Glycinin consists of five kinds of subunits, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). cDNAs for individual subunits were cloned by reverse transcription-polymerase chain reaction method and expressed in Escherichia coli using pET vector. The recombinant proglycinins were purified by ammonium sulfate fractionation and column chromatography in the form of homotrimers. Physicochemical properties such as molecular dimensions, solubility, surface hydrophobicity, thermal stability, and emulsifying ability of individual proglycinins were studied. Molecular dimensions were proportional to molecular size for all proglycinins except A2B1a. Solubility was intrinsic to each proglycinin. At the ionic strength of 0.5, all proglycinins except A1aB1b showed a very low solubility at acidic pH, but A1aB1b was soluble to higher than 60%. At ionic strength 0.08, all proglycinins exhibited isoelectric precipitation, although A2B1a and A1bB2 were not completely insoluble. The order of emulsifying ability (A1bB2 < A2B1a < A5A4B3 < A3B4 < or = A1aB1b) was not of the same for surface hydrophobicity (A5A4B3 < A1aB1b < or = A3B4 < A1bB2 < A2B1a) and thermal stability (A1bB2 << A2B1a < or = A5A4B3 < A3B4 < or = A1aB1b).

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1 bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly 1 -Ala 352 ) and light (Ser 353 -Pro 385 ) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T m ‫؍‬ 68.4°C, whereas cl-R339T refolded into the relaxed form with T m ‫؍‬ 85.5°C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure. The serpins2 are believed to fold in vivo into a metastable "stressed form" as they undergo a conformational change to the thermostable "relaxed form" by insertion of the reactive center loop (RCL) between strands 3A and 5A (see Fig. 1) (1-3). The metastability of the native serpin is essential for the inhibition of serine proteinases, although this molecular flexibility also results in aberrant intermolecular linkage and polymerization that bring about conformational diseases such as dementia (4, 5), hepatocirrhosis (6), angioedema (7), and emphysema (8, 9). Therefore a central target of serpin studies is how the serpin polypeptide can fold into a metastable structure avoiding thermodynamically more stable conformations. Several serpin folding models have been suggested by various unfolding experiments, whereas refolding experiments from a fully unfolded state are limited (10 -14) because of serpin susceptibility to misfolding and aggregation.Ovalbumin is a major component of egg white proteins, and although it is not an apparent protease inhibitor its membership of the serpin family of protease inhibitors is clear from the close similarity of its primary and tertiary structure (15, 16). We have elucidated its refolding process from a fully denatured state as (10, 17, 18)

Introduction of the extension region of beta-conglycinin alpha' subunit at the C-terminus of proglycinin A1aB1b results in the improvement of its emulsifying properties. To understand the basic for such improvement, we introduced the alpha' and alpha extension regions to the A2B1a C-terminus, and the alpha extension and A5A4B3 hypervariable regions, and an oligopeptide composed of 20 negatively or positively charged residues to the A1aB1b C-terminus, creating A2B1aalpha', A2B1aalpha, and A1aB1balpha, A1aB1bA4IV, A1aB1bNeg and A1aB1bPos, respectively. All the modified versions were produced in Escherichia coli. Their molecular size, thermal stability, surface hydrophobicity, solubility and emulsifying ability were studied. Analyses of molecular size and thermal stability suggested that all the modified versions formed the proper conformation similar to that of the wild type (WT). Solubility was intrinsic to each mutant. At ionic strength 0.5, the emulsifying abilities of all mutants were better than that of the WT except A1aB1bPos and A1aB1bNeg, and at ionic strength 0.08, all mutants especially A1aB1bPos exhibited better emulsifying ability than did the WT. The order of stability of the emulsion at both ionic strengths (0.08 and 0.5) was A1aB1balpha >or= A2B1aalpha > A1aB1balpha' >or= A2B1aalpha' >> A1aB1bPos > A1aB1bA4IV >or= A1aB1bNeg > A1aB1b, A2B1a. These results indicate that the emulsion stability of proglycinin mutants depends on length and hydropathy profile of the polypeptides added to the C-terminus of proglycinin.