Warren G. Bryson

Characterisation of photo-oxidation products within photoyellowed wool proteins: tryptophan and tyrosine derived chromophores

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.

Determination of Photo-oxidation Products Within Photoyellowed Bleached Wool Proteins

Abstract 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.

Cortical cell types and intermediate filament arrangements correlate with fiber curvature in Japanese human hair

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.

The differential expression of proteins in the cortical cells of wool and hair fibres.

Abstract:  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.

Application of proteomics for determining protein markers for wool quality traits.

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.

Covalent Modification of the Wool Fiber Surface: Removal of the Outer Lipid Layer

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.

Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool.

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.

Characterization of the exocuticle a-layer proteins of wool.

Abstract:  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 Allwörden 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.

Kynurenine Located within Keratin Proteins Isolated from Photoyellowed Wool Fabric

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.

Covalent Modification of the Wool Fiber Surface: The Attachment and Durability of Model Surface Treatments

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.