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Glycinin, one of the dominant storage proteins of soybeanseeds, has two disulfide bonds in eachconstituent subunit: Cys12-Cys45 and Cys88-Cys298 in the proglycinin Ala& subunit. To examine the effects of disrupting disulfide bonds on the formation and maintenance of structure and on the functional properties of proglycinin, we replaced the cysteine residues (Cysl2 and Cys88) by oligonucleotidedirected mutagenesis, giving mutant proglycinins Gly12, Ser88, and Gly12Ser88. The mutant proglycinins overproduced in Escherichia coli cells accumulated as soluble proteins and self-assembled into trimers like the native proglycinin. The functional properties of proglycinins Gly12 and Ser88 purified to near homogeneity were examined as models of modified glycinins. Proglycinin Ser88 formed a harder gel than native glycinin and unmodified expressed proglycinin even a t the protein concentrations at which native glycinin did not form a hard gel. On the other hand, proglycinin Gly12 formed a gel only at higher protein concentrations (>6% 1, the hardness of which was similar to that of the native glycinin. Both proglycinins Gly12 and Ser88 exhibited emulsifying activity similar to that of unmodified expressed proglycinin. These results suggest that the number and topology of free sulfhydryl residues are closely related to the heat-induced gel-forming ability and the gel properties of glycinin but not to its emulsification.