Effect of salt addition on the fractal structure of aggregates formed by heating dilute BSA solutions

The fractal dimension, Df, of aggregates in a dilute BSA system with added salt was evaluated by static light scattering (SLS). A fractal structure was observed for the system with NaCl addition. The values of Df increased with increasing heating time and ionic strength. The values of Df were larger than those (Df = 1.8 or 2.1) predicted by the conventional cluster-cluster aggregation model, probably due to a "restructuring" of aggregates during the aggregation process. On the other hand, a fractal structure was not apparent for the system with added CaCl2.     

Self-similarity properties of alpha-crystallin supramolecular aggregates.

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.     

Microbial growth patterns described by fractal geometry.

Fractal geometry has made important contributions to understanding the growth of inorganic systems in such processes as aggregation, cluster formation, and dendritic growth. In biology, fractal geometry was previously applied to describe, for instance, the branching system in the lung airways and the backbone structure of proteins as well as their surface irregularity. This investigation applies the fractal concept to the growth patterns of two microbial species, Streptomyces griseus and Ashbya gossypii. It is a first example showing fractal aggregates in biological systems, with a cell as the smallest aggregating unit and the colony as an aggregate. We find that the global structure of sufficiently branched mycelia can be described by a fractal dimension, D, which increases during growth up to 1.5. D is therefore a new growth parameter. Two different box-counting methods (one applied to the whole mass of the mycelium and the other applied to the surface of the system) enable us to evaluate fractal dimensions for the aggregates in this analysis in the region of D = 1.3 to 2. Comparison of both box-counting methods shows that the mycelial structure changes during growth from a mass fractal to a surface fractal.     

Amino acid sequence of porcine heart fumarase

The complete amino acid sequence of porcine heart fumarase (EC 4.2.1.2) has been determined from peptides produced by cyanogen bromide, endoproteinase Arg-C, S. aureus V8 protease, and trypsin. The enzyme is a tetramer of identical subunits with Mr = 50,015 and composed of 466 amino acid residues. Porcine heart fumarase displays 96% identity to human liver fumarase. Prediction of the secondary structural elements of porcine fumarase indicate that the enzyme contains a large amount of alpha helix with very little beta structure.     

Low frequency ultrasound induces aggregation of porcine fumarase by free radicals production

Hydrogen peroxide and hydroxyl-free radicals determine a diffuse aggregation of porcine fumarase and a loss of its enzymatic activity. In this study, hydroxyl-free radicals were generated "in situ" by irradiation with ultrasound (US) at 38 kHz. The structural characteristics of aggregated fumarase were studied using circular dichroism spectroscopy (CD) and steady state fluorescence spectroscopy. Enzyme aggregation is caused by the formation of intermolecular disufide bridges, originated by the oxidation of cysteine residues, together with a diffuse increase in beta-turn in the protein's secondary structure. These conformational changes lead to a fibrous, amyloid-like aggregation which appears ordered and regular under TEM microscopy.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.

The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.

During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.

Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.

This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.     

Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

Fractal dimensions and porosities of Zoogloea ramigera and Saccharomyces cerevisae aggregates

The fractal nature microbial aggregates is a function of the type of microorganism and mixing conditions used to develop aggregates. We determined fractal dimensions from length-projected area (D(2)) and length-number scaling (D(3)) relationships. Aggregates of Zoogloea ramigera developed in rotating test tubes were both surface and mass fractals, with fractal dimensions of D(2) = 1.69 +/- 0.11 and D(3)= 1.79 +/- 0.28 (+/-standard deviation), respectively. When we grew this bacteria in a bench-top fermentor, aggregates maintained their surface fractal characteristics (D(2) = 1.78 +/- 0.11) but lost their mass fractal characteristics (D(3) = 2.99 +/- 0.36). Yeast aggregates (Saccharomyces cerevisae) grown in rotating tests tubes had higher average fractal dimensions than bacterial aggregates grown under physically identical conditions, and were also considered fractal (D(2) = 1.92 +/- 0.08 and D(3) = 2.66 +/- 0.34). Aggregates porosity can be expressed in term of a fractal dimensions, but average porosities are higher than expected. The porosities of yeast aggregates (0.9250-0.9966) were similar to porosities of bacterial aggregates (0.9250-0.9966) cultured under the same physical conditions, although bacterial aggregates developed in the reactor had higher average porosities (0.9857-0.9980). These results suggest that that scaling relationships based on fractal geometry may be more useful than equations derived from Euclidean geometry for quantifying the effects of different fluid mechanical environments on aggregates morphology and characteristics such as density, porosity, and projected surface area.     

Colloidal properties of human transferrin receptor in detergent free solution

The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge.     

Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D2O. After a large number of cations were screened, it was found that (+)(R) alpha-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R) alpha-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site (i.e., the C3 carbon of malate). This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.     

Enzymatic removal of oxidized protein aggregates from erythrocyte membranes

Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.     

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.     

Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity

Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.     

Carbanilation of cereal beta-glucans for molecular weight determination and conformational studies

Cereal beta-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal beta-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal beta-glucans was developed. Wheat beta-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal beta-glucans were prepared by the reaction of cereal beta-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: approximately 2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal beta-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal beta-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d(f)=2.27) and the structure sensitive parameters (rho >2) suggested a porous globular structure for partially carbanilated beta-glucans.     

Decreased subunit exchange of heat-treated lens alpha A-crystallin

alpha A-Crystallin high-molecular-weight (HMW) aggregates were prepared by preheating at 80-90 degrees C and studied using spectroscopic measurements. Conformational differences were suggested based on data of increased bis-ANS (4,4(')-dianilino-1,1(')-binaphthalene-5,5(')-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW aggregated alpha-crystallin was more hydrophobic than the native alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. The two cysteines in alpha A-crystallin were mostly oxidized in HMW aggregates. The effects of HMW aggregation on the dynamic structure were studied with fluorescence resonance energy transfer; subunit exchange became slower. These results strongly suggest that HMW alpha A-crystallin aggregates result from exposure of buried beta-pleated sheets and increased hydrophobic interaction.     

毛竹杉木混交林土壤团粒结构的分形特征研究

运用分形理论对杉木纯林（19年生）、毛竹纯林（19年生）、6种杉木毛竹混交林（杉木19年生）的土壤团粒结构进行研究,建立了土壤团粒结构的分形维数与土壤团聚体含量、土壤结构体破坏率之间的回归模型,在此基础上,运用弹性分析与边际分析法进一步探讨了土壤团粒结构的分形维数对土壤性质变化所产生的影响效应。结果表明:土壤>0.25mm水稳定性团聚体含量越大,团粒结构的分形维数越小,土壤的结构与稳定性就越好;土壤团粒结构分形维数与水稳定性团聚体（>0.25 mm及>5.0mm）含量及结构体破坏率之间均存在极显著的回归关系;土壤团粒结构分形维数对土壤性质的变化产生较大的影响效应。本研究为毛竹杉木混交林的栽培管理,土壤肥力的科学评价提供依据。     

Cellular oligomerization of alpha-synuclein is determined by the interaction of oxidized catechols with a C-terminal sequence

The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.     

DNA-Triggered Aggregation of Copper,Zinc Superoxide Dismutase in the Presence of Ascorbate

The oxidative damage hypothesis proposed for the function gain of copper, zinc superoxide dismutase (SOD1) maintains that both mutant and wild-type (WT) SOD1 catalyze reactions with abnormal substrates that damage cellular components critical for viability of the affected cells. However, whether the oxidative damage of SOD1 is involved in the formation of aggregates rich in SOD1 or not remains elusive. Here, we sought to explore the oxidative aggregation of WT SOD1 exposed to environments containing both ascorbate (Asc) and DNA under neutral conditions. The results showed that the WT SOD1 protein was oxidized in the presence of Asc. The oxidation results in the higher affinity of the modified protein for DNA than that of the unmodified protein. The oxidized SOD1 was observed to be more prone to aggregation than the WT SOD1, and the addition of DNA can significantly accelerate the oxidative aggregation. Moreover, a reasonable relationship can be found between the oxidation, increased hydrophobicity, and aggregation of SOD1 in the presence of DNA. The crucial step in aggregation is neutralization of the positive charges on some SOD1 surfaces by DNA binding. This study might be crucial for understanding molecular forces driving the protein aggregation.     

Nutritional regulation of organelle biogenesis inEuglena: Photo- and metabolite induction of mitochondria

Exposure of dark-grown restingEuglena gracilis Klebs var.bacillaris Cori to light, ethanol, or malate produced an increase in the specific activity of fumarase (EC. 4.2.1.2) and succinate dehydrogenase (EC. 1.3.99.1) during the first 8–12 h of exposure to inducer, followed by a decrease in the specific activity of both mitochondrial enzymes between 12 and 72 h. The increased specific activity represented a net increase in the level of active enzyme, and it was dependent upon cytoplasmic protein synthesis. The photoinduction of fumarase required continuous illumination while the subsequent decrease in fumarase specific activity was independent of light. Light had little effect on the ethanol and malate induction of fumarase and succinate dehydrogenase. In the mutant W₃BUL, which has no detectable protochlorophyll(ide) and chloroplast DNA, light induced both mitochondrial enzymes and the kinetics of enzyme induction were similar to the induction kinetics in wild-type cells. The induction of mitochondrial enzymes appears to be controlled by a non-chloroplast photoreceptor. Dark-grown resting cells of the plastidless mutant W₁₀SmL have lost the ability to regulate fumarase levels. In this mutant, the specific activity of fumarase fluctuated and light had little effect on these fluctuations, indicating that fumarase synthesis was uncoupled from the nonchloroplast photoreceptor. Ethanol addition produced transient changes in fumarase specific activity in W₁₀SmL indicating that in this mutant, mitochondrial enzymes are still inductible by metabolites. Fumarase synthesis in wild-type cells was not induced in the dark by levulinic acid, a chemical inducer of the breakdown ofEuglena storage carbohydrates. Taken together, our results indicate that the photoinduction of mitochondrial enzyme synthesis is not a result of the photoinduction of carbohydrate breakdown. The mechanisms by which light and organic carbon induce the synthesis ofEuglena mitochondria may differ.