Performic acid cysteine oxidation

Na/NH3, -30°, 3 min, 1(X)% yield. This protective group is stable to acidic hydrolysis (4.5 N HBr/HOAc 1 N HCV, CF3CO2H, reflux). There is no evidence of S N acyl migration in 5-(A-ethylcarbamates) (RS = cysteinyl). Oxidation of 5-(A-ethylcarbamoyl)cysteine with performic acid yields cysteic acid. ... 

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

Oxidize methionine and cysteine to methionine sulfone and cysteic acid using performic acid prior to acid hydrolysis. 

Incomplete oxidation can be a problem. Higher recoveries of cysteine and cystine have been achieved by reduction of those amino acids with 2-mercaptoethanol followed by incubation with 4-vinylpyridine. This converts cysteine and cystine to S-(4-pyridylethyl)-L-cysteine, a derivative that can be separated by ion-exchange chromatography. Performic acid oxidation of methionine in the presence of phenol is a suitable method for analysis of cysteine. 

The two preparations from A. oryzae reportedly differ in amino acid and carbohydrate composition. The enzyme prepared by Minato contained 25% carbohydrate no cysteine was detected either by titration with p-mercuribenzoate in 6M urea or by cysteic acid analysis after performic acid oxidation (179). In contrast, Wolfenden et al. (92) reported 14 cysteine residues per mole of enzyme which reacted instantaneously with p-mercuribenzoate in the absence of urea. No explanation is available for this apparent discrepancy. 

The analysis of methionine and cysteine is problematic. The sulfur containing side chains of these amino acids are prone to oxidation. The standard hydrochloric acid hydrolysis will cause the partial conversion of these amino acids into cystine, cysteine, cysteine sulfinic acid, cysteic acid, methionine, methionine sulfoxide, and methionine sulfone. The classic strategy (79) for dealing with this problem is simply to drive the oxidative process to completion (i.e., convert all the cyst(e)ine to cysteic acid) and then to analyze chromatographically for cysteic acid and/or methionine sulfone. This is traditionally accomplished by a prehydrolysis treatment of the sample with performic acid. While this method has sufficed over the years, the typical recovery (85 -90%) and precision (4% intra- and 15% interlaboratory) have been poor (80). 

More recently, there have been numerous collaborative studies (81-84) that have attempted to improve the accuracy and precision of this method. A typical example by MacDonald et al. (81) reported a collaborative study by seven laboratories. Samples were oxidized with performic acid for 16 hours over ice bath. After oxidation, HBr was used to destroy excess performic acid. Samples were then roto-evaporated to dryness, dissolved in 6N HC1, nitrogen purged, and then hydrolysed for 18 hours at 100°C. Interlaboratory precision for cysteic acid determination in six food ingredients ranged from 7 to 10%. For methionine sulfone, interlaboratory precisions ranged from 1 to 13% for the same six food ingredients. The mean recovery of cysteine was 95% and of... 

An interesting study (85) explores the use of sodium azide as an oxidative agent instead of performic acid. The big advantage offered here is that the oxidation of cysteine to cysteic acid is effected concurrent with the hydrochloric acid hydrolysis. The authors claim that the presence of 0.2% (w/v) NaN3 in the HC1 digestion does not represent an explosion risk. Recoveries of cysteine as cysteic acid were typically —90% for pure proteins. 

Finally, it is very common for methionine to be determined as part of the standard hydrochloric acid hydrolysis. Indeed, methionine is not nearly as labile to oxidation as cysteine is. While this is appropriate for many samples, there are studies (27,91) that indicate that seriously flawed recoveries (10-40% low) may result from methionine determination by standard acid hydrolysis if the samples contain high levels of carbohydrate. For these sorts of samples, it is recommended that determination of methionine as methionine sulfone (by performic acid oxidation) be pursued. 

Oxidation of Cysteines. Treatment of a protein with performic acid cleaved all the disulfide bonds and converted all Cys residues to cysteic acid residues (Fig. 3-26). 

A final problem is to determine which cysteines are connected by disulfide links. Usually, the disulfide bonds are broken by either oxidative cleavage with performic acid or reductive cleavage with 2-mercaptoethanol before initial sequencing is undertaken ... 

Temperature is another parameter that must be dealt with carefully since it, too, may affect the microenvironment of reactive groups. Some competitive side reactions may be minimized or prevented by thoughtful choices of temperature. Performic acid oxidations of proteins generally are done at low temperatures — 10°C) to limit reaction to cysteines,... 

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. 

These 3 sulfur-containing amino acids and their derivatives are susceptible to oxidation and other destructive reactions. Even when great care has been taken to remove all oxygen from hydrolysis tubes, considerable losses of cysteine and cystine are found after acid hydrolysis, and this usually prevents direct quantitation of these amino acids in proteins. However, total cysteine plus half-cystine content may be determined as cysteic acid after performic acid... 

After oxidation of unmodified proteins with performic acid ( 3.8.1) the methionine will all be present as methionine sulfone, and the cysteine and cystine will be present as cysteic acid. The method for oxidizing proteins with performic acid on a preparative scale ( 3.8.1) has been modified for analytical studies (Moore 1963), to allow rapid destruction of excess performic acid. However, tryptophan is still destroyed by the performic acid in this modified procedure, and quantitation of tyrosine may be complicated by the formation of halogenated derivatives ( 2.2.3). 

Cysteic acid is the major product of performic acid oxidation of cysteine and cystine in proteins, and is usually produced in yields of more than 90%. Cysteic acid is not retarded by the resins generally used in amino acid analyzers and therefore elutes at the breakthrough volume (about 42% of the elution volume of aspartic acid). However, since other substances such as O-phosphoserine can also elute in this position, it is essential to analyze hydrolysates made both before and... 

Modification by performic acid oxidation Treatment of proteins with performic acid leads to the oxidation of cysteine and cystine residues to cysteic acid residues (Sanger 1949). Methionine residues are quantitatively converted to the sulfone (Hirs 1956), and tryptophan undergoes oxidative destruction (Toennies and Homiller 1942 Benassi et al. 1965). Other amino acids are not modified, provided that precautions are taken to avoid chlorination (Thompson 1954 Hirs 1956), or bromination (Sanger and Thompson 1963) of tyrosine residues. Cleavage of peptide bonds does not occur on performic acid oxidation at low temperature. 

The procedure described by Moore (1963) is the method of choice when performic acid oxidation is employed for the quantitative determination of the half-cystine plus cysteine content of proteins. This procedure is described in detail in 2.5.1. The yield of cysteic acid is 94 2% and that of methionine sulfone 100 +2%. 

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

The protein is completely hydrolyzed by acid (6 N HCl, 24 hours or longer at 110°C, under vacuum or inert gas) to its constituent amino acids and the resultant hydrolysate is evaporated to dryness. The amino acid composition is determined on protein hydrolysates obtained after 24,48, and 72 hours of acid treatment. The content of amino acids with bulky aliphatic side chains such as isoleucine, leucine, and valine, which undergo slow hydrolysis, is calculated from an extrapolation of the hydrolysate data to infinite time. The content of hydroxyl-containing amino acids, which are slowly destroyed during hydrolysis, is obtained by a corresponding extrapolation to zero time. Since cysteine, cystine, and methionine residues are somewhat unstable to hydrolysis, these residues are oxidized to cysteic acid and methionine sulfone, respectively, with performic acid before quantitative analysis. Cysteine, or half-cystine, is quantitated as a derivative such as carboxymethyl cysteine after reduction and alkylation, a necessary prerequisite to subsequent sequence analysis. Tryptophan... 

The molar ratios of the amino-acid residues were determined after erythrocuprein was subjected to acid hydrolysis at 110°C for 20 to 96 hours. The method of Moore and Stein was used throughout (88, 89). The sulfhydryl content of human erythrocuprein was determined using the spectrophotometric assay of Boyer (90). Total half-cystine was analysed as S-carboxy methyl cysteine in acid hydrolysates (91) of reduced and alkylated erythrocuprein, or as cysteic acid in acid hydrolysates oxidized with performic acid. The data are summarized in Table 2. 

On-Probe Oxidation In this procedure, thiol- and disulfide-containing peptides are treated with performic acid on the FAB probe to convert each cysteine residue to cysteic acid a concomitant increase of 98 Da in the mass of a peptide that contains an intramolecular disulfide bond results [9] ... 

In the case of amino acid analysis, the quantification of cysteine can be difficult because it is oxidized to cystine during acid hydrolysis. To circumvent this problem, cysteine can be oxidized to cysteic acid with performic acid prior to analysis. Alternatively, cysteine can be converted to the pyridyl ethyl derivative and subsequently detected by postcolumn reaction with ninhydrin. Still another method involves the production of carboxymethyl cysteine following alkylation with iodoacetic acid. All of these cysteine derivatives can be separated by either reversed-phase precolumn or ion-exchange postcoT umn methods. 

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