Abstract
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.
Highlights
Many milk protein components, the whey proteins and caseins, are known to be lipophilic under certain conditions
Intrinsic fluorescence excitation for ␣-LA was fixed at 280 nm, where, despite the fact that ␣-LA contains four tyrosines and four tryptophans, the Tyr contribution to total protein fluorescence is negligible [18], and excitation for Acrylodated intestinal fatty acid binding (ADIFAB) protein was fixed at 390 nm
We were unable to measure an accurate dissociation constant for 5-doxylstearic acid (5-DSA) binding to Ca2ϩ-loaded ␣-lactalbumin (Ca2ϩ)-␣-LA since the concentrations of either spin probe or protein required to observe any significant binding by ESR were essentially experimentally impossible
Summary
The whey proteins and caseins, are known to be lipophilic under certain conditions. § On leave from the Institute of Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia Another milk protein with potential roles in lipid transport is ␣-lactalbumin (␣-LA), an acidic whey protein of Mr Ϸ 14,200, which is expressed during lactogenesis in the mammary gland. Significant association between ␣-LA and fatty acid had not been observed with these lipids to date [3] This is perplexing because significant quantities of ␣-LA were found to be associated with milk fat globule membranes, suggesting a possible role for this protein in fat digestion or transport. ␣-LA and -lactoglobulin are structurally distinct, as ␣-LAs possess homologies to c-type lysozymes [14, 15] Another significant difference is that ␣-LA has a strong Ca2ϩ-binding site, which competitively binds Naϩ, Kϩ, Mg2ϩ (16 –19), and several distinct Zn2ϩ-binding sites [20, 21]. The present work examines the association of ␣lactalbumin with fatty acid spin-labeled analogs by several physical methods
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