Willy Herreman

Correlation between the order parameter and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and an evaluation of membrane fluidity

Abstract In this paper it is shown that for 1,6-diphenyl-1,3,5-hexatriene there exists a simple analytical relation between the orientational order parameter and the steady-state fluorescence anisotropy. This relation is derived on semi-empirical grounds. The order parameter and the true microviscosity for membranes as calculated from steady-state measurements are evaluated. For biological membranes the estimation of the order parameter from steady-state experiments is feasible, but the evaluation of the true microviscosity is not reliable. Also, the physiological relevance of the order parameter is discussed.

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. A new approximate solution of the rotational diffusion equation.

We discussed the time-dependence of fluorescent emission anisotropy of a cylindrical probe in membrane vesicles. We showed that, if the motion of the probe were described as diffusion in an anisotropic environment, it would be possible to determine not only the second-rank but also the fourth-rank orientational order parameter from the decay of the fluorescence anisotropy. The approximations involved were based on an interpolation of short-time and long-time behavior of the relevant correlation functions. A general expression was derived for the time dependence of the fluorescence anisotropy in closed form, which applies to any particular distribution model. It was shown to be in good agreement with previously reported results for the cone model and the Gaussian model. Finally, the applicability of the theory to time-resolved and differential phase fluorescence depolarization experiments was discussed.

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes

Various models for the analysis of time-dependent fluorescence anisotropy measurements were evaluated. The discussion was based on the analysis of pulsed experiments with 1,6-diphenyl-1,3,5-hexatriene embedded in small unilamellar vesicles of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine and in dimyristoylphosphatidylcholine/alpha-lactalbumin complexes. It was shown that a recently proposed model (Van der Meer, W., H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx, H. Schröder, 1984, Biophys. J., 46:515-523) described the data better than did the earlier suggested cone model (Kinosita K., Jr., S. Kawato, and A. Ikegami, 1977, Biophys. J., 20:289-305). This permitted the use of the new model for the estimation of the second- and fourth-rank order parameters on nonoriented systems. The results indicated that a fraction of the probes was oriented perpendicularly to the preferred direction of the lipids. An increase of the rotational correlation times of the fluorescent probe and a higher order of its environment were detected after the interaction of alpha-lactalbumin with the dimyristoylphosphatidylcholine vesicles at acidic pH at 24.2 degrees C.

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.

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.

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with alpha-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with alpha-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of alpha-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, delta H, of the interaction of the three vesicle types with alpha-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24 degrees C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic delta H values around 24 degrees C for large vesicles approximates the transition enthalpy of the pure phospholipid.

A new approach to polarized fluorescence using phase and modulation fluorometry

The usual method to obtain fluorescence anisotropy decay parameters from frequency domain measurements is to determine the phase angle difference between the perpendicular and parallel components of the polarized emission and the ratio of their modulated amplitudes (“differential method”). Here, an alternative method is introduced analogous to a time-resolved study of the fluorescence anisotropy (“Sine-Cosine Transform method”), in which the intensity difference is studied in the frequency domain. It is shown that the differential method is better at low steady-state fluorescence anisotropy, while the Sine-Cosine Transform method is preferable at high anisotropy values and even more preferable, if the probe rotation is strongly anisotropic or hindered, giving rise to multiple rotational diffusion phenomena.

The invisible made visible

An unexpected intense blue line appears when a white paper screen is used to demonstrate the spectral lines of a mercury lamp with a diffraction grating. The line is not present when a plastic screen is used and is due to the fluorescence of the whitening agents in the paper.

Correlation between Steady-State and Time-Resolved Fluorescence Anisotropy Data

The most common fluorescence depolarization measurement is a steady-state experiment. Continuous illumination with monochromatic polarized light is used to excite fluorescent probes embedded in the lipid regions of the membrane sample. One measures the fluorescence intensities parallel (I‖) and perpendicular (I⊥) to the polarization direction of the excitation light. The relevant parameter is the steady-state fluorescence anisotropy (FA), defined as