Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).     

Depolarized light scattering by amides and peptides

The Rayleigh ratios R_H have been measured for the depolarized scattering dilute solutions of N-ethylacetamide (EA), N-methylpropionamide (MP), and N-acetyl-pyrrolidine (AP) in P-dioxane, and dilute aqueous solutions of N-methylacetamide (MA), N,N-dimethylacetamide (DMA), EA, and MP. Squares of the optical anisot-ropies γ of the amides are obtained through extrapolation of these mesurements to infinite dilution. Values of γ² found for EA and MP in dioxane are in good agreement with calculations based principally on the previously evaluated polarizability tensor of the amide group in conjunction with C? C and C? H bond polarizabilities. The calculations also involve averaging over all conformations, each being weighted according to the estimated conformational energy. The mean-squared optical anisotropies (γ²) of the oligoglycines and oligoalanines are calculated by similarly averaging over all skeletal conformations. The anistropic polarizability tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \alpha $\end{document} for the prolyl structural unit is derived for γ² for AP. The much larger optical anisotropies exhibited by the amides when dissolved in water as compared with those observed in dixane are discussed.     

Correction of spectra for studying dye aggregates by resonance light scattering

Resonance light scattering (RLS), a phenomenon of abrupt enhancement of Rayleigh light scattering in close proximity to an absorption band, is easily detectable in solutions of strongly absorbing chromophores, which form large aggregates with strong π-electronic coupling among the chromophores. RLS spectra need to be corrected for the sensitivity of the spectrofluorimeter as well as for the effects of internal light filter. A method for correcting the measured RLS is described. It was shown by the method that addition of KCl induces formation of extended supramolecular aggregates (probably of H-type) of the anionic dye merocyanine 540 in water. The RLS spectra of a photosensitizer m-tetra(hydroxyphenyl)chlorin (Foscan®) indicate formation of J-aggregates of this dye in aqueous medium.     

Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering

Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified. The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering. Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight. Polydispersity and statistical parameters of how to interpret the analysis are also provided. Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations. It was shown that at pH 7.8–8 and ionic strength of 0.02–0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm. Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates. Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179. © 1994 John Wiley & Sons, Inc.     

Determination of proteins with fullerol by a resonance light scattering technique

Fullerol has been synthesized through the reaction of fullerene C60 with NaOH in aqueous solution by means of ultrasonic agitation and characterized by infrared and 1H-nuclear magnetic resonance spectroscopy. The fullerol obtained shows good solubility and excellent stability in water. A weak resonance light scattering (RLS) spectrum of fullerol was observed in aqueous solution. However, the intensity of the RLS signal could be enhanced in the presence of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep), and lysozyme (Lys). Based on the enhancement of the RLS, a sensitive method for the determination of proteins has been established. The quantitative conditions were considered with regard to the effects of the pH, the ion strength, and the concentration of the fullerol. Under the optimum conditions, the intensity of the RLS was proportional to the concentration of proteins with the limits of detection of 9.7, 10.9, 57.4, and 8.5 ng mL(-1) for BSA, HSA, Pep, and Lys, respectively. Almost no interference can be observed from some amino acids, nucleic acids, and most of the metal ions. The model samples and human serum samples were determined satisfactorily with the proposed method.     

Development of a sensitive and rapid nucleic acid assay with tetraphenyl porphyrinatoiron chloride by a resonance light scattering technique.

Based on the interaction between nucleic acids and tetraphenyl porphyrinatoiron chloride (FeTPPCl), a novel method for the determination of nucleic acids at the nanogram level has been developed. Under the optimum conditions, the weak resonance light scattering (RLS) intensity of FeTPPCl was greatly enhanced by nucleic acids and the enhanced RLS intensity was proportional to the concentration of nucleic acids in the range 0.02-2.8 microg/mL for calf thymus DNA, 0.05-3.3 microg/mL for fish sperm DNA and 0.07-4.5 microg/mL for yeast RNA. The detection limits (3sigma) were 2.9 ng/mL for calf thymus DNA, 3.9 ng/mL for fish sperm DNA and 5.2 ng/mL for yeast RNA. Almost no interference could be observed from proteins, nucleosides and most of the metal ions. The proposed method showed good reliability, sensitivity, rapidity and reproducibility when applied to the determination of nucleic acids in synthetic and biochemical samples. The time savings make this method suitable for large routine analyses.     

The relationship between light scattering and chlorophyll a fluorescence during oscillations in photosynthetic carbon assimilation

Light scattering, which can be taken as an indicator of the transthylakoid proton gradient, and the 518-nm rise, which can be regarded as a measure of the transthylakoid membrane potential, have been followed during oscillations in chlorophyll a fluorescence, which are known to be associated with corresponding changes in photosynthetic carbon assimilation. Both components oscillated in a manner which was broadly reciprocal to chlorophyll a fluorescence. However, there was a phase shift such that the light-scattering change usually anticipated fluorescence and often also the 518-nm shift. It is concluded that the proton motive force rises and falls slightly in advance of rises and falls in carbon assimilation. The relationship of these changes to a possible underlying mechanism is discussed.     

Dynamic light scattering of aqueous solutions of linear aggregates induced by thermal denaturation of ovalbumin

Dynamic light scattering measurements were performed on dilute aqueous solutions of native ovalbumin (OA) and on those of linear OA aggregates induced by thermal denaturation at low ionic strength and neutral pH. The weight-average molecular weight M_w of four aggregates tested ranged from 1,700,000 to 5,500,000. The translational diffusion coefficient D₀ of native OA at infinite dilution was estimated as 8.70 × 10 ^?7 cm²/s, which gave 56.0 Å as the diameter of the rigid spherical particle. The intensity autocorrelation function of linear OA polymers was analyzed with the cumulant method to obtain the first cumulant Λ_e. The dependence of Λ_e on the scattering vector q at very low polymer concentration was found intermediate between those of a flexible chain and a rigid rod. The translational diffusion coefficient D_tr [≡ (T_e/q²)_{q → 0}] was in proportion to M, and the magnitude was in good agreement with a value calculated from the wormlike cylinder model with values of three parameters determined in an earlier study, M_L = 1600 Å^?1, d = 120 Å, and Q = 230 Å, where M_L, d, and Q are the molecular weight per unit length, diameter, and persistence length, respectively. Based on these results, a new model, to be called as the dimer model, was proposed to interpret the formation mechanism of linear OA polymers induced by thermal denaturation. © 1993 John Wiley & Sons, Inc.     

Cell discrimination by multiangle light scattering.

Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.     

Desmin filaments studied by quasi-elastic light scattering.

We studied polymers of desmin, a muscle-specific type III intermediate filament protein, using quasi-elastic light scattering. Desmin was purified from chicken gizzard. Polymerization was induced either by 2 mM MgCl(2) or 150 mM NaCl. The polymer solutions were in the semidilute regime. We concluded that the persistence length of the filaments is between 0.1 and 1 microm. In all cases, we found a hydrodynamic diameter of desmin filaments of 16-18 nm. The filament dynamics exhibits a characteristic frequency in the sense that correlation functions measured on one sample but at different scattering vectors collapse onto a single master curve when time is normalized by the experimentally determined initial decay rate.     

Thermodynamic stability and formation of aggregates of human immunoglobulin G characterised by differential scanning calorimetry and dynamic light scattering

The final process step of polyclonal human immunoglobulin G is formulation with agents such as sugars, polyols, amino acid and salts. Often the most stable formulations were empirically identified. Physicochemical methods, such as differential scanning calorimetry and dynamic light scattering, provide a deeper insight on the biophysical properties of such a protein solution. The combination of these methods proved to be sensitive enough to detect fine differences in the properties relevant for the development of stable protein solutions. The influence of additives, such as maltose and glycine in combination with water or low concentrations of salts, on human immunoglobulin preparations was analysed. Differential scanning calorimetry illustrated that 0.2 M glycine had better stabilising effects compared to 10% maltose. Dynamic light scattering and differential scanning calorimetry revealed that solutions preventing aggregation were not optimal in terms of thermodynamic stability. Aggregation was minimised with increasing ionic strength, shown by dynamic light scattering, whereas thermodynamic stability for heat sensitive parts of human immunoglobulin G, analysed with differential scanning calorimetry, was decreased.     

Identification of cell asymmetry and orientation by light scattering.

Light scattering from chicken red blood cells has been used as a model system to identify the asymmetry of cells. The histogram for forward angle light scattering for these cells is bimodal, the signal size being dependent on the cell orientation. A dual orthogonal scatter system is used to conclusively demonstrate this orientational variation in signal. A third scattering system, using a single incident beam with two orthogonal detectors, is used to further characterize the orientational variation of the scatter signal. In this third system it is shown that the signal in a detector set 90 degrees from the incident beam collects light reflected from the cell surface. The optical selection of cells in specific orientations using these systems may circumvent the need to physically orient cell in flow systems.     

Quasi-elastic light scattering by calf thymus DNA and lamdaDNA.

The quasi-elastic light-scattering (homodyne) time-correlation functions of calf thymus and λDNA are shown to contain contributions from at least two relaxation processes. A method of asymptotic analysis is described and used to obtain an estimate of the longest relaxation time as well as the “average” relaxation time and the mean-squared dispersion in this average. Most theories of scattering from macromolecules in the limit of inifinite dilution predict that the longest relaxation time is due to translational self-diffusion. The data obtained, however, indicate that the longest time is not simply related to the translational self-diffusion coefficient of unaggregated macromolecules. It is also shown that the longest relaxation time of λDNA decreases in the later stages of the denaturation transition region. Some possible mechanisms for the origin of this long time are discussed, including a model of restricted motion of a molecule by its neighbors.     

Accumulation of protochlorophyll and chlorophyll a as controlled by photomorphogenically effective light

Summary Data are presented which indicate that the rate of synthesis and the pool size of photoconvertible protochlorophyll(ide) in the cotyledons of the mustard seedling are controlled by the active form of phytochrome (P_fr). Inductionreversion experiments show that formation of chlorophyll a through photoconversion of the protochlorophyll(ide) by repeated red pulses (5 min each) has no effect on synthesis of carotenoids and galactolipids. Since the protochlorophyll(ide)-converting activity of the standard far-red light used in this laboratory is very low, chlorophyll-a accumulation is very slow under continuous standard far-red light. It is concluded that photosynthesis (or photosynthetic phosphorylation) does not participate in the high irradiance reaction of photomorphogenesis.     

Superoxide photogeneration by chlorophyll a in water/acetone. Electron spin resonance studies of radical intermediates in chlorophyll a photoreaction in vitro

In this paper we report the use of DMPO (5,5-dimethyl-1-pyrroline-1-oxide) as spin trap in the ESR observation of O2-. photogeneration by in vitro Ch1 a in oxygen-saturated 50:50% (v/v) water/acetone. The observed hyperfine parameters for the spin adducts of DMPO are identical to those obtained from H2O2 decomposition, and to those reported by earlier workers for the formation of 02-. in oxygen-saturated preparations of spinach chloroplasts.     

Measurement of particle sizes by elastic and quasi-elastic light scattering.

A method of measuring an average particle radius in a highly polydisperse dispersion using the wavelength dependence of turbidity is described. For particles which are no larger than 0.3 of the wavelength of light used, a polynomial representation of the scattering cross-section can be used. For larger particles, more extensive numerical calculations are required. The use of the method is illustrated by determining the average particle radius of casein micelles by both elastic and quasi-elastic light scattering techniques. A polydisperse homogeneous sphere model is found to be a reasonably accurate representation of casein micelles. Several modifications of the model which would improve the agreement between the two techniques are mentioned briefly.