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.     

Physicochemical screening for chemical stabilizer of erythropoietin to prevent its aggregation

Recombinant protein aggregation is a problematic issue and can provoke immunological response. The aim of this study was to analyze the stability of erythropoietin (EPO), as a therapeutic protein expressed in mammalian cells, in the presence of different chemicals and find a specific stabilizer for EPO. The effects of several chemicals, including mannitol, betaine, trehalose, taurine, linoleic acid, beta-cyclodextrin, copper sulfate, spermidine, maltose, maltodextrin, sucrose, dextran, beta-alanine, myo-inositol, and cysteine, on protein stabilization through the thermally induced aggregation of EPO were monitored. Based on the results of turbidity assay for thermal aggregation, three different patterns were observed for protein stability of active pharmaceutical ingredient of EPO, namely, accelerated, dose-dependent, and inhibitory behaviors for aggregate formation due to treatment with spermidine, mannitol, and betaine, respectively. According to circular dichroism outcomes, EPO treatment with betaine and spermidine resulted in different helical contents of the secondary structure. Dynamic light scattering experiments indicated that treating EPO with betaine resulted in less protein aggregation due to freeze and thaw stresses. Betaine was able to stabilize EPO and inhibit its aggregation, as opposed to spermidine that induced protein aggregation.     

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.     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Toward a common aggregation mechanism for a β-barrel protein family: Insights derived from a stable dimeric species

Δ78Δ is a second generation functional all-β sheet variant of IFABP (intestinal fatty acid binding protein) corresponding to the fragment 29–106 of the parent protein. This protein and its predecessor, Δ98Δ (segment 29–126 of IFABP), were initially uncovered by controlled proteolysis. Remarkably, although IFABP and Δ98Δ are monomers in solution, Δ78Δ adopts a stable dimeric structure. With the aim of identifying key structural features that modulate the aggregation of β-proteins, we evaluate here the structure and aggregation propensity of Δ78Δ. The 2,2,2-trifluoroethanol (TFE) induced aggregation of this protein shows a primary nucleation–elongation mechanism, characterized by the stabilization of a dimeric nucleus. Its rate of production from the co-solvent induced aggregation prone state governs the kinetics of polymerization. In this context, the value of Δ78Δ lies in the fact that – being a stable dimeric species – it reduces an otherwise bimolecular reaction to a unimolecular one. Interestingly, even though Δ78Δ and IFABP display similar conformational stability, the abrogated form of IFABP shows an enhanced aggregation rate, revealing the ancillary role played on this process by the free energy of the native proteins. Δ78Δ share with IFABP and Δ98Δ a common putative aggregation-prone central peptide. Differences in the exposure/accessibility of this segment dictated by the environment around this region might underlie the observed variations in the speed of aggregation. Lessons learnt from this natural dimeric protein might shed light on the early conformational events leading to β-conversion from barrels to amyloid aggregates.     

Measurement of amyloid formation by turbidity assay—seeing through the cloud

Detection of amyloid growth is commonly carried out by measurement of solution turbidity, a low-cost assay procedure based on the intrinsic light scattering properties of the protein aggregate. Here, we review the biophysical chemistry associated with the turbidimetric assay methodology, exploring the reviewed literature using a series of pedagogical kinetic simulations. In turn, these simulations are used to interrogate the literature concerned with in vitro drug screening and the assessment of amyloid aggregation mechanisms.     

The crowd you're in with: Effects of different types of crowding agents on protein aggregation

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein–polymer interactions influence the structure and dynamics of proteins in crowded environments.     

A polymer physics perspective on driving forces and mechanisms for protein aggregation

Protein aggregation is a commonly occurring problem in biology. Cells have evolved stress-response mechanisms to cope with problems posed by protein aggregation. Yet, these quality control mechanisms are overwhelmed by chronic aggregation-related stress and the resultant consequences of aggregation become toxic to cells. As a result, a variety of systemic and neurodegenerative diseases are associated with various aspects of protein aggregation and rational approaches to either inhibit aggregation or manipulate the pathways to aggregation might lead to an alleviation of disease phenotypes. To develop such approaches, one needs a rigorous and quantitative understanding of protein aggregation. Much work has been done in this area. However, several unanswered questions linger, and these pertain primarily to the actual mechanism of aggregation as well as to the types of inter-molecular associations and intramolecular fluctuations realized at low protein concentrations. It has been suggested that the concepts underlying protein aggregation are similar to those used to describe the aggregation of synthetic polymers. Following this suggestion, the relevant concepts of polymer aggregation are introduced. The focus is on explaining the driving forces for polymer aggregation and how these driving forces vary with chain length and solution conditions. It is widely accepted that protein aggregation is a nucleation-dependent process. This view is based mainly on the presence of long times for the accumulation of aggregates and the elimination of these lag times with “seeds”. In this sense, protein aggregation is viewed as being analogous to the aggregation of colloidal particles. The theories for polymer aggregation reviewed in this work suggest an alternative mechanism for the origin of long lag times in protein aggregation. The proposed mechanism derives from the recognition that polymers have unique dynamics that distinguish them from other aggregation-prone systems such as colloidal particles.     

The multi-mode polarization modulation spectrometer: part 1: simultaneous detection of absorption, turbidity, and optical activity

Circular dichroism (CD) is an important spectroscopic technique for monitoring chirality and biological macromolecule conformation. However, during a CD measurement, absorbance, light scattering/turbidity, and fluorescence can also be detected. The simultaneous measurement of these different spectral features for a single sample is the basis of a multi-mode optical spectrometer. This allows time-efficient gathering of complementary information and provides a scheme to ensure that CD measurements are reliable. Aspects of circular polarization differential light scattering, pH, and temperature variation of a protein (antibody) solution are described. A procedure to help ensure that CD measurements are reliable is described.     

Effects of congenital cataract mutation R116H on αA-crystallin structure,function and stability

α-crystallin is a molecular chaperone that maintains the optical properties of the lens and delays the onset scattering caused by aging-related protein aggregation. In this research, we found that the missense mutation R116H resulted in an altered size distribution, impaired packing of the secondary structures and modified quaternary structure with great hydrophobic exposure. The mutant exhibited a substrate-dependent chaperone (aggregation–inhibition) or anti-chaperone (aggregation–promotion) effect. Equilibrium unfolding experiments indicated that the mutation stabilized an aggregation-prone intermediate which was not populated during the unfolding of the wild-type protein. The accumulation of this intermediate greatly promoted the formation of non-native large oligomers or aggregates during unfolding. These results suggested that both the aggregation of the mutant upon stress and co-deposition with the target proteins were likely to be responsible for the onset of cataract.     

Effects of turbidity and light intensity on foraging success of juvenile mandarin fish Siniperca chuatsi (Basilewsky)

This study aimed to evaluate the effects of turbidity and light intensity on foraging success of juvenile mandarin fish Siniperca chuatsi. Predation on crucian carp Carassius auratus by juvenile mandarin fish was tested at five levels of turbidity combined with two light intensities, imitating daylight and night in two turbidity types. Foraging success was significantly lower in clay-induced turbidity than in algal-induced turbidity. In clay-induced turbidity trials, there was a slight but insignificant increase in foraging success of mandarin fish with increasing turbidity under lighted conditions. In algal-induced turbidity trials, there were no significant differences in foraging success of mandarin fish among turbidity levels at both light and dark levels, but at 80 NTU turbidity level, foraging success was lower than in all the other turbidity levels. There was no significant difference in foraging success at different turbidities under darkness. These results suggest that piscivory of mandarin fish is influenced by different turbidity types but is not significantly influenced by increased turbidity combined with decreased light intensity.     

Stretched-Exponential Analysis of Heat-Induced Aggregation of Apo-Concanavalin A

The kinetics of heat-induced aggregation of apo-concanavalin A (aConA) was investigated as a function of temperature and protein concentration by circular dichroism and turbidity. Heat-induced aggregation, as well as conformational change, of aConA was fitted to stretched-exponential equations. The exponent of the conformational change maintained 0.5 despite the protein concentration and temperature, indicating the presence of a common intermediate during the conformational change. After the process, aggregates grew with increasing temperature and initial protein concentration. The reaction order of aggregation was 1.5, indicating that the rate-limiting steps of aConA aggregation involve both conformational change and aggregation.     

Concanavalin A aggregation and toxicity on cell cultures

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which–as a consequence–turns out to be non-toxic.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. I: Biotinylated liposomes and avidin

The aggregation of biotin-modified phospholipid vesicles (liposomes) induced by binding the protein avidin in solution is analyzed experimentally and theoretically. Avidin has four binding sites that can recognize biotin specifically, and is able to cross-link the liposomes to form large aggregates. The aggregation kinetics were followed using quasi-elastic light scattering (QLS) to measure the mean particle size, and by measuring the solution turbidity. The rate and extent of aggregation were determined as a function of vesicle concentration, protein concentration, and the biotin density on the surface of the liposomes. A model based on Smoluchowski kinetics, fractal concepts, and Rayleigh and Mie light scattering theory was developed to analyze the experimental observations. Small aggregates (<7800 A diameter) may be treated as globular; however, the fractal nature of larger particles must be taken into account. Parameters in the model are taken from molecular simulations, or fit to the experimental observations. The aggregation kinetics are primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration. Good agreement is found between the model and the experimental results. (c) 1996 John Wiley & Sons, Inc.     

Small heat shock protein Hsp27 protects myosin S1 from heat-induced aggregation, but not from thermal denaturation and ATPase inactivation

We applied different methods, such as turbidity measurements, dynamic light scattering, differential scanning calorimetry and co-sedimentation assay, to analyze the interaction of small heat shock protein Hsp27 with isolated myosin head (myosin subfragment 1, S1) under heat-stress conditions. Upon heating at 43 degrees C, Hsp27 effectively suppresses S1 aggregation, and this effect is enhanced by mutations mimicking Hsp27 phosphorylation. However, Hsp27 was unable to prevent thermal unfolding of myosin heads and to maintain their ATPase activity under heat-shock conditions.     

Arginine controls heat-induced cluster-cluster aggregation of lysozyme at around the isoelectric point

The process of protein aggregation has attracted a great deal of research attention, as aggregates are first of all a nuisance to preparation of high quality protein and secondly used as novel materials. In the latter case, the process of protein aggregation needs to be controlled. Here, we show how arginine (Arg) regulates the process of heat-induced protein aggregation. Dynamic light scattering and transmission electron microscopy revealed that heat-induced aggregation of lysozyme at around the isoelectric point occurred in a two-step process: formation of start aggregates, followed by further growth mediated by their sticking with diffusion-limited cluster-cluster aggregation. In the presence of Arg, the diffusion-limited regime changed to reaction-limited cluster-cluster aggregation. The data indicated that the solution additives that coexisted with proteins would affect the property of the formed product, such as morphology and mechanic strength.     

Light scattering and turbidity measurements on lipid vesicles

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 °C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the change in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Effect of inclusion body production on culture turbidity and cell dry weight in growing bacterial cultures.

An approach to the optimization of product yield in an inducible inclusion body-producing system is presented. Following induction by indoleacrylic acid (IAA) of a trpLE-HIVgp41 fusion protein, we found a large increase in culture turbidity and single-cell dry weight. After an initial transition phase, new and constant values for specific growth rate, single-cell light turbidity, and single cell dry weight were achieved, allowing for the determination of optimal conditions for product formation.     

Effects of arginine on kinetics of protein aggregation studied by dynamic laser light scattering and tubidimetry techniques

Prevention of undesirable protein aggregation is an extremely important strategy in protein science, medicine, and biotechnology. Arginine is one of the most widely used low molecular weight solution additives effective in suppressing aggregation, assisting refolding of aggregated proteins, and enhancing the solubility of aggregation-prone unfolded molecules in vitro. However, the mechanism of suppression of protein aggregation by arginine is not well understood. To address the mechanism, two model systems have been investigated: protection of alcohol dehydrogenase (ADH) and insulin from heat- and dithiothreitol-induced aggregation, respectively, in the presence of arginine. Using dynamic light scattering (DLS) technique, we have demonstrated the concentration-dependent suppression of light scattering intensity of both ADH and insulin aggregates upon addition of arginine to the incubation medium, a significant effect being revealed in the physiological concentration range of arginine (1-10 mM). DLS studies showed that arginine shifted the populations of nanoparticles with higher hydrodynamic radii to the lower ones, suggesting that the preventive effect of arginine on the protein aggregation process arises because it suppresses intermolecular interactions among aggregation-prone molecules. The results of turbidity measurements were also shown to be consistent with these findings.     

Dinoflagellate growth rate in water columns of varying turbidity as a function of migration phase with daylight

Photosynthetic dinoflagellates occasionally exhibit nonphototacticphase relationships between their diurnal vertical migrationand the daylight cycle. The cause of this variability in specificfield cases is generally unclear. A computer model, MIGTIM,is reported that incorporates light dependence of the initialslope () and maximum photosynthesis (P_max) of the photosynthesisversus light intensity (P-I) curve. MIGTIM is used to determinethe migration phase that optimizes growth rate of sinusoidallymigrating populations under different conditions of photosyntheticlight dependence, turbidity and migration depth. Migration phaseprovides little selective advantage for shallow populationsor for populations in low turbidity water columns. Migrationphase provides a strong selective advantage for deep populationsor for populations in high turbidity water columns. A comparisonbetween MIGTIM output and published field observations suggeststhat field populations experienced optimum growth rate underthe observed environmental and biological conditions. The analysissupports the contention that, under appropriate conditions,water column turbidity is an important component of the complexequation that determines migration phase and that this componentacts through the organism's cumulative diurnal photosyntheticresponse.