INTERACTIONS OF ALPHA-LACTALBUMINS WITH LIPID VESICLES STUDIED BY TRYPTOPHAN FLUORESCENCE

Complexes of alpha-lactalbumin (alpha-LA)(1) with dimyristoylphosphati dylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes a t pH 8 and at pH 2 have been obtained by means of gel filtration. Ther mal denaturation of alpha-LA complexes of DMPC or DPPC at pH 8 was fou nd to depend on the saturation of protein by metal cations. The intrin sic fluorescence of DMPC-alpha-LA and DPPC-alpha-LA was sensitive to t wo thermal transitions. The first transition corresponded to the T-c o f the lipid vesicles, while the-second transition arose from the denat uration of the protein. Fluorescence spectrum position suggested that at low temperature tryptophan accessibility increases upon protein-DMP C or protein-DPPC association. At temperatures above the protein trans ition (70 degrees C) tryptophan appears to interact significantly with the apolar phase of DMPC and DPPC, evidenced by spectral blue shifts. Whereas the free protein at pH 2 adopts the molten globule (MG) state and is characterized by the absence of a thermal transition, the rapi dly-isolated DMPC-alpha-LA complex was characterized by the appearance of a distinct fluorescence thermal transition between 50 and 60 degre es C. This result is consistent with a model of a partially-inserted f orm of alpha-LA which may possess some degree of tertiary structure an d therefore unfolds cooperatively.