Nonkeratinous Proteins Research Articles

Challenges in Wool Fiber Dyeing

This paper reviews the analysis of some properties of wool fiber and its dyeing challenges that can influence the outcomes of dyeing. It is relatively intricate to acquire the true color and the main source of a given problem is even more challenging. Its complexity is demonstrated by the different important chemical groups it has and the intermolecular forces of attraction between the linkages, the environmental demands on dyeing process, varied application areas, different important chemical groups present in the chain, fiber damage and permanent setting, presence of non-keratinous Proteins and due to the preparatory pretreatment process .Some approaches that could lead to progress in wool dyeing are discussed in this review and some alternative strategies are also proposed.

A Bacillus Strain Able to Hydrolyze Alpha- and Beta-Keratin

The ability to hydrolyze keratin, a rigid and strongly cross-linked protein in the waste of poultry feather and sheep wool, has made keratinase production by microorganisms highly important to the biotechnological industry. A proteindegrading bacterium (C4) was isolated from compost. Based on morphology and biochemical tests, along with 16S rRNA sequencing, the isolated C4 was tentatively identified as Bacillus sp. C4 (2008). The proteolytic activity of the Bacillus sp. C4 strain was broadly specific; it degraded keratinous and non-keratinous proteins to different degrees. Pea pods as substrate generated the highest protease production, followed by soybean meal and sheep wool. Notwithstanding, using wool keratin as a sole source of carbon and nitrogen yielded the highest level of soluble proteins. Furthermore, the C4 bacterium grew well, and produced a significant level of keratinase when using wool and feather as substrates. Supplementing the medium with yeast extract and peptone shortened the time required for feather degradation, but delayed the onset of the wool keratin hydrolysis with two days. The predominant amino acids released in feather hydrolysate were tyrosine, phenylalanine, and histidine. In contrast, the wool lysate was rich in aspartic acid, methionine, tyrosine, phenylalanine, histidine, and lysine. Results established that utilizing the C4 strain for keratin degradation in waste management holds considerable potential.

Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling

BackgroundThe classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets.Methodology/Principal FindingsWe present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279–modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively.Conclusions/SignificanceMass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals.

Exogenous proteins in exhaled human breath condensate

The analysis of the protein composition of exhaled breath to diagnose diseases of the respiratory system raises a problem of differentiation proteins of expressed in the tissues of the lungs and respiratory tract (endogenous) and got in the respiratory system from the ambient air in the process of respiration (exogenous). In this work an attempt was made to estimate a set of exhaled exogenic proteins by mass spectrometry coupled with nanoflow HPLC. Six-month isolation of healthy donors indoors with air cleaned of dust leads to removal from the spectrum of exhaled proteins of some keratins that are considered therefore to be exogenic. Non-keratin proteins may also circulate between the ambient air and human respiratory ways, but their concentration appears to be significantly lower the keratin concentrations (especially epidermis keratin). Among non-keratins dermcidin seems to be the most significant exogenic protein of exhaled air. The conclusion of the diagnostic value of exhaled proteins can be done only after careful comparison of the results of quantitative and qualitative analysis of their composition in norm and pathology for a statistically significant sample of donors.

Proteomic Analysis of Proteins Selectively Associated with Hydroxyapatite, Brushite, and Uric Acid Crystals Precipitated from Human Urine

The aim of this study was to compare the intracrystalline protein profiles of hydroxyapatite (HA), brushite (BR), and uric acid (UA) crystals precipitated from the same urine samples. HA, BR, and UA crystals were precipitated on two different occasions from the same pooled healthy urine. Crystals were washed to remove surface-bound proteins, and their composition was confirmed using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDAX). SDS-PAGE was used for visual comparison of the protein content of the demineralised crystal extracts, which were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HA comprised nanosized particles interspersed with organic material, which was absent from the BR and UA crystals. The number and type of individual proteins differed between the 3 minerals: 45 proteins were detected in the HA crystal extracts and 77 in the BR crystals, including a number of keratins, which were regarded as methodological contaminants. After excluding the keratins, 21 proteins were common to both HA and BR crystals. Seven nonkeratin proteins were identified in the UA extracts. Several proteins consistently detected in the HA and BR crystal extracts have been previously implicated in kidney stone disease, including osteopontin, prothrombin, protein S100A9 (calgranulin B), inter-α-inhibitor, α1-microglobulin bikunin (AMBP), heparan sulfate proteoglycan, and Tamm-Horsfall glycoprotein, all of which are strong calcium binders. We concluded that the association of proteins with HA, BR, and UA crystals formed in healthy urine is selective and that only a few of the numerous proteins present in healthy urine are likely to play any significant role in preventing stone pathogenesis.

Microstructural Analysis of Wool Fibers by SEM Images of Their Cross-Sections Etched by Alkali Treatment

We found that scanning electron microscopy (SEM) of alkali-etched cross-sections of wool fibers provides characteristic images consisting of stitch patterns (SP) and amoeba patterns (AP) at the cortex and that both the SP and AP appearing as convex structures are originated from cell membrane complexes, nuclear remnants, and intermacrofibril materials. When the sample fiber had primarily been extracted with chloroform/methanol, the SP became disintegrated, because of the removal of free lipids from CMC, particularly from the β-layers. When the fiber had been extracted with chloroform/methanol/water, the SP was almost completely lost. These results suggested that the SP and AP are derived from the internal lipids and some non-keratinous proteins involved in the cortex and that the extraction of wool fibers with the mixed solvents should disrupt both the β- and δ-layers to increase the susceptibility of the CMC to the alkaline hydrolysis. The post-washing of the alkali-etched cross-section with ethanol made the SP and AP clearer. The agglomerated and scattered AP detected in the outlines of the cortical cells were shown to be originated from the nuclear remnants and the intermacrofibril materials, respectively.

Heavy Water Labeling of Keratin as a Non-Invasive Biomarker of Skin Turnover In Vivo in Rodents and Humans

Measurement of skin turnover has been problematic in humans. Heavy water (2H2O) labeling has recently been developed as a safe, simple method to study in vivo kinetics of many biosynthetic processes, including DNA and protein synthesis. Here, we apply this approach to the measurement of 2H incorporation into skin keratin and show close agreement between keratin and keratinocyte turnover data in the epidermis of rodents. Elevated turnover rates of both keratin and keratinocytes were observed in the epidermis of the flaky skin mouse, although topical treatments effective in human psoriasis had no effect on either turnover rate in these mice. In humans, keratin turnover was monitored non-invasively by serial tape stripping during and after 2H2O labeling. Kinetic data were consistent with previous estimates of epidermal turnover, with a lag time of 18 days before label appeared at the skin surface and a transit time of 4-5 weeks. Variability in skin keratin turnover rates was present among healthy individuals. In summary, 2H2O labeling of skin keratin represents a non-invasive approach for assessing skin turnover dynamics in pre-clinical models and in human subjects.

Presence of putative histidine-rich proteins in the amphibian epidermis.

In amphibian epidermis mucus is thought to constitute the matrix material that links keratin filaments present in cells of the corneous layer. As contrast in mammals, and perhaps in all amniotes, histidine-rich proteins form the matrix material. In order to address the study of matrix molecules in the epidermis of the first tetrapods, the amphibians, an autoradiographic and electrophoretic study has been done after administration of tritiated histidine. Histological analysis of amphibian epidermis shows that histidine is taken up in the upper intermediate and replacement layers beneath the corneous layer. Ultrastructural autoradiographic analysis reveals that electron-dense interkeratin material is labeled after administration of tritiated histidine. Electrophoretic analysis of the epidermis shows labeled proteic bands at 58-61, 50-55, 40-45, and some only weakly labeled at 30 and 24-25 kDa at 4-48 hours after injection of tritiated histidine. Keratin markers show that bands at 40-61 kDa contain keratins. Most histidine is probably converted into other amino acids such as glutamate and glutamine that are incorporated into newly synthetized keratins. However, non-keratin histidine-incorporating proteins within the keratin range could also be formed. The bands at 30 and 24-25 kDa suggest that these putative histidine-rich proteins are not keratins. In fact, their molecular weigh is below the range of that for keratins. In contrast with the mammalian condition, but resembling reports for lizard epidermis, putative histidine-rich proteins in amphibians have no high molecular weight precursor. Although filaggrin is not detectable by immunofluorescence in sections of amphibian epidermis, protein extraction, electrophoresis and immunoblotting are more sensitive. In the epidermis of toad and frog, but only occasionally in that of newt, filaggrin cross-reactive proteic bands are seen at 50-55, 40-45, and sometimes at 25 kDa. This suggests that after extraction and unmasking of reactive sites in the epidermis of more terrestrial amphians (anurans), some HRPs with filaggrin-like cross-reactivity are present. The overlap that exists at 50-55 kDa between filaggrin-positive and AE2-positive keratins, but not that at 40-45 kDa further indicate that non-keratin, filaggrin-like proteins may be present in anuran epidermis. The present study suggests for the first time that very small amounts of histidine-rich proteins are produced among keratin filaments in upper intermediate, replacement and corneous layers of amphibian epidermis. Although the molecular composition of these proteins is unknown, precluding understanding of their relationship to those of mammals and reptiles, these cationic proteins might have originated in conjunction with the formation of a horny layer during the adaptation to land during the Carboniferous and were possibly refined later in the epidermis of amniotes.

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.

A Universal Structural Model for Human Hair to Understand the Physical Properties 1

Abstract A universal structural model for human hair has been proposed in order to understand the physical properties of hair. The model consists of two structural parts, both of which have two states (Two-part/two-state model). The transition temperature (Tc) between the two states of one part is about 70 °C, and that of the other part is about 0 °C in a water medium. These transition temperatures depend highly upon the water content of the hair. A higher transition temperature is observed in hair having a lower water content. Above the transition temperatures, both parts of the hair are soft and plastic (a melted state); they are hard and elastic (a solid state) below the transition points. One part of hair which has a higher transition temperature (ca. 70 °C in water) is assigned to be macrofibril and exo-cuticle consisting of keratinous proteins; the other part with a lower Tc (ca. 0 °C) is inter-macrofibrillar materials, a cell-membrane complex (CMC), and the endo-cuticle of non-keratinous proteins and lipids. This structural model was derived from the results of thermal-setting and relaxation experiments as well as TEM observations after permeating a dye with a high electron density (Erythrosin B).

Keratinase of Doratomyces microsporus.

The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8-9 and 50 degrees C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K.

Keratin expression in astrocytomas: an immunofluorescent and biochemical reassessment.

Several studies have shown that immunoenzymatic staining of formalin-fixed, paraffin-embedded astrocytomas with keratin antibodies frequently yields positive labelling, but no biochemical evidence of keratin expression in astrocytomas has been reported. We have investigated the presence of keratin in astrocytoma and normal brain tissues both by immunofluorescence on frozen sections and by 1D and 2D immunoblotting using seven monoclonal antibodies that, collectively, recognize most keratin polypeptides. Four of these antibodies did not stain neural tissues by immunofluorescence and were also negative by immunoblotting. The remaining three keratin antibodies stained normal brain and/or a high proportion of astrocytomas. Two of these three antibodies only stained glial fibrillary acidic protein (GFAP)-positive cells, while the third only stained GFAP-negative cells. 1D and 2D immunoblotting analysis showed that positive immunofluorescence staining of normal brain and/or astrocytomas seen with these three keratin antibodies was due to cross-reactivity with non-keratin proteins, such as GFAP. These results demonstrate that, contrary to earlier suggestions, keratin polypeptides are not frequently expressed in astrocytomas. Our studies also emphasize that keratin antibodies should be used cautiously for the differential diagnosis of undifferentiated gliomas from tumours of non-glial origin.

Reactions of β-sulphatoethyl sulphone crosslinking agent with wool. Part IV: Topochemistry of reactions by fractionation and extraction

Topochemical studies have indicated that the crosslinking reaction of 2-chloro-4,6-di( -aminobenzene-4-β-sulphato-ethylsulphone)-1,3,5- s-triazine (XLC) occurs mainly in the low-sulphur microfibrillar proteins of the wool fibre. This can be explained by a high content of lysine and histidine residues, which have been shown to be the major sites of the reaction. Modification of these residues was shown to be pH dependent. Where crosslinking was greatest, it was shown that the lysine and histidine side chains were bridged by the compound. The non-keratinous proteins derived from the cell membrane complex were shown to react with the XLC crosslinking reagent at pH 6.

The liquid retention of structurally modified wool in aqueous solutions.

The liquid retention properties of wool fibers were investigated for those structurally modified by chemical and mechanical processes, such as treating with solvents and enzyme, heating in 8.0M LiBr solution and stretching in steam.The extraction of nonkeratinous proteins with organic solvents and pronase was found to cause an increase in the liquid retension of wool fiber. Particularly, the removal of nonkeratinous proteins had marked effects on the retractive forces opposing the swelling pressure of the liquid in the cell membrane complex.The supercontracted wools in which the original oriented α-structure was transformed to disoriented β-structure in the microfibril, had high liquid retension values at pH2 and 8, distant from the iso-electric point, pH5, whereas the stretched wools in which the oriented β-structure was effected, was little affected in the liquid retention throughout the pH range examined. These results suggest that the extent of the liquid retention of wool is dependent not on the secondary structure of the microfibrillar peptide chains, but on the orientation of the peptide chains which is closely associated with the retractive forces opposing the swelling pressure of liquid in the microfibril.

Regulation of Trichophyton rubrum proteolytic activity

Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the stratum corneum. There are probably peptidases, as yet unidentified, that would cleave the peptides generated by the initial proteolysis into amino acids. These amino acids would provide the cell with a source of carbon, nitrogen, and sulfur. Under these conditions, the expression of general proteinases would be repressed, whereas specific keratinases would be induced in this nutrient-rich environment. Disease may occur when the fungus reaches stationary phase, when proteinases are secreted constitutively. These enzymes may directly or indirectly incite a host response, resulting in the inflammatory manifestations of dermatophytosis.

Role of Nonkeratinous Proteins in Crimp Formation of Wool Fibers by Draft and Immediate Relaxation

The role of nonkeratinous proteins in the crimp formation of wool fibers by draft and immediate relaxation was investigated. For this purpose, Australian Shropshire wool fibers were treated with formic acid and pronase (proteolytic enzyme) to modify the nonkeratinous proteins. Formic acid is known to remove some of the intercellular cement, one of the nonkeratinous proteins, from the cell membrane complex, while pronase removes all nonkeratinous proteins. Little crimp formation occurred in the wool fibers treated with formic acid and pronase, indicating that the nonkeratinous proteins, especially the intercellular cement of the cell membrane complex, play an important role in the crimp formation of wool fibers by the draft and immediate relaxation process. A similar effect on crimp formation also occurred when the wool fibers were steamed in a relaxed state.

Mechanism of Bilateral Staining of Merino Wool Fibers with Basic Dyes

The mechanism of bilateral staining of the cortex of wool fibers by basic dyes has been investigated in detail. Merino wool fibers were treated with formic acid and pronase, and their behavior in staining with basic dyes such as methylene blue and janus green was examined using light and electron microscopy. Formic acid is known to remove intercellular cement, one of the nonkeratinous proteins, from the cell mem brane complex, while pronase removes all nonkeratinous proteins. Bilateral staining was still distinctly observed after cuticle removal, but not for wool fibers treated with pronase, for fiber fragments obtained by treatment with formic acid with stirring, or for cortical cell particles recovered after fibrillation of the cortex in formic acid. The microfibril-matrix structure of the cortex remained unchanged, however, even after treatment with formic acid and with pronase. These results imply that bilateral staining with basic dyes occurs because of the difference in the network structure of nonker atinous proteins, especially the intercellular cement of the cell membrane complex between the orthocortex and paracortex.

Gewinnung und Charakterisierung von nichtkeratinarmer Wolle ? ein Modellpr�parat f�r die Grundlagenerforschung von textilchemischen Ausr�stungsprozessen

The nonkeratinous proteins of wool can be partly or completely dissolved by controlled enzymatic digestion. A comparison between the keratose content of intact wool and that of wool which has been hydrolysed for 96 h with pronase confirms that specifically all the nonkeratinous components are dissolved out of the wool composite by pronase. The influence of microbial infection can be excluded. The residue of wool after the hydrolysis, i. e., a wool low in nonkeratinous proteins, has a higher internal volume, as measured by mercury porosimetry. The centrifugal swelling test shows that these wool samples also have increased hygroscopy.

Morphological Composition of the Cuticle from Chemically Treated Wool

The cuticle plays a decisive part in wool processing, and changes in its morphological composition after industrial finishing are of particular interest. The known enzymatic method for determining the content of endo- and exocuticle is not applicable to chemically modified keratins. A new method was developed for calculating the com position of the cuticle from industrially finished wool on the basis of the results of amino acid analysis. The cuticle was isolated by mechanical treatment of wool in sodium dodecyl sulfate solution. A linear relationship was found between the content of the characteristic amino acids, aspartic acid and leucine, and the amount of en docuticle in isolated cuticle. The results were confirmed by quantitating the diagnostic compounds asparagine and N'-(γ-glutamyl)lysine by means of enzymatic hydrolysis. Shrinkproofing processes effected a specific destruction of the exocuticle, while chrome dyeing caused a loss of nonkeratinous proteins from the endocuticle.

5] Complete enzymatic hydrolysis of wool and its morphological components

Publisher Summary Enzymatic hydrolysis helps in assessment of the nutritional value of proteins in food science, the gentle and limited alteration of cells and cellular organelles in cell biology, to the chemical analysis of protein structure in biochemistry. Complete enzymatic hydrolysis of proteins allows quantitative recovery of amino acids that are unstable in acid or alkali, both primary amino acids and many of the co– and posttranslationally modified secondary amino acids. This chapter deals with the enzymatic hydrolysis of wool, wool cuticle, and its morphological subunits with respect to the distribution of diagnostic components in the fiber. Since, wool is subjected to drastic chemical treatments during industrial processing, therefore in some cases specific degradation of definite morphological components, such as exocuticle and nonkeratinous proteins is also observed. Keratins and nonkeratinous proteins have been defined on the basis of relative resistance to digestion by proteolytic enzymes and results from microscopic studies. The procedure for complete enzymatic hydrolysis of keratins is improved for application to wool keratins. Diagnostic components can be quantitated, permitting a correlation between morphological and chemical changes in the fiber to be made.