Keratins isolation

A direct detection method was recently developed for these adducts in stratum comeum of human skin based on immunofluorescence microscopy (30). Three partial sequences of keratins containing glutamine or asparagine, adducted with a 2-hydroxyethyl-thioethyl group at the omega-amide function, were synthesized and used as antigens for raising antibodies. After immunization, monoclonal antibodies were obtained with affinity for keratin isolated from human callus exposed to 50 xM sulfur mustard (see Plate 1). In contrast to the immunochemical... 

Roddick-Lanzilotta A et al (2007) New keratin isolates actives for natural hair protection. J Cosmet Sci 58(4) 405-411... 

Production by Isolation. Natural cysteine and cystine have been manufactured by hydrolysis and isolation from keratin protein, eg, hair and feathers. Today the principal manufacturing of cysteine depends on enzymatic production that was developed in the 1970s (213). 

Component of the myelin sheath surrounding the axons of nerve cells. Additional compounds of the myelin sheath are phospholipids, cholesterol, cerebrosides, and specific keratins. The myelin sheath constitutes an isolating barrier during electrophysiological axonal signaling. 

Z-Cystine has been obtained by the hydrolysis of a large number of proteins. However, the keratins are the only common proteins rich enough in cystine to serve as a source for this amino acid. Many investigators have devised methods for its isolation from the hydrolytic products of human hair,3 wool,2 horn,3 nail,3 feathers,3 and horse hair.4 The method of Folin5 is the basis for most of the others. The present method does not claim to give as high a yield as some of those reported in the literature, but is convenient and gives consistent results. 

The amino acid composition of keratin, the protein of hair and wool, includes a greater-than-average proportion of the sulphur-containing amino acid, cystine. Since this is the least soluble of the protein amino acids it can readily be isolated after carefully neutralising an acid hydrolysate of hair (Expt 5.187). Protein hydrolysis is usually effected by boiling for about 10-20 hours with 20 per cent hydrochloric acid. The hydrolysis of hair for the isolation of cystine is, however, best achieved using a mixture of hydrochloric and formic acids. 

Rajak, R. C., Parwekar, S., Malviya, H., and Hasija, S. K. (1991). Keratin degradation by fungi isolated from the grounds of a gelatin factory in Jabalpur, India. Mycopathologia 114, 83-87. 

In the past decade a number of physical techniques have been used to evaluate the unique barrier properties of mammalian skin [1]. This chapter deals with the use of another physical technique, fluorescence spectroscopy, to study the barrier properties of the human stratum corneum (SC), specifically with respect to the transport of ions and water. The SC is the outermost layer of the human epidermis and consists of keratinized epithelial cells (comeo-cytes), physically isolated from one another by extracellular lipids arranged in multiple lamellae [2]. Due to a high diffusive resistance, this extracellular SC lipid matrix is believed to form the major barrier to the transport of ions and water through the human skin [3-5]. The objective of the fluorescence studies described here is to understand how such extraordinary barrier properties are achieved. First the phenomenon of fluorescence is described, followed by an evaluation of the use of anthroyloxy fatty acid fluorescent probes to study the physical properties of solvents and phospholipid membranes. Finally, the technique is applied to the SC to study its diffusional barrier to iodide ions and water. 

Hydrolyzates of feathers and their constituent parts have also been analyzed, but there are fewer comparative data for this material. Skin keratin, horny materials, and the hard keratins of birds and reptiles have received little exact analytical investigation, although several studies of skin proteins have been reported. Difficulties in isolating the various skin structures and in defining the material studied have not encouraged precise analysis. 

The sequence of appearance, relative amounts, and final relationships of cell components that survive lysis are presented diagrammatically in Figure 11. The smallest filaments that have been isolated (35 A thick) have low sulfur content (32). They thicken to 60-90 A with addition of sulfur-rich protein (demonstrated by heavy metal staining) (32, 64) and histidine-rich protein (demonstrated with radioactive labeling) (49). Five to 10 of these thickened filaments aggregate to form fibrils that average 250 A in diameter (70). Meanwhile, KH and ER protein accumulate until the cell is lysed when they are mixed and dispersed (47) to coat the 250-A fibrils (70). The coated fibrils are submerged in a matrix that includes nucleoproteins and nonfibrous proteins these incorporate about 10 times more sulfur than the fibrils (32). The insoluble fibrils and matrix constitute about 65% of the cornified cell (66) other components include 10% soluble keratin, 10% dialyzable substances (amino acids, etc.), 7-9% lipids, and about 5% membrane protein (65, 66). 

Tonoiilament Assembly. The available information suggests that keratin is sequentially assembled around primary fibers that originate within the attachment plates of desmosomes (27). The dense cores of 50-A filaments (stained with uranyl acetate) represent such fibers (64, 84). These cores and their surrounding fibrous protein probably contain about 50% a helix (71). They contain even less sulfur (32, 64) than the high methionine (1.4 residues/100 amino acid residues), low cystine (1.1 residues/100 amino acid residues) fraction obtained by Baden after partial enzymatic hydrolysis (82). Studies with tritiated amino acids suggest that basal cells preferentially incorporate methionine, leucine, and phenylalanine within their elements (73, 74). In Figure 13A the primary rope is identified with the 35-A diameter of the smallest filaments that have been isolated (32). 

Characterization of the keratinized cells by classical histological and biochemical approaches has been difficult because of the intractable nature of the tissue. Yet it is precisely these properties of mechanical strength, insolubility, macromolecular character, and lack of metabolic activity along with its ease of isolation which makes stratum corneum amenable to analysis by physical methods. The extreme complexity of composition, molecular structure, and organization of stratum corneum make interpretation of these macroscopic properties in terms of molecular structure and events dependent heavily on analogous studies of model synthetic polymer systems and the more thoroughly characterized, keratin-containing wool. 

Keratin Structure and Orientation. Acute flattening of the flbrous protein-fllled cells in the flnal stages of keratinization establishes a biaxial orientation. As would be expected, no birefrigence is observed normal to the plane of the comeum surface, but significant birefrigence is observed parallel to the plane of the corneum surface (I, 42). The x-ray diflFraction pattern of this isolated epidermal protein, when highly drawn, exhibits the classical alpha pattern (7, 43). 

By means of careful hydrolysis with acids it is possible to isolate all the amino-acid components of keratin. According to Astbury J. Chem. Soc., 1942,337) the most probable composition is shown in Table 5.1. 

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