Designed chain architecture for enhanced migration resistance and property preservation in poly(vinyl chloride)/polyester blends

Blends of poly(vinyl chloride) (PVC) and poly(butylene adipate) (PBA) with varying degrees of branching were analyzed with respect to migration resistance during aging in water, preservation of material properties, and thermal stability. Gas chromatography-mass spectrometry, water absorption, weight loss, thermogravimetric analysis, Fourier transform infrared spectroscopy, contact angle, tensile testing, and differential scanning calorimetry were used to analyze the blends before and after aging in water for 6 weeks. Films plasticized with slightly branched polyester maintained their material and mechanical properties best during aging. High degree of branching was accompanied by poor miscibility, increased hydrophilicity, and polydispersity, and highly branched PBA was not favorable as a plasticizer. Strong intermolecular interactions reduced the water absorption and increased the migration resistance of the blends. Polymeric plasticizers with no, low, or moderate degree of branching improved the thermal stability of films compared to films plasticized with a traditional phthalate plasticizer. Proper design of plasticizer architecture led, thus, to improved migration resistance, long-term properties, and thermal stability in PVC/polyester blends.     

Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.     

The cytokine stimulating activity of (1-->3)-beta-D-glucans is dependent on the triple helix conformation

The immunomodulating properties of comb-like branched (1-->3)-beta-D-glucans scleroglucan, schizophyllan and lentinan depend on branching pattern, molecular weight and higher-order structure. The effect of weight average molecular weight Mw and higher order structure of scleroglucan, on stimulation of human monocytes cultured in vitro to secrete tumor necrosis factor-alpha (TNF-alpha) was investigated. The higher order structures of the scleroglucan samples were determined by electron microscopy. The data showed that the samples with a linear wormlike, triple helical structure with Mw less than 50 x 10(4) g/mol or larger than 110 x 10(4) g/mol stimulated the monocytes more efficiently than samples with Mw in the range (67-110) x 10(4) g/mol. The denaturation of the linear triple helices by NaOH (> 0.25 M), followed by neutralization yielded blends of linear and macrocyclic topologies with concomitant irreversible reduction of the cytokine inducing activity compared with the untreated scleroglucans. The dose-dependent ability to activate monocytes to cytokine production was not restored following annealing of the denatured-renatured samples, despite the fact that electron micrographs revealed similar structures of these annealed samples to the starting material. Pre-incubation of monocytes with antibodies against cluster of differentiation antigens CD14 or CD11b reduced the scleroglucan potency to stimulate TNF-alpha secretion mainly for mAb against CD14 in the presence of serum.     

Reactivity of Limulus amoebocyte lysate towards (1----3)-beta-D-glucans

The structure activity relationship for beta-D-glucans for the gelation of the amoebocyte lysates of the horseshoe crab (Limulus) has been investigated. beta-D-Glucans that had no (1----3) linkages induced little or no gelation. The (1----3)-beta-D-glucans curdlan (unbranched), grifolan (approximately 33% branched), schizophyllan (approximately 33% branched), lentinan (approximately 40% branched). SSG (approximately 50% branched), and OL-2 (approximately 66% branched) induced significant gelation. The optimum concentration for gelation was correlated with the content of branching. Single chain (rather than a triple helix) conformation and higher molecular weight were associated with higher reactivity.     

Exploring the (1 → 3)‐β‐D‐glucan conformational phase diagrams to optimize the linear to macrocycle conversion of the triple‐helical polysaccharide scleroglucan

The immunologically important (1 → 6) comb‐like branched (1 → 3)‐β‐D ‐glucans scleroglucan, schizophyllan, lentinan, and others, exist mainly as linear triple‐helical structures in aqueous solution. Partial interconversion from linear to circular topology has been reported to take place following conformational transition of the triple‐helical structure and subsequent regeneration of the triplex conformation. We here report on experimental data indicating that complete strand separation of the triple‐helical structure is required for this interconversion. NaOH or dimethylsulfoxide was used to induce dissociation of the triplex at combinations of concentrations and temperatures shown by calorimetry to yield a conformational transition of the triplex structures. For the alkaline treatment at 55°C, it is found that up to about 30% of the material readily can be converted to the cyclic topology. This fraction increased to about 60% when the subsequent annealing of the scleroglucan in aqueous solution at pH 7 was carried out at 100°C. Further increase of the annealing temperature yielded a smaller relative amount of cyclic species. The data indicate that the lower molecular weight fraction of the molecular weight distributions can be converted selectively to the macrocyclic topology by conditions that do not yield complete strand separation of the whole sample. These findings add to previous reports by providing more details about how the conditions required for the linear triplex to macrocycle interconversion relate to the conformational properties of the triple‐helical structure. © 1999 John Wiley & Sons, Inc. Biopoly 50: 496–512, 1999     

Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.     

A study of the glucofructofuranan from the New Zealand cabbage tree Cordyline australis

Extraction of the roots of the New Zealand cabbage tree Cordyline australis with water gave a glucofructofuranan in 60% yield (dry-weight basis). Viscosity measurements on aqueous solutions of the polysaccharide, and vapor pressure osmometry of the polysaccharide peracetate, showed the number average molecular weight of the glucofructofuranan to be 3000. Complete hydrolysis with dilute acid gave only -fructose and -glucose, in the ratio of 16:1. The polysaccharide was methylated by using dimethyl sulfoxide—sodium hydroxide—methyl iodide, and the methylated polymer was hydrolyzed to give 1,3,4,6-tetra-O-methylfructose (5.6 mol), 2,3,4,6-tetra-O-methylglucose (1 mol), 1,3,4-tri-O-methylfructose (8.4 mol), 2,3,4-tri-O-methylglucose (0.1 mol), and 3,4-di-O-methylfructose (2.7 mol). These results, supported by ¹³C-n.m.r. analyses, showed that the polymer is a highly branched glucofructofuranan containing mainly (1→2)-linked β- -fructofuranosyl residues, with branching at O-6 of 15% of the -fructosyl residues.     

NOSTOCACEAN MACRO‐MORPHOGENESIS

A poorly understood feature of nostocacean growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. Using macro‐photography, time‐lapse micro‐cinematography, light and electron microscopy of Nostoc species in pure culture, it has been possible to demonstrate how motility, adhesion and aggregation of photo‐induced hormogonia result in macro‐morphogenesis of dendroid forms. Red‐light induced hormogonia from synchronized cultures aggregate rapidly on agar as tight flowing streams, in patterns responsive to the direction and quality of incident light. Unlike the even textured cell surfaces of heterocystous filaments, the cell walls of swarming hormogonia are covered with a striate mucoid layer containing pili attached to cells of adjacent hormogonia. During differentiation to an aseriate phase, cell wall fusions occur and a gelatinous matrix forms around the enlarging sub‐globose cells. Liquid suspensions of hormogonia aggregate in a solid mass following the net centripetal movement of interlaced loops of curved hormogonia attached by adhesive pili. In darkness or dim white light, compressed hormogonial aggregates form erect tree‐like (dendroid) macro‐structures by photo‐tactic reversal of streaming motility. Hormogonia within the aggregates re‐organize into streams that push upward into the light, forming structured, positively phototropic protuberances, several millimeters in length. Under weak illumination, the structures become branched with crowns of waving hormogonia. The dendroid morphology is stabilized by deposit of gelatinous material derived from successive cycles of cell‐filament development, liberation of heterocysts and formation of dormant cells and trichomes.     

Disaggregation of high‐molecular weight species during downstream processing to recover functional monomer

The use of chaotropic agents to recover functional monomeric material was investigated for the downstream purification of an Fc‐fusion protein containing high levels of high‐molecular weight (HMW) species. In batch studies, chaotropic agents irreversibly disaggregated a majority of the aggregated protein. An integrated processing mode, termed as on‐column disaggregation, was developed in which the protein was captured on Protein A chromatography and then a chaotropic agent was used to simultaneously elute the bound protein and disaggregate the HMW species. On‐column disaggregation process resulted in protein recoveries of >95% and aggregation reduction of ～50%. Analytical results are presented showing that the recovered monomeric material was comparable to the reference protein in biochemical, biophysical, and pharmacokinetic properties. The kinetic and molecular mechanisms governing protein aggregation and disaggregation will also be elucidated. For the Fc‐fusion protein studied here, incorporation of the disaggregation strategy in both batch and on‐column modes led to an increase of >10% in overall downstream yield. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cell Wall Metabolism in Ripening Fruit: IV. Characterization of the Pectic Polysaccharides Solubilized during Softening of ;Bartlett' Pear Fruit

Fractionation of pectic polysaccharides from the juice of ripening `Bartlett' pears (Pyrus communis) gave two general types of polyuronides. The major type was a homogalacturonan (HGA) whose molecular weight decreased upon ripening. The other type comprised heteropolymers composed of various amounts of arabinose, rhamnose, and galactose. Treatment of the major arabinose-containing heteropolymeric fraction of high molecular weight (400,000) with a pear exo-polygalacturonase to degrade contaminating HGA gave a polyuronide which was inert to tomato endopolygalacturonase. Glycosyl-linkage analysis of this arabinosyl-polyuronide gave results expected from a rhamnogalacturonan I-like polysaccharide with large, highly branched araban side chains (RG-I). A linkage between HGA and RG-I was not found. RG-I, in ripening pears, appeared to be degraded with the initial loss of much of its arabinose.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.     

Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch‐like polyglucans

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water‐insoluble and semi‐crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water‐soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water‐insoluble, partly crystalline, amylose‐containing starch‐like polyglucan was restored in GlgB‐expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water‐soluble, poorly crystalline polyglucan.     

Starch biosynthesis: sucrose as a substrate for the synthesis of a highly branched component found in 12 varieties of starches

D-[14C]glucose was incorporated into starch when 12 varieties of starch granules were incubated with [14C]sucrose. Digestion of the 14C-labeled starches with porcine pancreatic alpha amylase showed that a high percentage (16.1-84.1%) of the synthesized starch gave a relatively high molecular weight alpha-limit dextrin. Hydrolysis of the 12 varieties of starch granules by alpha amylase, without sucrose treatment, also gave an alpha-limit dextrin, ranging in amounts from 0.51% (w/w) for amylomaize-7 starch to 8.47% (w/w) for rice starch. These alpha-limit dextrins had relatively high molecular weights, 2.47 kDa for amylomaize-7 starch to 5.75 kDa for waxy maize starch, and a high degree of alpha-(1-->6) branching, ranging from 15.6% for rice starch to 41.1% for shoti starch. ADPGlc and UDPGlc did not synthesize a significant amount (1-2%) of the branched component, suggesting that sucrose is the probable substrate for the in vivo synthesis of the component and that sucrose is not first converted into a nucleotide-glucose diphosphate intermediate.     

Bioengineering and Application of Novel Glucose Polymers

Abstract

Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value.     

Replica exchange molecular dynamics simulation of cross‐fibrillation of IAPP and PrP106‐126

Aggregation of proteins into amyloid is the central hallmark of a number of protein diseases. Most studies were carried out on the aggregation between proteins of similar species. However, it was observed that some patients with certain protein disease can easily acquire another unrelated protein disease. As such, it is also important to examine aggregation between proteins of different species. Usually aggregation between proteins of the same species can be attributed to the similarity between their respective amino acid sequences. In this article, we were motivated by an experimental study of aggregation between amylin (Islet Amyloid Polypeptide, IAPP) and prion_106‐126 (PrP106‐126) fragment (JACS, 2013, 135, 13582–9). It was found that the two non‐homologous peptides can aggregate quickly to form fibrils in the presence of negatively charged lipid bilayer. We attempted to elucidate the molecular mechanism of the early stage of dimerization of these two peptides through extensive replica exchange molecular dynamics simulations. Conformations consisting of various degrees of β‐sheets structures, both intra‐chain and inter‐chain, were found in the simulations. The conformations of the aggregated complex are very diverse, which suggests that the cross‐species fibrils formed between the two proteins are highly polymorphic. The driving forces are mainly hydrophobic interactions, including aromatic‐aliphatic interactions. The palindromic region of PrP106‐126 and SNNFGAIL region of IAPP were found to play important roles in the interaction. Our study sheds insight into the exciting research of protein cross‐fibrillation. Proteins 2016; 84:1134–1146. © 2016 Wiley Periodicals, Inc.     

Ultracentrifugal Analysis of Staphylococcal Alpha Toxin

Ultracentrifugal examination of staphylococcal alpha toxin at different stages of purification showed the presence of a major component having a sedimentation coefficient of 2.8S, present to the extent of more than 90% of the sample, and identifiable with active toxin. Several minor components having S(20,w) values of 11.5S, 8.5S, and 2.0S were detected. The 11.5S component presumably is identical with a toxin aggregate studied earlier and designated 12S; the 8.5S component appears to be delta toxin. A sedimentation equilibrium study of more highly purified material gave 32,700 as the best estimate of molecular weight of alpha toxin. Lowering the pH of the partially purified alpha toxin from 10.2 to 5.3 resulted in a small increase in S(20,w) of the 11.5S component and in the disappearance of the 8.5S component, whereas the S(20,w), molecular weight, and hemolytic activity of the toxin remained constant. Exposure of toxin to pH 3.5 irreversibly reduced the S(20,w) to 2.0S, the molecular weight to about 16,000, and caused irreversible inactivation. Raising the pH of acid-inactivated toxin and adding sodium dodecyl sulfate to 1% increased the S(20,w) to near its normal value (2.7S) but did not restore activity.     

Bioreducible poly(amido amine)s with different branching degrees as gene delivery vectors

Based on the knowledge that cationic polymers with different topographical structures behave differently in gene transfection process, herein, we synthesized three biodegradable poly(amido amine)s (PAAs) with the same repeating units and molecular weights except for degree of branching: linear PAA (LPAA), low‐branched PAA (LBPAA), and high‐branched PAA (HBPAA). We found that LBPAA could more effectively compact pDNA into positively charged nanoparticles than both HBPAA and LPAA. LBPAA polyplexes had the highest transfection efficiency among the three PAA polyplexes, and the difference in transfection efficiency is mainly attributed to the endocytosis rate. The cytotoxicity of PAAs was negligible at the transfection doses, probably due to the degradable disulfide bonds. Therefore, we could use branching as a parameter to simply tune a polymer's cellular uptake behavior and transfection efficiency. Biotechnol. Bioeng. 2013; 110: 990–998. © 2012 Wiley Periodicals, Inc.     

Effects of substrate branching characteristics on kinetics of enzymatic depolymerizaion of mixed linear and branched polysaccharides: I. Amylose/amylopectin alpha-amylolysis

Hydrolysis reactions of homopolysaccharides, which differ in their degree of branching, and mixtures of linear and branched polymers were carried out with alpha-amylase. The branching structures of both the original amylopectin substrate and the cluster domains of amylopectin, obtained by ethanol precipitation of the products of the action of alpha-amylase, were characterized via enzymatic digestion with debranching enzyme (i.e., isoamylase), followed by the fractions of the resulting products using gel filtration chromatography. The structural properties (i.e., molecular weight, molecular weight distribution, and branching characteristics) of the resulting products during depolymerization of amylose, amylopectin and their mixtures via alpha-amylase were characterized by size exclusion chromatography coupled with a low angle laser right scattering (SEC/LALLS) technique. It was determined that substrate branching characteristics strongly influence both the observed enzymatic activity as well as the enzyme's action pattern. A simplified kinetic model that represents the hydrolysis reactions of amylose and amylopectin mixtures via endo-acting alpha-amylase is proposed. We found that that reaction kinetics (i.e., enzyme affinity) was also governed by the substrate's conformation in solution. The relationships between the mass fraction of branched polymers and the kinetic parameters during alpha-amylolysis were compared with those predicted by the kinetic model. Excellent agreement was found between the model predictions and the experimental observations. The results reported here imply and interrelationship between enzyme action and polymeric substrate structural properties. (c) 1994 John Wiley & Sons, Inc.     

Effects of different organic waste amendments on soil microaggregates stability and molecular sizes of humic substances

Three soils which had been amended for several years with pig slurry, cattle slurry, and sewage sludge were dry-sieved to obtain microaggregates in the size range of 250–125, 125–50, and <50 μm. With amendments, aggregate size distribution of whole soils was shifted to larger sizes, especially for the most fragile soil, whereas percent content of microaggregates decreased except for the lower size aggregates of the fragile soil. Particle size distribution of microaggregates revealed an increase in percent sand and a reduction of percent silt and clay in the <50 μg size fraction for all soils. These results showed the aggregation effect induced by the organic waste additions. Aggregate stability of microaggregates revealed significant correlation with humic substances content (humic acids alone and humic plus fulvic acids) and non significant with total organic matter substantiating the belief that humic substances are the predominant binding agents in this aggregation range. Molecular weight distribution of humic acids extracted from microaggregates of unamended soils demonstrated that the lower the soil aggregate size distribution, the larger the contribution of the high molecular weight fraction. All microaggregates from amended soils showed a progressive increase of the high molecular weight humic acids with decreasing size, reaching a maximum in the <50 μm fraction. In this aggregate size a parallel enhancement of the aggregate stability was also evident. It is concluded that a close relationship exists between aggregate stability and high molecular weight humic substances. Additions to soils of organic material containing high molecular weight constituents would represent a useful management practice to improve aggregate stability.