Ignace Hanssens

Simple two-step procedure for the preparation of highly active pure equine milk lysozyme

A fast, simple two-step purification scheme is presented for the isolation of lysozyme at a high yield from equine milk. In the first step, fluidized bed technology, using the Streamline system, was exploited. In the following step, advantage was taken of Ca(2+)-induced conformational changes to obtain a pure, high specific activity, enzyme fraction by hydrophobic interaction chromatography.

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

alpha-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid. The enthalpy changes associated with the interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (delta H) are not only larger than at physiological pH, but also show a maximum at aobut 23 degrees C in the delta H vs. temperature graph. At pH 6 and 7, on the contrary, delta H increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of alpha-lactalbumin, while at pH 4 and 5 at 23 degrees C an extra peak appears in the gel-filtration graphs between the pure vesicles and alpha-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23 degrees C and a blue shift at 37 degrees C, both to 336 nm, are observed for lambda max of the fluorescence emission spectra at pH 4 when alpha-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure alpha-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs. The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles: II. A fluorescence polarization study

The interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles was studied as a function of temperature, pH and the molar ratio of phospholipid to protein. The method consisted of measuring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene used as a probe embedded in the vesicles. After incubation of the protein with the phospholipid for 2 h at 23 degrees C, the polarization of the light emitted by this probe shifted to higher values; the shift was greater at acidic pH than at neutral pH. After incubation at 37 degrees C, no shift in polarization was found at pH 7, 6 and 5 while a strong increase occurred at pH 4. Lowering the temperature, after incubation at 37 degrees C, had little effect on the polarization at neutral pH. At pH 5, however, and in the transition range of the phospholipid, the polarization increased greatly. A kinetic study of the interaction carried out around the transition temperature of dimyristoyl phosphatidylcholine as a function of pH shows that the speed of complex formation between alpha-lactalbumin and the lipid increases from neutral to acidic pH. From the present results and in agreement with our earlier calorimetric and fluorescence data (Hanssens, I., Houthuys, C., Herreman, W. and van Cauwelaert, F.H. (1980) Biochim. Biophys, Acta 602, 539--557), it is concluded that at neutral pH the interaction mechanism is probably different from that at acidic pH. At neutral pH and at all temperatures, alpha-lactalbumin is mainly absorbed electrostatically to the outer surface of the vesicle with little or no influence on the transition temperature of the phospholipid. At this pH, only around the transition temperature is penetration possible. At pH 4, however, the protein is able to penetrate the vesicle at all temperatures and to interact hydrophobically with the phospholipid fatty acid chains. As a result of this interaction, the transition temperature is increased by about 4 degrees C. This different behaviour changes progressively upon acidification: at pH 5, penetration seems to be impossible at temperatures far above the transition temperature but occurs rapidly around the transition temperature.

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

alpha-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine alpha-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.

A simplified purification procedure of α-lactalbumin from milk using Ca2+-dependent adsorption in hydrophobic expanded bed chromatography

The technique of expanded bed adsorption is originally designed for a direct recovery of proteins from fermentor feedstocks. In this article we describe the use of expanded bed adsorption for the recovery of α-lactalbumins from defatted milk using the hydrophobic Streamline Phenyl gel. α-Lactalbumins are Ca2+- binding proteins. Upon Ca2+ removal, they undergo a significant conformation change rendering them more hydrophobic. Based on this unique property we develop a protocol for fast and efficient purification of α-lactalbumin from milk. The use of this technique results in a reduction of the number of chromatographic purification steps.

Structural basis for difference in heat capacity increments for Ca2+ binding to two α-lactalbumins

Abstract Thermodynamic parameters for the unfolding of as well as for the binding of Ca 2+ to goat α -lactalbumin (GLA) and bovine α -lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (Δ C p ) offer the most direct information for the associated conformational changes of the protein variants. The Δ C p values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective Δ C p values for the binding of Ca 2+ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca 2+ -dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca 2+ -binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in Δ C p values for the binding of Ca 2+ is presumably in part related to this difference in charge distribution.

Shielding of phospholipid monolayers from phospholipase C hydrolysis by α-lactalbumin adsorption

Abstract α-Lactalbumin interacts more strongly with lecithin and cardiolipin monolayers at pH 3∼4 than at pH 7 to 10. At physiological pH this protein does not penetrate monolayers of DPPC and cardiolipin above pressures of 30 dynes/cm. Enzymatic hydrolysis of these monolayers by phospholipase C (Clostridium Welchii) is inhibited partially or totally when α-lactalbumin is injected in the subphase prior to the enzyme injection.

Hydrophobic interaction of lysozyme and α-lactalbumin from equine milk whey

From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca 2+ -dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca 2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca 2+ and in 2 mM Ca 2+ are also performed with equine α-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp→Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca 2+ conformers is clearly influenced by the mutation. The Ca 2+ -dependent exclusion of a hydrophobic domain is used in a new and simplified method for preparing lysozyme and α-lactalbumins simultaneously from equine milk whey.

Study of a hydrophobic site on bovine α-lactalbumin by labeling with [125I]-TID

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) labels apo-bovine alpha-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml-1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease the labeling of apo-alpha-lactalbumin with [125I]TID.

Ph-dependence of the α-lactalbumin structure : A fluorescence study

Summary The fluorescence parameters of demetallized α-lactalbumin in the range from pH 8 to 2 show an extremum around pH 5–4 (a minimum in quantum yield and wavelength and a maximum in polarization). This extremum is not due to a competition between Ca2+ and protons but rather to a stabilization of the conformation of the protein near the isoelectric pH by the ionic interactions between local positive and negative charges on the protein. The calcium-free protein has similar fluorescence characteristics at pH 2 and 8 but the thermal transition curve is different. The influence of 0.1 M NaCl is also considered.