Cystine analyses

The Association of Biomolecular Resource Facilities (ABRF) has been addressing the identification and ultimate resolution of such difficulties by collaborative trials [1-7]. The purpose of the 1994 trial was to discriminate between hydrolysis and the chromatographic analysis as a possible major source of errors, and additionally to test cystine analyses as part of a continuing effort. 

Table IV. Summary of errors in cystine analyses vS ABRF-94AAA...

Cysteine analyses showed a continuing trend of successful use of disulfide exchange reagents. Performic acid oxidation, although still successfully used, overall fared as well as the simple direct analyses of cystine. Direct cystine analysis of the pre-hydrolysate was as successful as that of the protein sample, suggesting that chromatography and derivatization and not hydrolysis are the most important factors in successful analysis of cystine by this simple methodology. 

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. 

S-sulfenylsulfonate 109 S-sulfonate 108 trimethylaminoethyl- 106 cystine, analysis 20 modification 101 oxidation 102 reduction 103 S-sulfo- 22... 

Halo mercury compounds such as methyl mercuric iodide also react readily with mercaptan in keratin fibers [90] and serve as the basis of Leach s method for cystine analysis. 

In terms of amino acids bacterial protein is similar to fish protein. The yeast s protein is almost identical to soya protein fungal protein is lower than yeast protein. In addition, SCP is deficient in amino acids with a sulphur bridge, such as cystine, cysteine and methionine. SCP as a food may require supplements of cysteine and methionine whereas they have high levels of lysine vitamins and other amino acids. The vitamins of microorganisms are primarily of the B type. Vitamin B12 occurs mostly hi bacteria, whereas algae are usually rich in vitamin A. The most common vitamins in SCP are thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, choline, folic acid, inositol, biotin, B12 and P-aminobenzoic acid. Table 14.4 shows the essential amino acid analysis of SCP compared with several sources of protein. 

The sulphite aftertreatment is particularly important with permonosulphuric acid treatment. Evidence for the underlying mechanism is available from analysis of sulphur oxidation products formed in the various processes (Table 10.34). It is evident from these results that the concentration of RSS()5 anionic groups necessary to change the hydration of the fibre surface is achieved by the reaction of bisulphite with cystine monoxide residues to give the required cysteine-S-sulphonate groups [311]. 

The iodoacetyl group of both isomers reacts with sulfhydryls under slightly alkaline conditions to yield stable thioether linkages (Figure 9.7). They do not react with unreduced disulfides in cystine residues or with oxidized glutathione (Gorman et al., 1987). The thioether bonds will be hydrolyzed under conditions necessary for complete protein hydrolysis prior to amino acid analysis. 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Figure 2.11 LC analysis of cystine DCL. Trace A shows the DCL templated with CaM, and trace B shows the blank DCL. The amplified peaks ( ) are dipeptides ce and cc. Reproduced from Reference 23 with permission of Wiley-VCH Verlag GmbH Co. KGaA. Copyright Wiley-VCH Verlag GmbH Co. KGaA.

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. 

Direct Analysis of Disulfide Topologies of Multiple-Cystine Peptides... 

Some of the multiple-cystine peptides are resistant to enzymatic and/or chemical cleavage, and thus the more simple methods described in Section 6.1.6.2 cannot be applied. In these cases assignment of their disulfide connectivities is generally attempted by NMR structural analysis. Thereby, if the two half-cystine residues are located on opposite sides of the NMR-derived 3D structure, a disulfide bond between them is improbable. 

Peptide bonds are cleaved in a nonselective, but not in a completely random manner. Based on anchimeric side-chain assistance, steric factors, and bond strains, acid-labile peptide bonds are predicted to include sites containing Asp, Glu, Ser, Thr, Asn, Gin, Gly, and ProJ22l The disulfide topologies of circulin B and cyclopsychotride, backbone-cyclized peptides with three disulfide bonds, were determined by partial hydrolysis for 5 hours.[22 Occasionally, the bond between adjacent half-cystine residues is cleaved due to the nonselective nature of the mechanism of partial acid hydrolysis.[21] By this procedure, in all cases, a complex mixture of peptide fragments is produced which requires careful chromatographic separation by RP-HPLC for subsequent analysis by mass spectrometry (see Section 6.1.6.2.7). 

Sequence analysis is one of the most useful tools for determining disulfide connectivities by detecting the phenylthiohydantoins (PTH) of either cystine or cysteine derivatives. Sequencing can be carried out on the intact peptide, although most frequently disulfide-bridged fragments are subjected to this type of analysis. 

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