Interactions of alpha-lactalbumin with fatty acids and spin label analogs.
Bovine alpha-lactalbumin (alpha-LA) has been shown by intrinsic protein fluorescence and electron spin resonance methods to interact with the spin-labeled fatty acid analog, 5-doxylstearic acid, as well as stearic acid. An intrinsic fluorescence titration of various alpha-LA forms with 5-doxylstearic acid causes first an increase and then a decrease in emission intensity with concomitant shifts in tryptophan emission wavelength. In some cases, up to three steps in the fluorescence titration curves were visible, which were fit to apparent binding steps from 10(-6) to 10(-4) M. The binding parameters of 5-doxylstearic acid for apo- and Ca2+-alpha-LA were an order of magnitude different from one another; the stronger one, apo-alpha-lactalbumin, exhibited a Kd of 35 microM. Electron spin resonance titrations of 5-doxylstearic acid-loaded apo-alpha-LA with stearate (micelles) seem to suggest separate binding loci if alpha-LA indeed binds stearate at these concentrations. The titration of alpha-LA by stearic acid results in a fluorescence emission red shift and an apparent stepped increase in fluorescence intensity. Lipid-protein association occurred at concentrations at which stearic acid micelles and aggregates begin to form in the absence of protein. Nonetheless, the relatively strong association between stearic acid and apo-alpha-LA was also confirmed by means of the fluorescent indicator acrylodated fatty acid binding protein, in which addition of alpha-LA to the stearate-loaded indicator protein reverses the decrease in fluorescence of the acrylodan chromophore conjugated to the protein.
Cawthern
K M
KM
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Narayan
M
M
Chaudhuri
D
D
Permyakov
E A
EA
Berliner
L J
LJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000378:metabolism*; D007768:Lactalbumin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D015032:Zinc / Q000378:metabolism
D005227:Fatty Acids; D013113:Spin Labels; D007768:Lactalbumin; D015032:Zinc; D002118:Calcium