HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sakurai, K. Articles by Goto, Y. Search for Related Content PubMed PubMed Citation Articles by Sakurai, K. Articles by Goto, Y. Social Bookmarking                   What's this?

Salt-dependent monomer–dimer equilibrium of bovine ß-lactoglobulin at pH 3

¹ Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan
² Faculty of Science and Technology, Meijo University, 1-501, Shiogamaguchi, Tenpaku-ku, Nagoya 468-8502, Japan

Reprint requests to: Yuji Goto, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan; e-mail: ygoto{at}protein.osaka-u.ac.jp; fax: 81-6-6879-8616.

Although bovine ß-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer–dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO₄ was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO₄, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (H_D) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric ß-lactoglobulin at pH 3.0 with NaClO₄. The net heat effects after subtraction of the heat of salt dilution, corresponding to H_D, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the H_D value at 20°C and the heat capacity change (C_p) of dimer formation. In both NaCl and NaClO₄, the obtained C_p value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed C_p values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer–dimer equilibrium of ß-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Keywords: ß-Lactoglobulin; monomer; dimer equilibrium; ultracentrifuge; heat capacity change

Abbreviations: ASA, accessible surface area • ß-LG, ß-lactoglobulin • CD, circular dichroism • Cp, heat capacity change of dimer formation • Cp,h, hydration heat capacity change of dimer formation • GD, Gibbs free energy change of dimer formation • HD, enthalpy change of dimer formation • SD, entropy change of dimer formation • Gdn-HCl, guanidine hydrochloride • HSQC, heteronuclear single quantum coherence • ITC, isothermal titration calorimeter • KD, dimerization constant • NMR, nuclear magnetic resonance

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?