Cereal Chem 65:248-252   |  VIEW ARTICLE

Characterization of Acetic Acid-Soluble and Insoluble Proteins Isolated from Doughs Mixed in the Presence of N-Ethylmaleimide.

K. Okada, Y. Negishi, and S. Nagao. Copyright 1988 by the American Association of Cereal Chemists, Inc. 

Acetic acid-soluble and insoluble proteins were isolated from doughs mixed in the presence of N- ethylmaleimide (NEMI). Reduced-pyridylethylated acetic acid-insoluble protein (AF) and reduced- pyridylethylated acetic acid-soluble protein (BF) were sequentially fractionated into an acetic acid-insoluble fraction (F-0), an acidic 70% ethanol-insoluble fraction (F-1), a neutral 70% ethanol-insoluble fraction (F- II), and a neutral 70% ethanol-soluble fraction (F-III). Only about 30% of AF dissolved in 0.1N acetic acid solution, whereas BF dissolved completely. The AF-I fraction was recovered in trace amounts and could not be characterized. The fractions AF-0, AF-II, and AF-III contained higher contents of aspartic acid, alanine, lysine, and arginine and lower contents of glutamic acid and proline than the BF fractions (BF-I, BF-II, and BF-III). The AF and BF fractions differed in reversed-phase high-performance liquid chromatography (RP- HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) subunit patterns. Subunits of AF-0 eluted later by RP-HPLC than those of other fractions, indicating that they have higher surface hydrophobicities. Distribution of S-succinyl-L-cysteine in the protein fractions showed that NEMI reacted with AF-O and BF-I more than with the other protein fractions during dough mixing. S-succinyl-L- cysteine was present at similar high levels in peaks separated by RP-HPLC from AF-O, whereas it was present in variable amounts in peaks separated by RP-HPLC from the other protein fractions. We speculate that dough breakdown in the presence of NEMI is caused by partial depolymerization of glutenin and changes in protein surface hydrophobicities, mainly in AF-O and BF-I, induced by modification of the key disulfide bonds by NEMI. RP-HPLC, SDS-PAGE, and amino acid analysis showed that acetic acid- insoluble protein is composed of different subunits than acetic acid-soluble protein.