title: Thermostabilization of ovalbumin by an alkaline treatment: examination for the possible implications of an altered serpin loop structure.
abstract: Ovalbumin, a member of the serpin superfamily, is transformed via an intermediate state into a non-cleaved, thermostabilized form (S-ovalbumin) during either the storage of unfertilized eggs or development of fertilized eggs; essentially the same thermostabilization also occurs upon in vitro incubation of isolated ovalubumin under alkaline conditions. To investigate the implications of a partial insertion of the alpha-helical serpin loop into beta-sheet A that has been proposed as a conformational mechanism for S-ovalbumin production, we examined the thermostabilization process of ovalbumin with different loop structures. When the thermostabilization processes were compared for the intact, P1-P1'-cleaved and P1-P1'/P8-P7-cleaved forms of egg white ovalbumin, both the rates for the conversion from the native to intermediate and from the intermediate to S-ovalbumin were almost indistinguishable among the three protein forms. Furthermore, the fully loop-inserted form of recombinant ovalbumin mutant R339T that had been thermostabilized by P1-P1' cleavage with Tm values from 72 to 88 degrees C was further thermostabilized by an alkaline treatment, yielding a final product (loop inserted S-ovalbumin) with a Tm value of 93 degrees C. No significant difference was found between native ovalbumin and S-ovalbumin in respect of the rate of proteolytic cleavage of the loop by elastase and subtilisin. These data strongly suggest that S-ovalbumin is produced by a mechanism other than that of the partial loop insertion model.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamasaki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Arii
authors.forename: Masaaki
authors.initials: M
authors.lastname: Hirose
mesh_terms: D000468:Alkalies / Q000737:chemistry; D000818:Animals; D002645:Chickens; D007700:Kinetics; D008956:Models, Chemical; D010047:Ovalbumin / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015843:Serpins / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000468:Alkalies; C104026:S-ovalbumin; D015843:Serpins; D010047:Ovalbumin; D010447:Peptide Hydrolases