Keratin , hydrolysis

The minimum in swelling is consistent with the observation of Steinhardt and Harris [106]. In the absence of added electrolyte, there is no combination of wool hber with mineral acid or alkah from pH 5 to 10. This is in the vicinity of the isoionic point of hair. The large increase in swelhng above pH 10 is largely due to ionization of diacidic amino acid residues in the hair and partly due to keratin hydrolysis. The increase in swehing from pH 3 to 1 is due to the combination of acid with the dibasic amino acids. Brener and Prichard [57] attribute the decrease in swelhng below pH 1 to an irreversible structural change. 

The differences in the amino acid chemistry of the hide coUagen and the hair keratin are the basis of the lime-sulfide unhairing system. Hair contains the amino acid cystine. This sulfur-containing amino acid cross-links the polypeptide chains of mature hair proteins. In modem production of bovine leathers the quantity of sulfide, as Na2S or NaSH, is normally 2—4% based on the weight of the hides. The lime is essentially an unhmited supply of alkah buffered to pH 12—12.5. The sulfide breaks the polypeptide S—S cross-links by reduction. Unhairing without sulfide may take several days or weeks. The keratin can be easily hydrolyzed once there is a breakdown in the hair fiber stmcture and the hair can be removed mechanically. The coUagen hydrolysis is not affected by the presence of the sulfides (1—4,7). 

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). 

Keratin, 5, 39 Ketene, 4, 3g 8, 124 Ketone hydrolysis, 7, 60 Ketone-splitting of acetoacetic ester, 7, 60... 

Preparation by acidic proteine hydrolysis (e. g. of keratines) with following fractionated crystallization (obtained in commonly with i.-cystine). 

Cystine is one of the units from which the proteins are built up. It is prepared by acid hydrolysis of keratin (from hair or horns). 

Alpenfels and coworkers studied the hydrolysis of glycoproteins and keratin fibers with 1 or 2 M hydrochloric acid for various periods of time at 100°. These investigators found that the concentration of neutral monosaccharides from hard keratin reached a maximum after hydrolysis with 2 M hydrochloric acid for 2 h at 100°, and the yield of the neutral monosaccharides was linear up to 25 mg of hair per mL of hydrochloric acid solution. The latter fact shows that a relatively large amount of protein does not interfere with the analysis of a relatively small amount of carbohydrate. 

The separation of cystine and tyrosine as they are obtained by hydrolysis with hydrochloric acid was described by Morner in I901. The protein—hair, keratin from horn, eggshells, etc.—was boiled with five times its quantity of 13 per cent hydrochloric acid under a reflux condenser on a water bath for six to seven days. The solution was then decolorised with charcoal and evaporated in vacuo, and the residue dissolved in 60 to 70 per cent, alcohol. The two acids then crystallised out on neutralising with soda, and were separated by fractional crystallisation from ammonia if much tyrosine was present it separated out first, but if cystine exceeded tyrosine in quantity this compound crystallised out first the remainder was only separated with difficulty. Embden separated the mixture of the two acids by means of very dilute nitric acid, in which tyrosine is very easily soluble, but cystine with difficulty. Their separation may also be effected by precipitation with mercuric sulphate in 5 per cent, sulphuric acid solution in which the mercury compound of tyrosine is soluble (Hopkins and Cole). 

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. 

These processes provide a complete destruction of the structure of the hair keratin by the complete hydrolysis of proteins that compose it. Hydrolysis can occur enzymatically or chemically. 

The hair treatment with concentrated solutions of sodium hydroxide allows the complete dissolution of the keratin structure by chemical hydrolysis of proteins in about 1 h. Under these conditions, some drugs such as amphetamines are volatile and thus there may be losses of the analyte. In more basic solutions occurs the complete hydrolysis of molecules such as cocaine, heroin, and 6-MAM [51]. Concentrated solutions of hydrochloric acid are also used it eliminates the problem of volatile basic compounds, but increases the time of dissolution. 

Such isopeptides also have been found naturally present in keratin (77,78,85) and in polymerized fibrin (79). Their quantitative determination requires an enzymic hydrolysis using pepsin, pronase, amino-peptidase, and prolidase as described by Cole et al. (135) followed by a chromatographic separation using an amino acid analyzer under very specific conditions (80). 

Scleroproteins. Insoluble in water and neutral solvents and resistant to enzymic hydrolysis. These are fibrous proteins serving structural and binding purposes. Collagen of muscle tissue is included in this group, as is gelatin, which is derived from it. Other examples include elastin, a component of tendons, and keratin, a component of hair and hoofs. 

Determination of cystine as such after elution from a chromatogi aphic column (Simmonds 1954) gave values about 28 % lower than the results of the micro-Shinohara method. Corfield and Robson (1956), on the other hand, obtained a satisfactory sulfur balance by combining analyses for methionine with the results of cystine analysis by the Shinohara method, but considered this to be suspect in view of the known degradation of cystine during hydrolysis. Earlier, Cuthbertson and Phillips (1945) had reported a similar result for analyses on wool, whereas Lindley (1948), using similar methods, found major discrepancies for other keratins such as calf hair and cow hair. 

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). 

Derivation Hydrolysis of protein (keratin), organic synthesis. Occurs as small hexagonal crystals in urine. 

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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