A semiautomated hemolysis microassay for membrane lytic proteins

A simple, rapid, semiautomated microassay for hemolysis using a microtiter plate spectrophotometric system is described. The assay relies on the differences in light scattering (turbidity) properties of intact and of lysed erythrocytes. Lysis of erythrocyte suspensions in 96-well plates is determined by absorbance at 690 nm. A linear correlation between the percentage of hemolysis and the turbidity decrease is observed, indicating that this assay may be used for both rapid screening and quantitation of the hemolytic activity. This assay allows screening of 300 samples in less than 6 min. Small samples derived from protein fractionation columns (HPLC, for example) can be rapidly screened. This assay has been used in the successful isolation of a cytolytic membrane-lytic protein from the granules of cloned cytotoxic T lymphocytes and NK cells.     

Relationship of turbidity to the stages of platelet aggregation

The process of platelet aggregation as detected by turbidity changes in the platelet aggregometer was studied relative to light scattering by large particles. For latex beads a plot of light scattering intensity/unit mass versus particle size gave increased light scattering intensity for small particle sizes but decreased scattering at large particle size. This behavior is predicted by Rayleigh-Gans theory. These results were related to the platelet aggregometer, an optical instrument used to measure the association of small particles (monomeric platelets) to large particles (platelet aggregates). Formalin-fixed platelets do not show changes in light transmission due to energy-requiring processes, such as shape change, so that turbidity changes in the presence of aggregating agents could be attributed to a change in platelet aggregation state. Small platelet aggregates showed increased turbidity compared to a similar mass of monomeric platelets. In fact, very large platelet aggregates that were visible to the unaided eye were needed to produce a decrease in light scattering intensity. Thus, turbidity can either increase or decrease with platelet aggregation depending on the size of the aggregates. Studies of platelet aggregation that show no initial increase in turbidity must be characterized by dominance of large platelet aggregates and monomeric platelets.     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Optical characterization of liposomes by right angle light scattering and turbidity measurement

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.     

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beer's law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

Light scattering study of phosphatidylcholine vesicles at the pretransition

Laser light scattering has been used to investigate the thermal pretransition of dipalmitoylglycerophosphocholine vesicles with variable radius as obtained by the mild sonication method. Intensity changes in 90° scattered light are observed at the pretransition for larger vesicles and actually increase with increasing vesicle size, reaching a constant value.This constant value is in good agreement with the value calculated from the refractive index data.The intensity ratio of scattered light at temperatures of 30°C and 40°C (I₄₀/I₃₀) approaches unity at a radius of small single-bilayer vesicle. This result is interpreted as no pretransition for small vesicles in agreement with the calorimetric results. An expression of the particle scattering factor is also presented for multilayered shells composed of anisotropic elements. It is shown numerically, using this expression, that changes in the lipid layer thickness and the tilting angles at the pretransition have no effects on the scattering factor. Therefore it is concluded that the intensity changes in scattered light reflect the changes in the refractive index of the vesicle originating in the polar head groups.     

Cooperativity of the phase transition in single- and multibilayer lipid vesicles.

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Dynamic light scattering study of suspensions of purple membrane

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.     

Visual feeding of fish in a turbid environment: Physical and behavioural aspects

Turbidity has both positive and negative effects on prey detection, by increasing or diminishing the contrast between prey and background due to the scattering of light. The positive effect of turbidity on prey contrast depends on the optical properties, scattering properties of suspended particles and the visual sensitivity of the predator.

The positive effect of turbidity is pronounced for larval fish, given that their visual field is short, leaving fewer particles between them and their prey to scatter light and interfere with detection. This relationship, together with a decreased risk of predation, makes turbid environments more optimal for some species and size groups of fish (planktivores and fish larvae) and less so for others (adult piscivore fish). Thus, turbidity might have a structuring effect on a fish community. Recently it has been demonstrated that UV light might have positive effects on prey detection and consumption. How UV light might interact with different kinds of particles producing turbidity is not well documented.     

Electrooptic study of the deionized form of bacteriorhodopsin

Electric field induced light scattering by suspensions of cation-depleted purple membranes, obtained by deionization of purple membrane (PM) suspensions on a cation exchange column or by electrodialysis at a pH around 6, shows a strong drop (more than 5 times) in the value of the permanent dipole moment relative to that of PM fragments. The membrane dipole moments were measured both at low dc and ac electric fields as well as by using electric field pulses with reversing polarity. Some slight changes in the dispersion of the electric polarizability were also observed.Microelectrophoretic measurements showed that the electric charge of the membrane fragments is increased by 30% after deionization. The importance of these data for the understanding of the blue membrane properties and subsequently for the mechanism of proton pumping are discussed.     

Visual feeding of fish in a turbid environment: Physical and behavioural aspects

Turbidity has both positive and negative effects on prey detection, by increasing or diminishing the contrast between prey and background due to the scattering of light. The positive effect of turbidity on prey contrast depends on the optical properties, scattering properties of suspended particles and the visual sensitivity of the predator. The positive effect of turbidity is pronounced for larval fish, given that their visual field is short, leaving fewer particles between them and their prey to scatter light and interfere with detection. This relationship, together with a decreased risk of predation, makes turbid environments more optimal for some species and size groups of fish (planktivores and fish larvae) and less so for others (adult piscivore fish). Thus, turbidity might have a structuring effect on a fish community. Recently it has been demonstrated that UV light might have positive effects on prey detection and consumption. How UV light might interact with different kinds of particles producing turbidity is not well documented.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.     

Core-Shell Modeling of Light Scattering by Vesicles: Effect of Size,Contents, and Lamellarity

Having a fast, reliable method for characterizing vesicles is vital for their use as model cell membranes in biophysics, synthetic biology, and origins of life studies. Instead of the traditionally used Rayleigh-Gans-Debye approximation, we use an exact extended Lorenz-Mie solution for how core-shell particles scatter light to model vesicle turbidity. This approach enables accurate interpretations of simple turbidimetric measurements and is able to accurately model highly scattering vesicles, such as larger vesicles, those with multiple layers, and those with encapsulated material. We uncover several surprising features, including that vesicle lamellarity has a larger effect on sample turbidity than vesicle size and that the technique can be used to measure the membrane thickness of vesicles. We also examine potential misinterpretations of turbidimetry and discuss when measurements are limited by forward and multiple scattering and by the geometry of the instrument.     

Light scattering from intact cells reports oxidative-stress-induced mitochondrial swelling

Angularly resolved light scattering measurements were performed on suspensions of EMT6 cells and on mitochondria isolated from rabbit liver. Mie theory analysis of the scattering from intact cells indicated that mitochondrial-sized organelles dominated scattering in the range 5-90 degrees . This interpretation was supported by the analysis of scattering from isolated mitochondria. Intact cells were subjected to oxidative stress by photodynamic insult. After 3 h of incubation in the heme precursor aminolevulinic acid hexylester, EMT6 cells accumulated abundant protoporphyrin IX, an endogenous photosensitizer formed in mitochondria. Irradiation of aminolevulinic acid/protoporphyrin IX-sensitized cells with 10 J cm(-2) of 514 nm light led to pronounced changes in angularly resolved light scattering consistent with mitochondrial swelling. Electron microscopy of similarly treated EMT6 cell monolayers showed significant changes in mitochondrial morphology, which included distension of the outer unit membrane and bloating of the internal mitochondrial compartment. Informed by these electron microscopy results, we implemented a coated sphere model to interpret the scattering from intact cells subjected to oxidative stress. The coated sphere interpretation was compatible with the scattering measurements from these cells, whereas simpler Mie theory models based on homogenous swelling were dramatically unsuccessful. Thus, in this system, angularly resolved light scattering reports oxidative-stress-induced changes in mitochondrial morphology.     

A study of the kinetics and mechanism of ADP-triggered platelet aggregation

The time-course of ADP-triggered aggregation of human blood platelets has been followed by sensitive right-angle light scattering intensity measurements as a function of the platelet and fibrinogen concentrations. Rayleigh-Gans light scattering theory has been combined with the Smoluchowski aggregation model to predict the dependence of the right-angle scattering intensity on particle size and concentration as well as the time-dependent changes during aggregation. The validity of the calculations was confirmed by measuring the scattering intensity with suspensions of polystyrene microspheres of known radius, as well as the time-dependent changes in the 90 degrees scattering intensity during aggregation of these particles. However, in contrast to the predictions of the model, the time-course of the scattering intensity changes during platelet aggregation was characterized by single exponential decay with a rate constant which reached a limiting value of 0.017 s-1 at high platelet concentrations. The value of kagg was also independent of the fibrinogen concentration over a 30-fold range. Covalently cross-linked fibrinogen dimers and Fragment D-inhibited fibrin protofibrils yielded aggregation rates that agreed with those measured with fibrinogen. The results indicate that the rate of platelet aggregation is not limited by either the rate of fibrinogen binding or the frequency of platelet-platelet collisions under these conditions.     

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments

Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.     

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersions of dimyristoyl phosphatidylcholine, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, $\text{2,2,6,6-}\text{tetramethylpiperdine}\text{-N-}\text{oxyl}$, phosphatidylcholine and stearic acid, and also 90° light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526–535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/?σ, is a measure of the mean number of molecules, per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

Effect of shear stress and free radicals induced by ultrasound on erythrocytes

The present study was undertaken to elucidate the mechanism of hemolysis induced by ultrasound. Ar or N2O gas was used to distinguish between cavitation with or without free radical formation (hydroxyl radicals and hydrogen atoms). Free radical formation was examined by the method of spin trapping combined with ESR. After sonication of erythrocyte suspensions, several structural and functional parameters of the erythrocyte membrane--hemolysis, membrane fluidity, membrane permeability, and membrane deformability--were examined. Although free radical formation was observed in the erythrocyte suspensions sonicated in the presence of Ar, no free radical formation was observed in the presence of N2O. However, the hemolysis behavior induced by ultrasound was similar in the presence of Ar or N2O. The membrane fluidity, permeability, and deformability of the remaining unlysed erythrocytes after sonication in the presence of Ar or N2O were unchanged and identical to those of the control cells. On the other hand, after gamma irradiation (700 Gy), the hemolysis behavior was quite different from that after sonication, and the membrane properties were significantly changed. These results suggest that hemolysis induced by sonication was due to mechanical shearing stress arising from cavitation, and that the membrane integrity of the remaining erythrocytes after sonication was the same as that of control cells without sonication. The triatomic gas, N2O, may be useful for ultrasonically disrupting cells without accompanying free radical formation.