The isolation of a toxic substance from agenized wheat flour / by P.N. Campbell and T.S. Work and E. Mellanby.

  • Campbell, P. N. (Peter Nelson)
Date:
[1951?]
Catalogue details

Licence: In copyright

Credit: The isolation of a toxic substance from agenized wheat flour / by P.N. Campbell and T.S. Work and E. Mellanby. Source: Wellcome Collection.

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    mixture of unknown substance and y-aminobutyric acid separated into two ninhydrin-positive spots; the mixture of unknown and a-aminobutyric acid could not be separated. Methionine gave a-aminobutyric acid under similar condi¬ tions. Fig. 1. Phase-contrast photograph of the crystalline toxic substance from agenized wheat flour ( x 180). Phenol/NH] Toxic substance (^) Homocysteic acid BuOH/ HOAc/H2Q Methionine sulphoxide O / Homoserine- opo Methionine sulphone O a -Aminobutyric acid Fig. 2. Diagrammatic representation of the position of the toxic substance and its degradation products (acid hydrolysis) in a two-dimensional chromatogram. Treatment of some methionine peptides with NC13 The following peptides were treated with NC13 in CHC13 : methiony lgly cine, carbobenzyloxymethionylmethionine amide, methionylmethionine, carbobenzyloxymethionyl- methionine, carbobenzyloxy-L-methionine amide, carbo- benzyloxyglycyl-L-methionine and carbobenzyloxy-L-meth- ionylglycine. A sample (20 mg.) of each was suspended in approximately 10-15 ml. of CHC13. Air containing NC13 from approximately 50 ml. of Cl2 was passed into each. The solutions were left at room temperature for about 1 hr. when the remaining NC13 was removed by sucking a vigorous stream of air through the solutions until no further CHC13 remained. Each specimen was dissolved in 6n-HC1 (0-2- 0-3 ml.) and heated in a sealed tube for 11 hr. at 100-105°. The amino-acid composition of each hydrolysate was studied by two-dimensional paper-partition chromato-j graphy using butanol acetic acid and phenol/NH3as solvents.! Wherever a component was detected which resembled the , toxic factor in RF values a second chromatogram was run with a mixture of peptide hydrolysate and crystalline toxic > substance. In every case it was possible to separate the, toxic substance from ail the peptide degradation products; and it was, therefore, concluded that in no case had the NClJj treatment of a peptide given rise to the toxic substance. RESULTS AND DISCUSSION Isolation In order to isolate the toxic substance from wheat flour it was obviously desirable to obtain a flour possessing the maximum activity. As a result of many experiments it was concluded that it was not) practicable to treat wheat flour with more than about; ten times the amount of nitrogen trichloride which was normally used commercially. In the later stages; of the work commercial gluten itself was treated with nitrogen trichloride, since the toxic substance; is associated with the gluten fraction of the flour, bub the product was only slightly more active than thaii 1 obtained when the flour itself was treated wittf nitrogen trichloride. The digestion of agenized; gluten with pepsin .and trypsin resulted in a very; considerable reduction of the average moleculail weight, for about 80 % of the nitrogen was dialysable: at this stage. Since on a nitrogen basis the non! dialysable material was as toxic as the dialysable ii it was apparent that the toxic substance was ari integral part of the protein and was not a low-mole cular-weight impurity. Although it is possible that the non-dialysabki residue from the enzymic digestion could have beer * satisfactorily hydrolysed by acid, it was felt that the; elimination of possibly ‘resistant’ non-dialysable; peptides at this stage was an advantage. The acid hydrolysis caused a destruction of aboui j 30% of the toxic substance (see Table 1), but thel proportion of amino nitrogen to total nitrogen wasi raised from 25% to approximately 60%; thus m further substantial reduction in the average mole!; cular weight was achieved. It was felt that fraction: ation would be so simplified by this reduction ir! molecular weight as to make an acid hydrolysis ar; essential step in spite of the ensuing loss of activity ) Electrodialysis achieved a very considerabb I purification of the toxic substance with negligible|i loss. Table 1 shows that the toxic dose containeci 2300 mg. nitrogen before electrodialysis and 500 mgi nitrogen after electrodialysis and removal o crystalline material from the neutral dialysatef About 36% of the total amino-acids in hydrolysecf gluten consist of glutamic acid. Thus most of the! nitrogen in the anode compartment was glutamid acid. The basic amino-acids, arginine, lysine am 1