Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).
|Titolo:||pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B|
|Parole Chiave:||FATTY-ACID-BINDING ; SODIUM DODECYL-SULFATE ; GEL-ELECTROPHORESIS ; TANFORD TRANSITION ; ESCHERICHIA-COLI ; PALMITIC ACID ; C-13 NMR ; GRADIENT ; RETINOL ; UREA|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore CHIM/03 - Chimica Generale e Inorganica
|Data di pubblicazione:||2002|
|Digital Object Identifier (DOI):||10.1021/bi020493f|
|Appare nelle tipologie:||01 - Articolo su periodico|