Performic acid methionine oxidation

Methionine sulfone is a product of oxidation of proteins with performic acid ( 2.5.1) or other strong oxidizing agents. Milder oxidation of methionine gives the sulfoxide derivatives ( 2.5.9.), which are also converted to the sulfone by performic acid. In general, quantitative yields of methionine sulfone are obtained by oxidation with performic acid. Methionine sulfone is stable to acid hydrolysis and elutes from most analyzer columns immediately after aspartic acid. Since the elution position of methionine sulfone is more sensitive to temperature changes than that of aspartic acid, these two amino acids may not be well resolved in some systems. However, temperature adjustments of the columns usually allow better separations to be obtained with the... 

Partially oxidized cystine residues are intermediates in the reaction. Some hydrolysis of peptide bonds occurs during oxidation with peracetic acid, less with performic acid. Methionine and tryptophan residues are also oxidized. Oxidized wool is soluble in alkaline solutions. When these solutions of dissolved, oxidized wool proteins are acidified, a low-sulfur protein, a-keratose, is precipitated. A high-sulfur protein, -y-keratose, remains in solution. (P-keratose is the residue of oxidized wool that is not soluble in alkali.)... 

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. 

Methionine and half-cystine were determined in duplicate as methionine sulfone and cysteic acid on the performic acid-oxidized protein [8. Moore, JBC 238, 235 (1963)). 

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... 

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. 

M Spindler, R Stadler, H Tanner. Amino acid analysis of feedstuffs determination of methionine and cystine after oxidation with performic acid and hydrolysis. J Agric Food Chem 32 1366-1371, 1984. 

If appropriate precautions have been taken in the preparation of a protein, and if oxygen is completely removed before hydrolysis, methionine will usually be recovered from acid hydrolysates in yields greater than 95 %. However, in some proteins (particularly those that are chemically modified) and in many peptides the methionine may be at least partially oxidized to the sulfoxide or sulfone forms, and even though these may be analyzed with amino acid analyzers (see below), the total yield of methionine (and oxidized products) is usually somewhat low. A good check on total methionine content in a peptide or protein is obtained by analyzing for methionine sulfone after performic acid oxidation, since methionine and its sulfoxides are quantitatively converted to the sulfone by this procedure. 

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). 

The method described above gives direct analysis of methionine sulfoxide content in proteins. Another method makes use of carboxy-methylation of methionine at acid pH to give the carboxymethyl-sulfonium derivative ( 3.5). Methionine sulfoxide, which is not affected by the carboxymethylation reaction, is then oxidized to the sulfone which is stable to acid hydrolysis and can easily be quantitated. This is possible because methionine carboxymethyl-sulfonium salts are not affected by performic acid oxidation, although they are degraded by acid hydrolysis. Therefore, the methionine sulfone content is equal to the methionine sulfoxide content plus any sulfone that may have been initially present (shown by analysis before oxidation). 

Methionine carboxymethylsulfonium salts These derivatives of methionine (isomers) are prepared by treating proteins with iodoacetic acid the reaction is most specific for methionine at acid pH ( 3.5). These derivatives are not affected by performic acid oxidation (see under methionine sulfoxide), but are degraded by acid hydrolysis to give methionine, carboxymethyl-homocysteine, homoserine and homoserine lactone (Gundlach et al. 1959). 

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%. 

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... 

Amino acids Acid hydrolysis (HCI 6N) followed by chromatography. There are many methods with variants in duration (24-48 hours) and temperature (110-I45°C). Methionine and cystine are obtained after performic acid oxidation and tryptophan after alkaline hydrolysis. 

Strong oxidizing agents such as performic acid convert methionine to the acid-stable sulfone. As a result, procedures that generate cysteic acid also produce this derivative, which serves as an independent check on the methionine content of a sample. 

One drawback of this procedure is that methionine and tryptophan residues are also prone to oxidation by performic acid. 

Fig. 207. One-dimensional separation in a chamber for continuous development [13] (see p. 76) for detection of leucine and isoleucine in the presence of 18 protein amino acids + j -alanine + y-amino-n-butjrric acid 0.5 Xg of each amino acid applied in a total of 0.5 xl O.IN hydrochloric acid 4.6 h nm detection with ninhydrin oxidation with performic acid [44] is necessary if methionine is present. Identification of leucine and isoleucine is unambiguous if a standard sample of each is chromatographed in a parallel run on the same layer...

Methodfor sulfur-containing amino acids. Moore (12) determined cysteine and cystine as cysteic acid by performic acid oxidation. However, methionine can also be determined as methionine S,S-dioxide. 

---