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The bovine milk lipocalin, beta-Lactoglobulin (beta-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC10PE) to beta-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, KB, was (1.2+/-0.2)x10(6) M(-1) at 25 degrees C, pH 7.4 and I=0.15 M. Dependence of KB on pH and on the monomer-dimer equilibrium of beta-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the beta-barrel in the protein. The monomer-dimer equilibrium of beta-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, KD, was (7.0+/-1.5)x10(5) M(-1) at 25 degrees C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC10PE between beta-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. beta-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.

A novel method is described to demonstrate inaccessibility to the bulk aqueous phase of the microinterface between pig pancreatic phospholipase A2 and lipid bilayers to which this protein is bound. The method is based on the fact that the fluorescence emission quantum yields of the tryptophan residue of the protein and of a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore attached to a lipid are lower in water as compared to that in deuterated water. The fluorescence emission quantum yield of these chromophores is measured in water and in deuterated water under conditions where the protein is either bound or not bound to the surface of a lipid bilayer containing the dansyl chromophore. Under conditions where the protein is tightly bound to the surface of the bilayer, desolvation of both fluorophores abolishes the observed effect of deuterated water. The tryptophan residue in the bound phospholipase A2 also becomes inaccessible to fluorescence quenching by acrylamide or succinimide. Desolvation of the microinterface is observed only under conditions that are significant for the catalytic action of phospholipase A2 in the scooting mode and not in the hopping mode. Also, under similar conditions, binding of pro-phospholipase A2 to anionic vesicles does not cause dehydration of the microinterface. The mechanistic significance of these observations for lipid-protein interactions, in general, and for interfacial catalysis and interfacial activation, in particular, is discussed.

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37 degrees C to 570 cP at 5 degrees C, as reported for di(1-pyrenyl(propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37 degrees C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40 degrees C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol (epsilon - 13.3) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol (epsilon = 17.5), whereas a polarity comparable to that of methanol (epsilon = 32.7) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.

The influence of sucrose, between 0 and 70% in the aqueous phase, upon multilamellar liposomes of dimyristoylphosphatidylcholine was examined by differential scanning calorimetry and X-ray diffraction analysis. Increasing concentrations of sucrose increase the temperatures of both the main transition and the pretransition of the lipid. The effect is greater on the pretransition than on the main transition. At 35 degrees C the interlamellar spacing in the multilamellar liposomes is reduced by increasing sucrose concentration in the aqueous phase and no significant effects are seen in the chain lattice of the bilayers. This result is interpreted as a dehydrating effect of sucrose upon the bilayer-water system at 35 degrees C. At 5 degrees C the interlamellar spacing is increased and this increase is, at high (70%) sucrose concentrations, attributable to an untilting of the lipid acyl chains with no change in the thickness of the aqueous layers in the multilamellae.

The interaction of La3+ with phosphatidylserine vesicles is elucidated by binding studies, differential scanning calorimetry, X-ray diffraction, freeze fracture electron microscopy, and release of vesicle contents. La3+ effectively competes with Ca2+ for phosphatidylserine binding sites. The saturation level is close to a La/lipid ratio of 1:3. A concentration of 0.1 mM of La3+ is sufficient to induce fusion between sonicated vesicles.

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.

5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.