The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.     

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.     

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed.     

Structural evolution of human recombinant alpha B-crystallin under UV irradiation

External stresses cause certain proteins to lose their regular structure and aggregate. In order to clarify this abnormal aggregation process, a structural evolution of human recombinant alphaB-crystallin under UV irradiation was observed with in situ small-angle neutron scattering. The abnormal aggregation process was identified to fall in three time zones: incubation, aggregation, and saturation. During the incubation time, the size of aggregates was almost unchanged but a deformation in the local structure was developing. After the incubation time, abnormal aggregation proceed. When the volume of the aggregates reached around twice the size as that of the initial aggregates, the aggregation rate slowed down, which marked the onset of saturation.     

Non-native aggregation of alpha-chymotrypsinogen occurs through nucleation and growth with competing nucleus sizes and negative activation energies

The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.     

Aggregation in cooked maize starch

This paper focuses on the aggregation of starch granules. The starch was cooked in a limiting amount of water and dispersed in different solvents. The size of the resulting aggregates was measured using light scattering. The solvents employed in this study (water, glycerol, ethanol, and 2-propanol) dispersed the aggregates to different degrees and resulted in different size distributions for the starch aggregates. It was observed that the size distribution of the cooked starch dispersed in the less polar solvents showed a higher representation of larger aggregates. Higher cooking temperatures resulted in larger aggregates. The methodology here presented proved to be a reproducible and practical way to study aggregation of starch.     

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed.     

Modeling aggregation and growth processes in an algal population model: analysis and computations

 Aggregation, the formation of large particles through multiple collision of smaller ones is a highly visible phenomena in oceanic waters which can control material flux to the deep sea. Oceanic aggregates more than 1 cm in diameter have been observed and are frequently described to consist of phytoplankton cells as well as other organic matter such as fecel pellets and mucus nets from pteropods. Division of live phytoplankton cells within an aggregate can also increase the size of aggregate (assuming some daughter cells stay in the aggregate) and hence could be a significant factor in speeding up the formation process of larger aggregate. Due to the difficulty of modeling cell division within aggregates, few efforts have been made in this direction. In this paper, we propose a size structured approach that includes growth of aggregate size due to both cell division and aggregation. We first examine some basic mathematical issues associated with the development of a numerical simulation of the resulting algal aggregation model. The numerical algorithm is then used to examine the basic model behavior and present a comparison between aggregate distribution with and without division in aggregates. Results indicate that the inclusion of a growth term in aggregates, due to cell division, results in higher densities of larger aggregates; hence it has the impact to speed clearance of organic matter from the surface layer of the ocean. Received 1 July 1994; received in revised form 23 February 1996     

Formation of fibrous aggregates from a non-native intermediate: the isolated P22 tailspike beta-helix domain.

In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel beta-helix, is essentially structured in this species. We used the isolated beta-helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model for fluorescence quenching in light harvesting complex II in different aggregation states

Low-temperature (77 K) steady-state fluorescence emission spectroscopy and dynamic light scattering were applied to the main chlorophyll a/b protein light harvesting complex of photosystem II (LHC II) in different aggregation states to elucidate the mechanism of fluorescence quenching within LHC II oligomers. Evidences presented that LHC II oligomers are heterogeneous and consist of large and small particles with different fluorescence yield. At intermediate detergent concentrations the mean size of the small particles is similar to that of trimers, while the size of large particles is comparable to that of aggregated trimers without added detergent. It is suggested that in small particles and trimers the emitter is monomeric chlorophyll, whereas in large aggregates there is also another emitter, which is a poorly fluorescing chlorophyll associate. A model, describing populations of antenna chlorophyll molecules in small and large aggregates in their ground and first singlet excited states, is considered. The model enables us to obtain the ratio of the singlet excited-state lifetimes in small and large particles, the relative amount of chlorophyll molecules in large particles, and the amount of quenchers as a function of the degree of aggregation. These dependencies reveal that the quenching of the chl a fluorescence upon aggregation is due to the formation of large aggregates and the increasing of the amount of chlorophyll molecules forming these aggregates. As a consequence, the amount of quenchers, located in large aggregates, is increased, and their singlet excited-state lifetimes steeply decrease.     

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.     

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.     

Concentration dependent Cu2+ induced aggregation and dityrosine formation of the Alzheimer's disease amyloid-beta peptide

The Amyloid beta peptide (Abeta) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu2+ ions can induce the de novo aggregation of the Abeta peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu2+ mediates the change from amyloid material toward Cu2+ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Abeta1-42 at neutral pH is governed by the Cu2+:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu2+ switching effect centered at equimolar Cu2+:peptide ratios. At sub-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms both small spherical oligomers approximately 10-20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu2+ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu2+ induced aggregates are toxic when compared to Abeta1-42 aged in the absence of Cu2+. Importantly, the formation of dityrosine crosslinked Abeta, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu2+ induced aggregates. These results define the role Cu2+ plays in modulating the aggregation state and toxicity of Abeta1-42.     

Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).     

Fractal aggregation of porcine fumarase induced by free radicals

The present study demonstrates that H(2)O(2) and OH(.-) cause fibril aggregation and catalytic inactivation of porcine fumarase. In the aggregated (oxidized) enzyme, modifications in both secondary and tertiary protein structure occur and the enzyme aggregation obeys to fractal geometry. We then collected information on the fractal dimension and on the size and shape of fumarase aggregates by using Synchrotron Radiation (SR) Small Angle X-ray Scattering (SAXS) analysis. The geometrical self-similarity assessment of aggregates has been revealed by both AFM and SEM measurements at different scale of magnification. Micrographs collected remarkably demonstrate that the oxidized enzyme shows dendritic fractal structure over a large range of sizes.     

How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.     

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag‐time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size‐exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.     

Viral aggregation: mixed suspensions of poliovirus and reovirus.

The aggregation of mixtures of two dissimilar viruses, poliovirus I (Mahoney) and reovirus III (Dearing), was followed by electron microscopy under conditions known to induce either aggregation or dispersion of each virus separately. Neither virus aggregated at pH 7 in an appropriate buffer, and no mixed aggregates were formed. Under conditions of lowered ionic strength (by dilution into distilled water) poliovirus became aggregated, whereas reovirus did not, and again no mixed aggregates were formed. At pH 6, however, poliovirus again aggregated and, although reovirus did not, it attached to poliovirus aggregates. Thus, some inducement toward aggregation was necessary to cause formation of mixed aggregates. This inducement probably took the form of a reduction of the ionic double layer surrounding the particles, which is known to occur at low pH. At pH 5 and below both viruses aggregated severely, and large mixed aggregates were formed. These mixed aggregates could be broken up by neutralization of the suspension, although small aggregates of poliovirus remained. Reovirus showed a marked tendency to attach to large clumps of poliovirus, but the reverse tendency was not observed. The results indicate that mixed aggregates may be of significance in the isolation of viruses from water or wastewater.     

Viral aggregation: mixed suspensions of poliovirus and reovirus.

The aggregation of mixtures of two dissimilar viruses, poliovirus I (Mahoney) and reovirus III (Dearing), was followed by electron microscopy under conditions known to induce either aggregation or dispersion of each virus separately. Neither virus aggregated at pH 7 in an appropriate buffer, and no mixed aggregates were formed. Under conditions of lowered ionic strength (by dilution into distilled water) poliovirus became aggregated, whereas reovirus did not, and again no mixed aggregates were formed. At pH 6, however, poliovirus again aggregated and, although reovirus did not, it attached to poliovirus aggregates. Thus, some inducement toward aggregation was necessary to cause formation of mixed aggregates. This inducement probably took the form of a reduction of the ionic double layer surrounding the particles, which is known to occur at low pH. At pH 5 and below both viruses aggregated severely, and large mixed aggregates were formed. These mixed aggregates could be broken up by neutralization of the suspension, although small aggregates of poliovirus remained. Reovirus showed a marked tendency to attach to large clumps of poliovirus, but the reverse tendency was not observed. The results indicate that mixed aggregates may be of significance in the isolation of viruses from water or wastewater.