A method for the reduction of methionine sulphoxide to methionine using titanium trichloride is described. This method, coupled with the gas chromatographic determination of rnethionine after reaction with CNBr, measures methionine sulphoxide by the increase in methionine values after reduction with titanium trichloride. Results obtained for total methionine by gas chromatography after reduction and CNBr reaction, showed close agreement with those obtained by ion-exchange chromatography after performic acid oxidation. This method was applied to pure proteins which had been subjected to hydrogen peroxide oxidation, and to a wide range of food proteins. An evaluation of other, but less effective, reducing agents is briefly discussed.
Peptide-bound methionine when reacted with CNBr releases methyl thiocyanate which is quantitatively measured by gas-liquid chromatography. Seeds of varieties of legume contain y-glutamyl-S-methylcysteine, which also reacts with CNBr to give methyl thiocyanate, which results in elevated apparent methionine values. An extraction procedure for its removal was developed and applied to many legume varieties. Methionine values agreed with those obtained by ion-exchange chromatography after performic acid oxidation; an exception was Phaseolus mungo (black gram). Samples of Ph. rnungo from several sources all contained substantial amounts of free methionine but, as no y-glutamyl-S-methylcysteine was present, the need for extraction was eliminated. For the determination of methionine in cereals, conditions were adapted to decrease gel formation in the presence of formic acid and CNBr, and to eliminate interference at the gas chromatography stage. This was achieved by a mild acid hydrolysis prior to CNBr reaction, conditions for which are described.
Intact methionine residues in food proteins, including a limited number of fish meals, have been determined using gas chromatography to measure methyl thiocyanate released after CNBr reaction. However, methionine values for certain fish products determined by that method have been found to be low and variable compared with those determined by ion-exchange chromatography after performic acid oxidation. This was traced to the presence of a component of many fish meals that, when reacted with CNBr, gave a product which co-eluted with the internal standard, ethyl thiocyanate. A method was developed to eliminate this interference and allow methionine determinations to be carried out using the same gas chromatographic procedure.
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