Cystine isolation

The first evidence that methionine was the precursor of cystine came from the nutritional observations that methionine could replace cystine in the diet of rats. Direct proof for the transfer of methionine sulfur to cystine was furnished by Tarver and Schmidt with S -labeled methionine. These investigators observed that the cystine isolated from the new-grown hair of the animals given the labeled methionine contained radioactive sulfur. Subsequently, definite proof that methionine contributes only the sulfur atom to cystine was supplied by du Vigneaud and co-workers. The latter fed methionine-3,4-C —S and found that about 80% of the cystine sulfur was derived from the ingested methionine, whereas none of the isotopic carbon appeared in the cystine chain. 

Further evidence that serine was involved in the reaction that forms cystine came from the observation of Stetten that the N concentration in the cystine isolated from rats fed serine-N was higher than could be accounted for by transamination. 

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). 

Cysteine [52-90 ] is a thiol-bearing amino acid which is readily isolated from the hydrolysis of protein. There ate only small amounts of cysteine and its disulfide, cystine, in living tissue (7). Glutathione [70-18-8] contains a mercaptomethyl group, HSCH2, and is a commonly found tripeptide in plants and animals. Coenzyme A [85-61-0] is another naturally occurring thiol that plays a central role in the synthesis and degradation of fatty acids. 

On several occasions the product isolated by the submitters was contaminated with L-cystine dihydrochloride, which was not easily removed by recrystallization. In this event the product was converted to the zwitterionic form and recrystallized in the following manner. The pH of a solution of the product in water was adjusted to 6 with aqueous 2.5N potassium hydroxide. The neutralized solution was evaporated to dryness under reduced pressure at ca. 40°. The residue was dissolved in a minimum amount of hot water, and two volumes of 95% ethanol were added to precipitate S-acetamidomethyl-L-cysteine monohydrate, dec. 187°, [a] 9 — 42.5° (c = 1, water). 

With the exception of cystine, a, a -diamino acids represent a class of compounds not found naturally in biological samples. However, it should be noted that such compounds have been isolated previously from certain... 

Small molecules containing disulfide bonds (such as cystine-containing peptides) may be reduced and isolated simply by removing the immobilized reductant. Separation of reduced molecules from reductant is much more difficult if a soluble reducing agent is used with low-molecular-weight disulfides. 

As early as 1905 Abderhalden (Al) isolated from the hydrolyzate of the nondiffusible fraction of human urine four amino acids, i.e., leucine, alanine, glycine, and glutamic acid, and detected two others phenylalanine and aspartic acid. Some amino acids derived from this fraction have been quantitatively determined by Albanese et al. (A3) who found in the amount of the nondiffusible fraction corresponding to one liter of urine as much as 32.8 mg tryptophan, 18.0 mg phenylalanine, 16.2 mg methionine, 15.2 mg cystine, 13.1 mg arginine, 6.7 mg histidine, and 3.9 mg tyrosine. 

Plant defensins are cystine-rich, cationic peptides ranging in size from 45 to 54 amino acids, of which eight are cysteine. They were first discovered in wheat and barley ° and were proposed to form a novel subclass of thionins, the 7-thionins. As it became clear that they closely resemble mammalian and insect defensins in primary and secondary structure, the term plant defensins was introduced to describe these peptides. It is generally assumed that all plants express plant defensins " and that they are expressed in a wide range of plant tissue, that is, leaves, floral tissue,tubers,bark, root, pods, and seeds,with seeds in particular being from where most plant defensins have been isolated. ... 

The arsenal of plant defense peptides contains members capable of binding carbohydrate residues, namely /31-4 linked A -acetyl glucosamine residues that form the biopolymer chitin. The actual mode of action remains unclear. Antifungal and antimicrobial activity has been shown in vitro. For example Ac-AMP2 is a small disulfide-rich chitin-binding peptide isolated from the seeds of Amaranthus caudatus with antimicrobial activity. It differs from Ac-AMP 1 by one additional arginine residue at the C-terminus. The structure was determined by NMR and contains a cystine knot motif. Ac-AMP2 displays a so-called hevein domain partly... 

The unambiguous formation of disulfide bonds by the synthetic strategy discussed in this section is also well suited for determining the cystine networks of newly isolated natural peptides or isomers 1231... 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

It is a single chain peptide hormone, isolated in pure form and contains tyrosine, tryptophan, cystine, arginine, methionine of approximately 25,000 molecular weight. It has a direct effect upon the breasts immediately after the delivery of baby and in conjunction with other hormones, it stimulates the breast to secrete milk. 

Isoelectric precipitation results in loss of whey protein as well as undesirable non-protein components. Gillberg (35) has shown that the cystine composition of the isolate is lower than for the meal extract because non-precipitated whey protein has a relatively larger proportion of the total sulfur amino acids. This observation is reinforced by Mattil (36), who showed that the range for amino acid composition was different for several commercial concentrates as compared to commercial isolates. He found methionine to be lower for isolates than for concentrates. 

De Groot and Slump (40) studied the influence of alkali on soy protein isolates, monitoring the production of lysinoalanine and changes in amino acid content. They found that above pH 10, treatment at 40 C for 4 hours resulted in decreased cystine and increased LAL (Figure la). They also found that at pH 12.2 for 4 hours, lysine and cystine content steadily decreased with increasing temperatures from 20° to 80 C, and LAL content increased dramatically. At pH 12.2 and 4O C they reported that the greatest loss in cystine and increase in LAL occurred in the first hour (Figure lb). Thus they concluded that exposure of soy protein isolate at pH 12.2 for only a short time would destroy some cystine and decrease the nutritive value. 

Cystine was first isolated from a urinary calculus by Wollaston in 1805. it was shown to be a component of protein by Morner in 1899 and independently by Embden in 1900. Proof of its stiuctiiie was given by Friedman in 1902, See also Amino Acids Coenzvmes Proteins and Vitamin. 

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. 

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