Horst Ruf

[21] Dynamic laser light scattering to determine size distributions of vesicles

Publisher Summary This chapter describes dynamic laser light scattering to determine size distributions of vesicles and presents a survey of the underlying principles with regard to vesicle suspensions. From a size distribution, it can be decided whether vesicles are homogeneous in size. Its characteristics can be used for controlling the stability of a sample and for studying processes such as aggregation and fusion. The nonmicroscopic methods requiring rather homogeneous populations with a well-defined shape are most appropriately applied to small unilamellar vesicles. Electron microscopy is used for vesicles having an extended size range. Because of the remarkable sensitivity of parameters to the size distribution of vesicles based on the composition of a medium, it is important to characterize and analyze each vesicle preparation.

Data accuracy and resolution in particle sizing by dynamic light scattering

The noise levels and the decay characteristics in experimental data from dynamic light scattering measurements determine the number of details of continuous size distributions that can be resolved. Here, a procedure is described that allows one to obtain resolution limits empirically from experimental data on hand. This is done by comparing differences of autocorrelation functions associated with size distributions of different complexity with noise in the experimental data. Size distributions of rather different complexity but related to autocorrelation functions that describe the experimental data nearly equally well are obtained from series of regularized inversions carried out by the size distribution algorithm CONTIN. The procedure is demonstrated with two data sets of different noise content. It is shown that a monomodal size distribution with a relative width (standard deviation/average radius) of about 0.2 can be determined with a high accuracy, when the residuals in data of the first order autocorrelation function are of the order of 10−4. A bimodal size distribution with narrow peaks of relative widths of about 0.05 and a size ratio of about 3:2, the autocorrelation function of which describes these data equally well, cannot be resolved from this level of noise. If data accuracy is increased about ten fold, it should be possible to distinguish between these two quite different size distributions and to make a decision on whether the underlying size distribution is actually a monomodal or a bimodal one. To obtain data of such high statistical accuracy would imply a rather long measurement duration yet.

Size distributions of chylomicrons from human lymph from dynamic light scattering measurements

Abstract Chylomicrons, the vehicles for the transport of exogeneous triglycerides and cholesterol in the lymph and the blood, were characterized by their size from dynamic light scattering measurements. To achieve an appropriate resolution, correlation data were collected over several hours. Analysis was performed with an extended version of the regularization method CONTIN, and special attention was given to errors in the experimental baseline and to randomness of the residuals. The solutions selected by means of Fishers F-test by CONTIN agreed with those obtained with the stability plot of Schnablegger and Glatter, when in the case of data of lower statistical accuracy the solution was taken from the lower part of the confidence interval of the F-test. The intensity-weighted size distributions indicated two classes of particle, their mean diameters being 100–140 nm and 330–350 nm. The ability to resolve two peaks of such a size ratio is demonstrated. The numbers of particles associated with the two peaks were estimated by means of the scattering properties of the particles, which showed that the overwhelming majority were small ones. This estimation also suggested that the mean size of the first peak of the number distribution is significantly smaller than the typical size of chylomicrons. This was consistent with the finding that the sample contained not only apolipoprotein B-48 but also a similar amount of apolipoprotein B-100, which is associated with lipoproteins of smaller size. The larger particles of the second peak are probably dietary triglyceride-rich chylomicrons.

Determination of internal volume and volume distribution of lipid vesicles from dynamic light scattering data

Abstract The total internal volume, enclosed by the spherical, unilamellar membranes of a lipid vesicle dispersion, is calculated from the vesicles size distribution according to the relationships derived here. The size distribution of phosphatidylcholine vesicles is determined from dynamic light scattering measurements. Mean vesicle radii of about 100 nm and rather symmetric distributions with standard deviations of about 25 nm are characteristic of vesicles being prepared by dialysis of mixed octyle glucoside/egg yolk phosphatidylcholine micelles. A total internal volume of about 6 ml per mmol of phospholipid has been determined from the dynamic light scattering data. This is in good agreement with the results obtained from determinations of the amount of 5(6)-carboxyfluorescein trapped by the vesicles employing absorption- and fluorescence spectroscopy, belonging to the hitherto commonly used methods. In addition, the new procedure is applied to determine the density distribution of the volume enclosed by the vesicles of a given radius as well as the number density distribution of vesicles of a given internal volume, required for a detailed interpretation of flux measurements. The latter distribution with standard deviations around 3 · 106 nm3 is rather unsymmetric with respect to the corresponding mean value of the internal volume enclosed by a single vesicle, which is here typically about 4 · 106 nm3.

Reassignment of cation-induced population of main conformational states of FITC-Na+/K+-ATPase as detected by fluorescence spectroscopy and characterized by equilibrium binding studies

To link proposed features of the Na+/K+-ATPase reaction cycle to the molecular structure, spectroscopic studies mainly concerning the elucidation of partial reactions are carried out. This can be done by introducing fluorescence labels such as the F1TC-group (1) which enables the study of alkali ion binding as a key feature of this ion pump. According to earlier studies, a correlation between the fluorescence emission intensity and the position of the E1/E2 equilibrium is suggested: low fluorescence intensity in the presence of K+ has been attributed to E2, high intensity to E1(1–5). Evidence will be presented that this correlation concerning E1 is not fulfilled. Membrane-bound Na+/K+-ATPase has been prepared from pig kidney according to (6), the FITC-enzyme according to (1); details are given in (7).

Use of dynamic light scattering for the determination of size distributions of chylomicrons from human lymph

A sample of chylomicrons from human lymph was characterized using dynamic light scattering measurements. The stability of the sample was checked by carrying out series measurements at 3 different days, and was found to be very high during this period. The size distribution was determined by means of an extended version of the algorithm CONTIN that allowed normalization errors to be taken into account. We applied here a special evaluation scheme, where the data were corrected for these errors before final analysis. In addition to the intensity weighted size distribution obtained from this method we derived the size distributions of number and mass, utilizing the scattering amplitude functions of Mie. For these calculations the intensity-weighted size distribution was refined using an interpolation scheme to reduce problems associated with the use of mean values of the scattering amplitude functions for the intervals of the original size grid. The results showed that the chylomicron sample contained two classes of particles with intensity-weighted mean diameters of 107 nm and 320 nm. Number and mass distributions derived for the two major peaks indicated that practically all particles were small ones, but also that the few remaining large ones were carrying about 10 percent of the total mass.

Asymmetrie Reconstitution of the Erythrocyte Anion Transport System in Vesicles of Different Curvature: Implications for the Shape of the Band 3 Protein

Abstract The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposom es of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be “right-side-out” in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein’s intramembrane domain and that this domain has the form of a truncated cone or pyramid

Na+/K+-ATPase: Probing for a High Affinity Binding Site for Na+ by Spectrofluorometry

The transfer of Na+ and K+ across membranes by the Na+/K+-pump is coupled to the transition between two conformational states of the enzyme denoted E1 and E2 (1,12). In the E1 state the enzyme binds preferentially Na+, while in the E2 state it binds preferentially K+. Fluorescent probes like fluorescein-isothiocyanate (FITC) covalently bound to the protein (8) or potential-sensitive styryl dyes such as RH 421 incorporated into the membrane (2,9,13) change their fluorescent properties in the presence of Na+ or K+, and thus offer the possibility of studying the binding of these ions and conformational transitions of the enzyme spectroscopically. FITC-labeled Na+/K+-ATPase is strongly fluorescent in the presence of high Na+ concentrations, but exhibits a much lower fluorescence if K+ or one of its congeners is bound. Accordingly the two fluorescence emission intensity states have been assigned to the conformational states E1 and E2 (6,8,11). Na+ binding alone can also be studied by means of the fluorescence changes of RH 421, which has been used to study phosphorylation of the native enzyme by ATP in the presence of Na+ and Mg++ under conditions where no K+ is present (2,9,13). Titrations of the enzyme with Na+ in the presence of these dyes showed that the two different fluorescent labels report binding of Na+ to two different sites (4). The implications of these findings to the assignment of fluorescent states to conformations of the enzyme will be discussed here in more detail.