Characterization of reacting solutions of macromolecules by laser light scattering and Airfuge centrifugation

Simple, fast and accurate measurements combining centrifugation in a table top Airfuge and laser light scattering in the Airfuge tube are described. The procedure achieves quantitative separation of particles according to their sedimentation coefficient in microliter volumes. By scanning through the sedimenting boundary association equilibrium constants are evaluated.     

Marine microalgae attack and feed on metazoans

Free-living microalgae from the dinoflagellate genus Karlodinium are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills. Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species Karlodinium armiger and Karlodinium veneficum have caused fast paralysis and mortality of finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass in-situ. Here we show that a strain of K. armiger (K-0688) immobilises the common marine copepod Acartia tonsa in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of K. veneficum did not attack or affect the motility and survival of the copepods. Copepod immobilisation by the K. armiger strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of K. armiger at densities below 1000, cells ml⁻¹, but above 3500 cells ml⁻¹ the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to reduced grazing pressure, promoting the persistence of blooms at high densities. K. armiger also fed on three other metazoan organisms offered, suggesting that active predation by mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web.     

Bacteria, diatoms and detritus in an intertidal sandflat subject to advective transport across the water-sediment interface

This study focused on organic particles withrespect to their transport and sedimentarymineralisation in a North Sea intertidalsandflat previously characterised as stronglyinfluenced by advective transport across andbelow the water-sediment interface. Measuredpermeabilities of the sandy sediment rangedfrom 5.5 to 4110^–12 m², andpermeabilities calculated from granulometricdata exceeded the measured values by a factorof 4.4 2.8. Bacteria (2–9% of the POC)were highly variable in space and time. Theywere less mobile than interstitial fine (<70 µm) organic and inorganic particles, aspart of the population lived attached to large,heavy sand grains. The vertical distribution ofbacteria was closely related to the organiccarbon content of the fine-grained interstitialmaterial. In winter, bacterial numbers in theuppermost 5 cm amounted to 39–69% of thesummer ones. Carbon mineralisation rates rangedbetween 20 mg C m^–2 d^–1 in winter and580 mg C m^–2 d^–1 in summer, keepingstep with finer-grained sediments thatcontained an order of magnitude more organiccarbon. Sedimentary carbohydrates were mainlyintracellular or tightly bound to particles,and their concentrations were depth-invariantin winter, but exponentially decreasing withdepth in summer. Below 5 cm depth, the meanconcentration was (1590 830) µg cm^–3, without major downcoreor seasonal changes. Phytobenthos andphytodetritus were dominated by diatoms andcomprised merely minor amounts of other primaryproducers. Planktonic diatom depth profileswere related to weather and phytoplanktonconditions, and benthic diatoms showed similardepth distributions due to passive and activemotion. The penetration of relatively freshphytodetritus down to at least 5 cm, shown bychloropigment composition, emphasised the closecoupling between water column and sandysediment, facilitated by advective interfacialand subsurface flows.     

Conformational change and aggregation of κ-carrageenan studied by flow field-flow fractionation and multiangle light scattering

The relatively novel combination of flow field-flow fractionation (FFF) and multiangle light scattering (MALS) was employed to study a nondegraded κ-carrageenan in different 0.1M salt solutions. The applicability of the technique was tested, and the effects of salt type and salt composition on the molar mass and radius of gyration were studied. A conformational ordering was induced at room temperature by switching the solvent from 0.1M NaCl (coil form) to 0.1M NaI (helix form). An approximate doubling of the average molar mass and an increase in radius of gyration was then observed, in agreement with results obtained previously using size exclusion chromatography–MALS. This increase in size was attributed to conformational ordering and to the formation of double helices. Severe aggregation was observed above 40% CsI in the 0.1M mixed salt solution of CsI and NaI. This was ascribed to the association of helices into large aggregates. For these large associates, having molar masses of several millions, a reversal of the elution order in flow FFF was detected. © 1998 John Wiley & Sons, Inc. Biopoly 45: 85–96 1998     

Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering

The feasibility of size exclusion chromotography (SEC)-multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, and antibodies under various conditions such as addition of detergent, changes in pH, and variation of protein concentration and heat stress temperature was examined. Separation of proteins and their aggregates was performed using SEC-high-performance liquid chromatography. Detection of analytes was carried out with on-line UV, refractive index, and multiangle laser light-scattering detectors. Quantification and molecular weight determination were performed using commercial software. Aggregation and degradation were examined under various conditions and quantitative results are presented for bovine serum albumin, choriogonadotropin, glyceraldehyde-3-phosphate dehydrogenase, Herceptin, and ReoPro. This method can simultaneously determine both the quantities and the molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids misleading results caused by molecular shape or interactions with the column matrix. This technique is valuable not only for assessing the extent of aggregation but also for effectively monitoring molecule degradation as evidenced by molecular weight reduction and change in monomer amount.     

Using polarization analysis to separate the coherent and incoherent scattering from protein samples

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006 < Q < 0.04 Å^− 1) to the wide-angle region (up to ≈ 2.35 Å^− 1). Quantitative information was obtained on the fraction of coherent to spin-incoherent scattering from different protein samples: deuterated and protonated protein powders at different hydration levels and solutions of protonated proteins in D₂O at different concentrations. The results obtained are discussed in the context of the validity of the assumptions generally made when interpreting quasielastic neutron scattering experiments performed without polarization analysis.     

Solution properties of starch nanoparticles in water and DMSO as studied by dynamic light scattering

Solution properties of starch nanoparticles dispersed in DMSO and in water were studied using dynamic light scattering. The particle size distribution had two main peaks in both solvents at all scattering angles studied. They were at around 40 and 300 nm, ascribed to isolated starch nanoparticles and their aggregates, respectively. From the excess scattering intensity by the 40-nm particles, the molecular weight of the nanoparticle was estimated as 2.2–2.6×10⁶ g/mol. When the concentration was increased, another peak appeared at around 1 μm. Raising the temperature from 25 to 65 °C did not change the distribution, indicating a purely entropic process in dynamic equilibrium of the aggregation. In DMSO, an oscillatory behavior was observed in the autocorrelation function at high temperatures.     

Analysis of the conformational properties of κ‐ and ι‐carrageenan by size‐exclusion chromatography combined with low‐angle laser light scattering

The conformational properties of κ‐carrageenan in 0.2M LiI and ι‐carrageenan in 0.2M LiCl were analyzed by size exclusion chromatography combined with low‐angle laser light scattering. Fractionated samples with narrow molecular weight distributions (M_w/M_n ∼ 1.4) were used, and M_w in the disordered states were 35,000 (κ‐35) and 200,000 (κ‐200) for κ‐carrageenan and 65,000 (ι‐65) and 170,000 (ι‐170) for ι‐carrageenan, respectively. The analyses were performed across a temperature range where the conformational transitions occurred, and at extremely low concentrations (2–50 μg/mL) due to low amounts of samples injected and the subsequent dilution occurring during the separation. The results indicate that a twofold increase of the molecular weight (M_w) occurs for κ‐carrageenan upon inducing the ordered conformation. For ι‐carrageenan an additional increase in M_w may take place, which is attributed to the strong tendency for aggregation of ordered chains especially at high molecular weights. The results thus suggest that both κ‐ and ι‐carrageenan are double (or multiple) stranded in their ordered conformations, within the concentration range studied here. © 1999 John Wiley & Sons, Inc. Biopoly 49: 71–80, 1999     

Changes in pig diet particle size profile and nutrient content during on-farm storage and distribution to the feeders

The present study assessed the effect of silo emptying and feed transport by conveyor systems on particle size and nutrient content of the feed delivered to the pigs. Experiment 1 sampled feed from four feeders along the conveyor system of two barns. Samples were taken immediately after filling the feed silo (Begin) and when the silo was almost empty (End). In Experiment 2, three barns with drag-type conveyors, three with auger-type conveyors and two with spiral-type conveyors were sampled. Along the different conveyors, samples at 10, 20, 50 and 85 m distance from the feed silo were taken from the feeders. In each barn, sampling was repeated for two subsequent batches of feed delivered. In all samples, particle size profile was determined and nutrient content was analysed. In Experiment 2, mineral content was also determined. In Experiment 1, the size of the different particle fractions decreased from Begin to End. An interaction (p < 0.05) between sampling time and conveyor type was detected for the 10% smallest particles. In Experiment 2, an effect of sampling time on the 10% largest particles was detected (p < 0.05). The effect of sampling time on several nutrients was observed in Experiments 1 and 2, but the affected nutrients differed between both experiments. Results implied that it was mainly emptying of the silo that affected mash particle size profile and nutrient content. The potential impact of these changes on pig performance requires further investigation.     

Coupling of laser excitation and inelastic neutron scattering: attempt to probe the dynamics of light-induced C-phycocyanin dynamics

Excitation energy transfer (EET) in light-harvesting antennae is a highly efficient key event in photosynthesis, where light-induced dynamics of the antenna pigment-protein complexes may play a functional role. So far, however, the relationship between EET and protein dynamics remains unknown. C-phycocyanin (C-PC) is the main pigment/protein complex present in the cyanobacterial antenna, called "phycobilisome". The aim of the present study was to investigate light-induced C-PC internal thermal motions (ps timescale) measured by inelastic neutron scattering. To synchronize the beginning of the laser flash (6 ns duration) with that of the neutron test pulse ( approximately 87 mus duration), we developed a novel type of "time-resolved" experimental setup on MIBEMOL time-of-flight neutron spectrometer (LLB, France). Data acquisition has been modified to get quasi-simultaneously "light" and "dark" measurements (with and without laser, respectively) and eliminate many spurious effects that could occur on the sample during the experiment. The study was carried out on concentrated C-PC ( approximately 135 g/L protein in D(2)O phosphate buffer), contained in an aluminium/sapphire sample holder (almost "transparent" for neutrons) and homogeneously illuminated inside an "integrating sphere". We observed very similar incoherent dynamical structure factors of C-PC with or without light. The vibrational density of states showed two very slightly increased vibrational modes with light, at approximately 30 and approximately 50 meV ( approximately 240 and approximately 400 cm(-1), respectively). These effects have to be verified by further experiments before probing any temporal evolution, by introducing a time delay between the laser flash and the neutron test pulse.     

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase

(1) The natural ATPase inhibitor (IF₁) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF₁ polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF₁. The higher the salt concentration, the lower the aggregation state. Aggregation of IF₁ was decreased at slightly acidic pH. It increased with the concentration of IF₁ as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF₁ in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF₁ (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547–2551) was shown to have a limited effectiveness as a trap for IF₁. The reason was that IF₁ released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF₁ which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF₁ preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336–1344). The particles recovered in the sediment were deprived of IF₁ and could therefore be used for preparation of F₁-ATPase. The advantage of this method is that both IF₁ and F₁-ATPase can be prepared from the same batch of mitochondria.     

Cascading Trophic Interactions from Fish to Bacteria and Nutrients after Reduced Sewage Loading: An 18-Year Study of a Shallow Hypertrophic Lake

The effects of major reductions in organic matter, total phosphorus (TP), and total nitrogen (TN) loading on the chemical environment, trophic structure, and dynamics of the hypertrophic, shallow Lake Søbygård were followed for 18 years. After the reduction in organic matter loading in 1976, the lake initially shifted from a summer clear-water state, most likely reflecting high grazing pressure by large Daphniaspecies, to a turbid state with extremely high summer mean chlorophyll a (up to 1400 μg L^{? 1}), high pH (up to 10.2), and low zooplankton grazing. Subsequently, a more variable state with periodically high grazing rates on phytoplankton and bacteria was established. Changes in zooplankton abundance and grazing could be attributed to variations in cyprinid abundance due to a fish kill (probably as a consequence of oxygen depletion) and pH-induced variations in fish recruitment and fry survival. The results suggest strong cascading effects of fish on the abundance and size of zooplankton and phytoplankton and on phytoplankton production. A comparatively weak cascading effect on ciliates and bacterioplankton is suggested. Due to high internal loading, only minor changes were observed in lake-water TP after a reduction in external TP loading of approximately 80% in 1982; net retention of TP was still negative 13 years after the loading reduction, despite a short hydraulic retention time of a few weeks. TN, however, decreased proportionally to the TN-loading reduction in 1987, suggesting a fast N equilibration. Only minor improvement in the environmental state of the lake has been observed. We suggest that another decade will be required before the lake is in equilibrium with present external P loading.