Reduced Wool Fibres, their Preparation and Alkylation

Maclaren, JA; Kilpatrick, D. J.; Kirkpatrick, Alan

doi:10.1071/bi9680805

Cited by 25 publications

(7 citation statements)

References 10 publications

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“…However, even after the disruption of the disulphide bonds, only about 8% of the protein can be extracted with dilute salt solutions or water at neutral pH. An acceptable level of extraction (70-800/0) is obtained at very alkaline pH in the presence of high concentrations of chaotropic agents, such as urea [3]. Reduction, oxidation and sulfitolysis have been used to cleave disulphide bonds in keratin extractions [4].…”

Section: Extraction and Chemical Factionation Of Wool Proteinsmentioning

confidence: 99%

Immobilised ph gradient isoelectric focusing of wool proteins

Herbert¹,

Woods²

1994

Electrophoresis

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Conventional carrier ampholyte-isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient-isoelectric focusing (IPG-IEF) for wool proteins, S-amidomethyl wool proteins were analysed using IPG-IEF. Over twenty bands were visible on a pH 4-7 IPG-IEF gel. IPG-IEF was combined with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG-IEF appears to be ideally suited to the separation of wool proteins. The resolution of two-dimensional electrophoresis using IPG-IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.

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Section: Extraction and Chemical Factionation Of Wool Proteinsmentioning

confidence: 99%

Immobilised ph gradient isoelectric focusing of wool proteins

Herbert¹,

Woods²

1994

Electrophoresis

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“…10,11) However, the detergents in protein solution and the S-alkylation of reduced cysteine interfered with the chemical and physical analyses and their complete removal was difficult. 12,13) To avoid the above mentioned problems, another approach is protein extraction from the cells of the lower part of hair follicles as pre-keratinized material. 2,14) However, using this method it was difficult to obtain an amount of protein sufficient for analysis and, furthermore, the obtained proteins were not originated from the full-keratinized matured structure.…”

mentioning

confidence: 99%

A Rapid Extraction Procedure of Human Hair Proteins and Identification of Phosphorylated Species.

Nakamura

Arimoto

Takeuchi

et al. 2002

Biological & Pharmaceutical Bulletin

202

155

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The protein content in a keratinized structure, including animal hair, nail, horn and feather, is approximately 80% of the total mass.1,2) Two large groups of human hair proteins are known. One is hard a-keratins forming microfibrous intermediate filaments and the other is matrix proteins forming a nonfilamentous matrix as intermediate filaments-associated proteins. The hard a-keratins are further resolved into two subfamilies, consisting of at least 4-9 distinct type I acidic (40-50 kDa) and 4-6 type II neutral/basic (55-65 kDa) members.3) Matrix proteins are classified into high-sulfur proteins (10-20 kDa) and high-tyrosine proteins (6-9 kDa). N-terminal acetylations have been reported for a posttranslational modification of animal hair a-keratins and the related proteins. 4,5) Little is known, however, about posttranslational modifications of human hair a-keratins and the related proteins, while epithelial cytokeratins or soft a-keratins have been well studied. 6)A number of procedures have been reported to isolate hard a-keratins and their related proteins for analyses. 7,8) It is difficult to obtain them in the native state, because the hard akeratins are highly cross-linked with each other by disulfide bonds, enabling intermediate filaments to covalently crosslink with matrix proteins. Proteins extracted from keratinized structures are generally prepared by reduction in the presence of denaturing agent and S-alkylation under extremely low or high pH conditions. Using a combination of these reagents, protein yields were not uniform and protein hydrolysis was liable to occur. 9) Other denaturing agents, such as anionic detergents and guanidine hydrochloride, are also used for the extraction.10,11) However, the detergents in protein solution and the S-alkylation of reduced cysteine interfered with the chemical and physical analyses and their complete removal was difficult.12,13) To avoid the above mentioned problems, another approach is protein extraction from the cells of the lower part of hair follicles as pre-keratinized material. 2,14) However, using this method it was difficult to obtain an amount of protein sufficient for analysis and, furthermore, the obtained proteins were not originated from the full-keratinized matured structure.In this paper, we describe a new and convenient extraction procedure of proteins containing keratins from human hair in the absence of detergent called the 'Shindai method,' because the procedure was developed in Shinshu University. It is also effective for extraction of protein components from wool, chicken feathers, rat hair and human nails. Moreover, we present evidence for the first time that phosphorylated species are contained in human hair hard a-keratin and matrix proteins. MATERIALS AND METHODS Extraction of Hair ProteinsHuman hair (of a Japanese woman) was washed with ethanol; external lipids were removed using a mixture of chloroform/methanol (2 : 1, v/v) for 24 h. The delipidized hair (20 mg) was mixed with a solution (5 ml) containing 25 mM Tris-HCl, pH 8.5, 2.6...

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“…The results of the amino acid analyses (Table II) show that within the limit of the analytical method, the only residue to be affected (apart from 1/2-cystine values, which have been excluded from this comparison) is histidine. Histidine (and tyrosine) is known to be modified by prolonged treatment of reduced wool with iodoacetate [ 15 ] , but the reduced sample without ABD-F treatment did not show any loss of histidine. One possible explanation of this finding is that ABD-F reacts with the nucleophilic imidazole ring of histidine; however, treatment of histidine with ABD-F in borate buffer at pH 8 and examination of the mixture after 24 hours at 50°C failed to show any new fluorescent peaks in the liquid chromatogram.…”

Section: Specificity Of Abd-f For Wool Thiol Groupsmentioning

confidence: 78%

A Method for Determining the Penetration of Reducing Agents into Wool Using Fluorescence Microscopy

Evans

1989

Textile Research Journal

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The fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) has been used to label thiol groups in partially reduced wool fibers. In fibers in which 10% of the disulfide bonds were reduced with either dithiothreitol or tributylphosphine, up to 66% of the thiol groups were specifically labeled in 2 hours at pH 8. The extent of the labeling was determined by analysis of ABD-labeled wool for cysteine-S-ABD. Specificity of labeling was confirmed by comparing the amino acid analyses of untreated wool with reduced wool before and after treatment with ABD-F. Fibers were reduced with a variety of reducing agents, and sections were examined by fluorescence microscopy. From the distribution of the fluorescence. reduction was largely confined to the outer regions of the fiber for 10% reduction at pH 9 (40°C, 30 minutes) with dithiothreitol, tributylphosphine, and tetrakis(hydroxymethyl)-phosphonium chloride. With sodium thioglycollate, however, there was more even penetration of the fibers under the same conditions. The application of this method is discussed for assessing the effect of reducing agents used in conjunction with polymer shrink-resist treatments.The cleavage of disulfide bonds in wool to form thiol groups is one of the most important chemical reactions in wool processing. Generally the extent of reduction required to produce useful changes in fiber properties is small, and for particular applications such as shrinkproofing, it is desirable that the reaction be confined to the surface to minimize fiber damage. In principle, reduction will be confined to the surface regions of the fiber if the diffusion of the reductant into the fiber is slow relative to the rate of disulfide cleavage and if treatment conditions limit the amount or availability of the reducing agent to the fiber. In practice this is difficult to achieve, and little work has been published on how to elucidate the best reducing agents and reaction conditions for limiting reduction to the surface regions of wool. Studies on the reduction of hair with trishydroxymethyl phosphine [8] and dithiothreitol [24] have established, however, that under certain conditions, reduction can be confined to the surface region.To date the only available method for determining the penetration of reducing treatments into wool has been to study sections of reduced fibers treated with reagents such as methylene blue [24] or radiolabeled methyl iodide [8]. Methylene blue is not specific for thiol groups [ 18 ] , however, while methylation of thiol groups in wool with tritiated methyl iodide is accompanied by side reactions that can lead to loss of the radioactive label as volatile dimethyl sulfide [ 4 ] .Fluorescence microscopy is a more sensitive means for studying the distribution of reduction within wool than is transmitted light microscopy of dyed samples. Two different approaches are feasible: reduction can be done using a fluorescent derivative of the reducing agent, or alternatively, a fluorescent label can be used that reacts specifically ...

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Reduced Wool Fibres, their Preparation and Alkylation

Cited by 25 publications

References 10 publications

Immobilised ph gradient isoelectric focusing of wool proteins

Immobilised ph gradient isoelectric focusing of wool proteins

A Rapid Extraction Procedure of Human Hair Proteins and Identification of Phosphorylated Species.

A Method for Determining the Penetration of Reducing Agents into Wool Using Fluorescence Microscopy

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