Frans Van Cauwelaert

Stability of equine lysozyme : I. thermal unfolding behaviour

The thermal denaturation of Ca(2+)- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase of the ellipticity at 222 nm and 287 nm, coincides with the transition detected by fluorescence and occurs at 30-50 degrees C for the apo-form and at 50-60 degrees C for the Ca(2+)-form of lysozyme. It seems to correlate with the transfer of some tryptophan residues to a more hydrophobic environment and with a local rearrangement of the tertiary and secondary structures. The unfolding transition detected by the decrease of the ellipticity at all wavelengths occurs nearly in the same temperature region for the apo- and Ca(2+)-forms, i.e. 50-80 degrees C and 55-80 degrees C, respectively. The presence of a Ca(2+)-binding loop in equine lysozyme may be partly responsible for the drastic destabilization of its structure as a whole both in the presence but especially in the absence of Ca2+ in comparison with hen and human lysozymes.

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.

Comparison of the binding of Ca2+ and Mn2+ to bovine α-lactalbumin and equine lysozyme

Abstract The enthalpy change of the binding of Ca 2+ and Mn 2+ to equine lysozyme was measured at 25°C and pH 7.5 by batch microcalorimetry: ΔH° Ca 2+ = −76 ± 5 kJ mol −1 , ΔH° Mn 2+ = −21 ± 10 kJ mol −1 . Binding constants, log K Ca 2+ = 6.5 ± 0.2 and log K Mn 2+ = 4.1 ± 0.5, were calculated from the calorimetric data. Therefore, ΔS Ca2+ ° = −131 ± 20 JK −1 mol −1 and ΔS° Mn 2+ = 8 ± 44 JK −1 mol −1 . Removal of Ca 2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine α-lactalbumin.

Molecular structure of carrageenans and kappa oligomers: a Raman spectroscopic study

The Raman spectra of kappa and iota carrageenan, a desulphated furcellaran and a series of oligomers have been compared in the region 700-1500 cm-1. Spectral differences depending on the amount and the location of the sulphate group on the ring, the chain length, the nature of the counterion and the conformation are discussed. Indications that the ionic interactions in the Na+ salts of the oligomers are different from those in K+ and Rb+ salts are given. On the macromolecular level it is found that the vibrational movements of the skeleton are related to the chain flexibility and the conformation. In gels of K+ and Rb+ kappa carrageenan spectral evidence is given for the existence of structural order.

Calorimetric experiments of Mn2+-binding to α-lactalbumin

Abstract We measured by batch microcalorimetry the standard enthalpy change Δ H ° of the binding of Mn 2+ to apo-bovine α-lactalbumin; Δ H ° = −90 ± 4kJ·mol −1 . The binding constants, K Mn 2+ , calculated from the calorimetric and circular dichroism titration curves, are (4.6±1) · 10 5 M −1 , respectively. Batch calorimetry confirms the competitive binding of Ca 2+ , Mn 2+ and Na + to the same site. The relatively small enthalpy change for Mn 2+ binding compared to Ca 2+ binding favours a model of a rigid and almost ideal Ca 2+ -complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an α-helix which is directly connected to the complexating loop.

Raman spectroscopic analysis of the sodium salt of kappa-carrageenan and related compounds in solution

Abstract Laser-Raman spectra of Na + kappa-carrageenan, Na + neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm −1 are reported for solutions in H 2 O and D 2 O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm −1 , close to the maximum of the symmetrical COS stretching (∼850 cm −1 ). The symmetrical SO stretch is proposed to occur near 1040 cm −1 and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm −1 . The intense band in the region 730–740 cm −1 is ascribed to a complex ring vibration.

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.

Preparative and analytical separation of oligosaccharides from κ-carrageenan

Abstract The separation of the enzymic degradation products of κ-carrageenan, as studied by size-exclusion chromatography on Bio-Gel P-6 and reversed-phase high-performance liquid chromatography μBondapak C 18 , resulted in a homologous series of oligomers in addition to non-homologues substances. On the former column steric exclusion competed with ion exclusion originating from solute—solute and solute—gel interactions. Competition was reduced with increasing ionic strength of the eluent. Separation on μBondapak C 18 , was influenced by the type of salt, indicating that the reversed-phase separation was not solely based on solute—column interactions.