring during photoyellowing.

The three-dimensional orientation and arrangement of intermediate filaments in Romney wool ortho-, meso-, and paracortical cells has been revealed using single axis high voltage electron tomography. Modelled tomograms confirm that intermediate filaments in orthocortical cells are arranged helically, with the helical angle progressively increasing from the centre to the periphery of macrofibrils. Intermediate filaments in meso- and paracortical cells display parallel arrangements differing mainly in packing density, with the mesocortex packed more tightly than the paracortex. The intermediate filament arrangements observed confirm expectations based on earlier two-dimensional transmission electron microscopy observations by the authors and other researchers. It is expected that these findings will contribute to a better understanding of the biological and structural basis of wool fibre curvature.

Using the stress-strain relationship of wool cells, a three-component model (cuticle, ortho-, and para-/mesocortex) was developed to model the bending properties and behaviour of the wool. The bending rigidity varied with not only the elastic moduli and geometry, but also the direction of the applied moment, whereas bending stiffness is insensitive to the direction of the load. The simulations indicated that the cuticle might contribute 25% of the bending stiffness in extreme cases and should not be ignored, as has been the case in previous studies. Single fibre curvature (SFC), as a particular bending behaviour associated with the removal of moisture, was illustrated using finite element analysis. The physical properties of the three components (cuticle, ortho-, and para-/mesocortex) of Romney wool fibres are estimated using the stress-strain relationship models. The geometric configuration of the samples is built from true fibre images. The simulations are validated to be qualitatively consistent with the observations of SFC. The displacement, stress and strain energy of the wool fibre due to moisture desorption are mapped using coloured figures.

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

The keratin proteins from wool can be divided into two classes: the intermediate filament proteins (IFPs) and the matrix proteins. Using peptide mass spectral fingerprinting it was possible to match spots to the known theoretical sequences of some IFPs in web-based databases, as enzyme digestion generated sufficient numbers of peptides from each spot to achieve this. In contrast, it was more difficult to obtain good matches for some of the lower molecular weight matrix proteins. Relatively few peaks were generated from tryptic digests of high-sulfur proteins because of their lower molecular weight and the absence of basic residues in the first two-thirds of the sequence. Their high sequence homology also means that generally only a few of these peptides could be considered to be unique identifiers for each protein. Nevertheless, it was still possible to uniquely identify some of these proteins, while the presence of two peptides in the matrix-assisted laser desorption/ionization time-of-flight mass spectrum allowed classification of other protein spots as being members of this family. Only one major peptide peak was generated by the high-glycine tyrosine proteins (HGTPs) and there were relatively few sequences available in web-based databases, limiting their identification to one HGTP family.

The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

Nanomechanical properties of biological fibers are governed by the morphological features and chemically heterogeneous constituent subunits. However, very little experimental data exist for nanoscale correlation between heterogeneous subunits and their mechanical properties. We have used keratin-rich wool fibers as a model of composite biological fibers; a wool fiber is a simple two component cylindrical system consisting of a core cellular component surrounded by an outer cell layer and their ultrastructure and chemical composition are well-characterized. The core is 16-40 micrometer in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and distinctly rich in disulfide bonds. We used an atomic force microscope (AFM) to examine the nanomechanical properties of various structural components using complementary techniques of force-volume imaging and nano-indentation. AFM images of transverse sections of fibers were obtained in ambient environment, and the mechanical properties of several identified regions were examined. The outer cell layer showed a significantly higher mechanical stiffness than the internal cellular core region. Chemical reduction of disulfide bonds eliminated such dichotomy of mechanical strengths, indicating that the higher rigidity of the outer layer is attributed primarily to the presence of extensive disulfide bonding in the exo-cuticle. This is the first detailed correlative study of nano-indentation and regional elasticity measurements in composite biological systems, including mammalian biological fibers.

In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents.

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5°. The length of one turn of the helical path of an IF was calculated to be approximately 1μm for an IF tilted at 30° and positioned 100nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.

Crimp and bulk, important wool fiber properties, are thought to be related to differences in the protein composition of the orthocortex and paracortex. Fiber morphological studies have demonstrated that the paracortex has a higher proportion of matrix and cysteine than the orthocortex. While there is some evidence for the differential expression of genes between these cell types in the follicle, this has not been demonstrated satisfactorily in the mature fiber. Using proteolytic digestion of wool fibers, followed by ultrasonic disruption to obtain relatively pure fractions of both cell types, the KAP3 high sulfur protein family was found to be present in higher concentrations in the paracortex. This significant finding provides an explanation for the higher cysteine content reported in the paracortex. This represents an advance in our understanding of protein expression variation in the orthocortex and paracortex, and how this relates to key physical and mechanical properties of wool fibers.

Naturally straight and curved human scalp hairs were examined using fluorescence and electron microscopy techniques to determine morphological and ultrastructural features contributing to single fiber curvature. The study excluded cuticle and medulla, which lack known bilateral structural asymmetry and therefore potential to form curved fibers. The cortex contained four classifiable cell types, two of which were always present in much greater abundance than the remaining two types. In straight hair, these cell types were arranged annularly and evenly within the cortex, implying that the averaging of differing structural features would maintain a straight fiber conformation. In curved fibers, the cell types were bilaterally distributed approximately perpendicular to fiber curvature direction with one dominant cell type predominantly located closest to the convex fiber side and the other, closest to the concave side. Electron tomography confirmed that the dominant cell type closest to the convex fiber side contained discrete macrofibrils composed of helically arranged intermediate filaments, while the dominant cell type closest to the concave side contained larger fused macrofibrils composed of intermediate filament arrangements varying from helical to hexagonal arrays approximately parallel to the longitudinal fiber axis. These findings concur with the current hypothesis of hair curvature formation and behavior.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods have been used to provide high-resolution separation of wool intermediate filament proteins (IFPs). An improved method of extraction was developed based on a previously published method. The improved method for extraction eliminates the use of dialysis and freeze-drying between the extraction and rehydration steps, allowing the extraction and rehydration for the first dimension gel to be achieved in one day. Improvements to the method for maintaining reducing conditions and chaotrope constitution, combined with low %T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern of higher intensity, with numerous minor spots not previously observed, and a marked improvement in the vertical resolution. Further work to analyse the composition of each of the protein spots has been made much easier by being able to separate the IFPs into discrete spots.

The outermost protein layer of wool cuticle cells is known as the exocuticle a-layer. This layer is a resistant barrier to the degradation of the fibre and, as a result, little is known of its proteinaceous composition. Merino wool fibres were subjected to both proteolytic and chemical digestion and the resulting material was found by transmission electron microscopy to be highly enriched in a-layer. Amino acid analysis revealed a high cysteine and glycine content, with a close, but not exact, match to the Allworden membrane. Subsequent digestion of the a-layer preparation by 2-nitro-5-thiocyano-benzoic acid produced a large number of short peptides, and analysis by mass spectrometry revealed peptides with strong homologies to cuticular ultra-high sulphur proteins of sheep wool and cuticular ultra-high and high-sulphur proteins of human hair, thus supporting other evidence for the presence of these sulphur-rich proteins in the a-layer.

Understanding the photodegradation of complex protein systems represents a significant goal in protein science. The photo-oxidation and resultant photoyellowing of wool in sunlight is a severe impediment to its marketability. However, although some photomodifications have been found in irradiated model amino acid systems, direct identification of the chromophoric photoproducts responsible for photoyellowing in irradiated wool itself has proved elusive. We here describe the direct characterisation and location of yellow chromophores and related photomodifications within the proteins of photoyellowed wool fabric, utilising a quasi-proteomic approach. In total, eight distinct photoproducts were characterised. Of these, five were derived from tryptophan; namely hydroxytryptophan, N-formylkynurenine, kynurenine, residues consistent with the dehydration of kynurenine, and hydroxykynurenine, while three were derived from tyrosine; namely dihydroxyphenylalanine, dityrosine, and a cross-linked residue consistent with a hydroxylated dityrosine residue. Fourteen modified peptide sequences were identified and the positions of modification for thirteen of these were located within the primary structure of known wool proteins. The nature of the photoproducts characterised offer valuable insight into the reaction pathways followed in the UV-induced photoyellowing of wool proteins.

Photo-oxidative processes occurring in wool can lead to significant photoyellowing of the fiber. In particular, wool that has been chemically bleached photoyellows more rapidly and to a greater degree than untreated wool. Direct identification of the chromophores responsible for such yellow discoloration in irradiated wool has proven to be elusive for many years. This article describes the characterization and location of yellow photo-oxidation products within the proteins of photoyellowed bleached wool fabric, using advanced protein chemistry techniques. The discolored fabric was enzymatically digested and chromatographed by high-pressure liquid chromatography, with monitoring at 400 nm, to select out fractions containing yellow chromophoric species. Thorough tandem mass spectrometric analysis was then used to sequence peptides and, in turn, to characterize modifications to key amino acid residues that had resulted in yellow chromophore formation. In total, 11 separate yellow chromophoric species were identified, ten derived from tryptophan residues and one from tyrosine. The tryptophan-derived modifications characterized included hydroxytryptophan, N-formylkynurenine, hydroxyformylkynurenine, kynurenine, hydroxykynurenine, carbolines, tryptophandiones and nitrotryptophan. The tyrosine-derived modification of tyrosine to dopa was also identified. The range of photomodifications we observed provides insight into the photo-oxidation pathways occurring within irradiated fibrous proteins leading to the formation of yellow chromophores.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.

Nano-mechanical properties of composite biomaterials are governed by the morphological features and chemical distribution of heterogeneous multiple subunits. However, very little is known about such structure-function correlation at the nanoscale. We have used wool fibers as a model of composite biomaterials for such study. Wool fibers provide an ideal two component cylindrical model for such study as their ultrastructure and chemical composition have been well characterized, they have a core cellular component surrounded by an outer cell layer. The core is 16-40 micron in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and are distinctly rich in disulfide bonds. In this study, we have used an atomic force microscope (AFM) to examine the micromechanical properties of the different structural components using complementary techniques of nano-indentation and force-volume imaging. AFM images of transverse sections of wool fibers were obtained in ambient environment (Figure 1).The exo-cuticle showed a significantly higher mechanical stiffness than the internal cellular core region (Figure 2).

The usual spectrophotometric method for determination of chromium involves permanganate oxidation of chromium(III) to chromium(VI), followed by complex formation with diphenylcarbazide. When chromium-spiked liver tissue samples were mineralised by acid digestion, permanganate oxidation was not complete even on a boiling water-bath, but cerium(IV) oxidised chromium(III) completely at room temperature, the rate of oxidation being dependent on pH and matrix composition. Phosphate and iron(III) ions interfered. Precipitation of cerium(IV) by phosphate was avoided by addition of sulphuric acid. The coloured complex formed by iron(III) with diphenylcarbazide could be extracted with dichloromethane. High concentrations of some other metals and complexing agents interfered with the determination of chromium standards, but these substances did not occur in interfering concentrations in mouse liver digests. With the modified assay, standard chromium(III) and chromium(VI) solutions and mineralised liver samples containing standard additions of chronium(III) or chromium(VI) could be measured in the range 2.00 × 10-5–4.00 × 10-3 g dm-3. The four analytical curves were linear, and the superposition of standard and sample curves indicated that chromium was completely recovered from the tissue digestion step.

The determination of iron(III) in aqueous solution is investigated by e.s.r. techniques. Hydrated Fe(III) gives rise to a broad spectrum for which the analytical range is 5.0 × 10 -1 – 5.0 × 10 -4 mol dm -3 iron(III) with a precision of ± 0.98%. Iron(III) in 2.0 mol dm -3 NH 4 F solution gives a spectrum with a much narrower line width through the formation of the FeF 6 3- complex. The resulting increase in signal amplitude leads to an improved analytical range of 1.0 × 10 -2 –6.0 × 10 -7 mol dm -3 iron(III) with a precision of ± 0.45%. There is also a significant decrease in the time required to determine Fe(III) in this system. The effects of variation in instrumental variables, pH, and stability on the spectra of both systems are considered.

The flow cell decreases the time required to change samples to 1 min, and a typical relative standard deviation of measurement is improved from about 0.3% to 0.1%.