Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance

Prokaryote-expressed polyomavirus structural protein VP1 with an N-terminal glutathione-S-transferase tag (GST-VP1) self-assembles into pentamer structures that further organize into soluble aggregates of variable size (3.4 × 10²–1.8 × 10⁴ kDa) [D.I. Lipin, L.H.L. Lua, A.P.J. Middelberg, J. Chromatogr. A 1190 (2008) 204]. The adsorption mechanism for the full range of GST-VP1 soluble aggregates was described assuming a dual-component model [T.Y. Gu, G.J. Tsai, G.T. Tsao, AICHE J. 37 (1991) 1333], with components differentiated by size, and hence pore accessibility, rather than by protein identity. GST-VP1 protein was separated into two component groups: aggregates small enough to access resin pores (LMW: 3.4 × 10²–1.4 × 10³ kDa) and aggregates excluded from the resin pores (HMW: 9.0 × 10²–1.8 × 10⁴ kDa). LMW aggregates bound to resin at a higher saturation concentration (29.7 g L⁻¹) than HMW aggregates (13.3 g L⁻¹), while the rate of adsorption of HMW aggregates was an order of magnitude higher than for LMW aggregates. The model was used to predict both batch and packed bed adsorption of GST-VP1 protein in solutions with known concentrations of HMW and LMW aggregates to Glutathione Sepharose HP resin. Asymmetrical flow field flow fractionation with UV absorbance was utilized in conjunction with adsorption experimentation to show that binding of HMW aggregates to the resin was strong enough to withstand model-predicted displacement by LMW aggregates. High pore concentrations of LMW aggregates were also found to significantly inhibit the diffusion rate of further protein in the resin pores. Additional downstream processing experimentation showed that enzymatic cleavage of LMW aggregates to remove GST tags yields more un-aggregated VP1 pentamers than enzymatic cleavage of HMW aggregates. This model can be used to enhance the chromatographic capture of GST-VP1, and suggests an approach for modeling chromatographic purification of proteins that have a range of quaternary structures, including soluble aggregates.     

Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.     

Ab initio quantum mechanical study of the binding energies of human estrogen receptor alpha with its ligands: an application of fragment molecular orbital method

We have theoretically examined the relative binding affinities (RBA) of typical ligands, 17beta-estradiol (EST), 17alpha-estradiol (ESTA), genistein (GEN), raloxifene (RAL), 4-hydroxytamoxifen (OHT), tamoxifen (TAM), clomifene (CLO), 4-hydroxyclomifene (OHC), diethylstilbestrol (DES), bisphenol A (BISA), and bisphenol F (BISF), to the alpha-subtype of the human estrogen receptor ligand-binding domain (hERalpha LBD), by calculating their binding energies. The ab initio fragment molecular orbital (FMO) method, which we have recently proposed for the calculations of macromolecules such as proteins, was applied at the HF/STO-3G level. The receptor protein was primarily modeled by 50 amino acid residues surrounding the ligand. The number of atoms in these model complexes is about 850, including hydrogen atoms. For the complexes with EST, RAL, OHT, and DES, the binding energies were calculated again with the entire ERalphaLBD consisting of 241 residues or about 4000 atoms. No significant difference was found in the calculated binding energies between the model and the real protein complexes. This indicates that the binding between the protein and its ligands is well characterized by the model protein with the 50 residues. The calculated binding energies relative to EST were very well correlated with the experimental RBA (the correlation coefficient r=0.837) for the ligands studied in this work. We also found that the charge transfer between ER and ligands is significant on ER-ligand binding. To our knowledge, this is the first achievement of ab initio quantum mechanical calculations of large molecules such as the entire ERalphaLBD protein.     

Improved selection of LMW over HMW proteins from human plasma by mesoporous silica particles with external modification

Selective extraction of low molecular weight (LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. Various methods, such as solid-phase extraction (SPE), ultrafiltration, and size-exclusion chromatography have been developed for such extraction purpose. In this study, we present, to our knowledge, the first demonstration of alkyl-diol@SiO₂ mesoporous material MCM-41 (alkyl-diol group on the external surface of mesoporous material) for selective extraction of LMW proteins and peptides from complex biological samples. The adsorption kinetics of LMW proteins, the influence of pH on adsorption and the desorption recovery by different elution solvents were investigated by using standard proteins as model samples. It was demonstrated that the modification of alkyl-diol group on the external surface could efficiently decrease the adsorption of HMW protein and increase the desorption recovery of LMW protein. Furthermore, the mesoporous materials were applied to selectively extract LMW proteins and peptides (<10 kDa) from crude human plasma. And the modified MCM-41 material had much better extraction selectivity and efficiency for LMW proteins and peptides than unmodified one.     

Bulk Metathesis Polymerization of Cyclooctene

The bulk ring-opening metathesis polymerization (ROMP) of cyclooctene initiated with the WCI₆/Sn(CH₃)₄ catalytic system was investigated at 40, 100, and 160°C using high vacuum techniques. The polymerizations were followed over a period of several days. Detailed analyses of the polymerization products by gel permeation chromatography (GPC) and ¹H and ¹³C NMR were carried out. Along with unsaturated high molecular weight (HMW) polymer (polyoctenamer), low molecular weight (LMW) polymer was found, the proportion of the latter increasing with time. The LMW fraction contains saturated LMW polymer together with ring polymer. The results are explained in terms of kinetic and thermody-namic arguments.     

Comparative Analysis of the Distribution of Copper in Green and Brown Brazilian Propolis Extracts

We proposed here a novel analytical procedure for copper speciation in green and brown propolis extracts using SEC—HPLC—GFAAS with 0.5% m v^?1 SDS in 2.5 m mol L^?1 Tris–HCl (pH 7.4) as the mobile phase buffer solution. Both basic (0.05 mol L^?1 NaOH) and acid (0.05 mol L^?1 HCl) conditions were evaluated for sample extraction. Depending on the extraction procedure, differences in copper distribution were identified. Copper was mainly associated with high-molecular-weight (HMW) fractions in green propolis extract when extracted with basic solution, whereas with acid extraction solution, only low-molecular-weight (LMW) fractions were obtained in both samples. Furthermore, combined analysis of results obtained using SEC-UV and GF AAS confirmed the association of copper with LMW and HMW species.     

Reconstituted olfactory receptors in bilayer lipid membranes

Bullfrog olfactory receptors were reconstituted in bilayer lipid membranes (BLMs). Three odorants were presented to the reconstituted system. The three structurally related odorants were diethylsulfide (DES), thiophene (THP) and diethanolsulfide (DOS). The ordorants were presented in pairs. DOS induced a response in the presence of either of the other two odorants. DES and THP did not induce a response in the presence of either of the other two odorants. These observations suggest that there are two substructures, one common to the three odorants and one that is unique to DOS. The results support the notion that olfactory receptors detect certain molecular segments of odorants.     

Does the supplementation of metal ions modify the intrinsic distribution of element species in food extracts?

Summary A combination is presented of gel permeation chromatography for the separation of proteins with atomic absorption spectrometry for the detection of protein-bound metals. For the evaluation of the effects resulting from sample spiking with free metal ions, flame AAS in the direct coupling mode was used. Besides sample spiking we also evaluated the effects of adding traces of metals to the eluent. The separation material was TSK HW-55(S), the bed volume was 27 ml. The investigations were carried out on the example of an aqueous soybean flour extract that had been characterized before in view of its intrinsic metal distributions. The supplementation experiments were focused on zinc and nickel, both having essential as well as toxic properties. For both metals a selective enhancement of metal concentrations was observed after sample spiking in distinct fractions due to free binding capacities of certain proteins. It was also possible to detect hydrated (free) metal ions. Zinc is bound to the medium molecular weight (MMW) fraction and to the high molecular weight (HMW) fraction, nickel to the HMW fraction only. A depletion of metal ions from the mobile phase was observed in case of the elution of proteins with free binding capacities.Published in extracts as a poster contribution on the occasion of the conference ANAKON 89, Baden-Baden, Federal Republic of Germany     

Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient

Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.     

The role of peptides and proteins in melanoidin formation

High‐molecular‐weight (HMW) coloured compounds called melanoidins are widely distributed, particularly in foods. It has been proposed that they originate through the Maillard reaction, a non‐enzymatic browning reaction, due to the interaction between protein or peptide amino groups and carbohydrates. The melanoidin structure is not definitively known, and they have been generally defined as HMW nitrogen‐containing brown polymers. In order to gain information on the nature of melanoidins, a simple in vitro model was chosen to investigate the products of the reactions between sugars and peptide/proteins. This approach would elucidate whether melanoidin formation is due to the binding of different sugar units to a peptide/protein or vice versa. With this aim, the reactivity of two different peptides, EPK177 and physalaemin, and a low‐molecular‐weight (LMW) protein, lysozyme, was tested towards different saccharides (glucose, maltotriose (MT), maltopentaose and dextran 1000) in aqueous solutions at different temperatures. The incubation mixtures were analysed at different reaction times by MALDI/MS. Furthermore, in order to verify the possible role of sugar pyrolysis products in melanoidin formation, the products arising from the thermal treatment at 200 °C of MT were incubated with lysozyme, and the reaction products were analysed by the same MS approach. The obtained results allowed the establishment of some general views: melanoidins cannot simply originate by reactions of sugar moieties with proteins. In fact, the reaction easily occurs, but it does not lead to any coloured product, as melanoidins have been described to be; melanoidins cannot originate from the thermal degradation products of glycated proteins. In fact, the thermal treatment of glycated lysozyme leads to a severe degradation of the protein with the formation of LMW species, far from the view of melanoidins as HMW compounds; experimental evidence has been gained on the melanoidin formation through reaction of intact protein with the pyrolysis products of MT. This hypothesis has been supported either from MALDI measurements or from spectroscopic data that show an absorption band in the range 300–600 nm, typical of melanoidins. Copyright © 2009 John Wiley & Sons, Ltd.     

Unexpected molecular weight dependence of shish kebab in water‐assisted injection molded HDPE

As a part of continuous efforts to systematically understand the morphological development in water‐assisted injection molding, high density polyethylene with different molecular weights was molded in this study. Unexpectedly, it was found that shish kebab with high lamellar and molecular orientations was formed in the sample with a lower molecular weight (LMW) rather than in the higher one, especially in the water channel layer. Present finding is obviously inconsistent with the general consensus, that is, higher molecular weight (HMW) polymer is much easier to form preferential orientation in flow field than LMW one. Such anomalous phenomenon is explained by the fact that even though melts experienced the same processing, lower shear rate is practically achieved in HMW sample due to its high viscosity. The result indicates that the flow history in industrial processing method is far from that in laboratory one. Copyright © 2012 John Wiley & Sons, Ltd.     

A quadruplet electrochemical platform for ultrasensitive and simultaneous detection of ascorbic acid,dopamine, uric acid and acetaminophen based on a ferrocene derivative functional Au NPs/carbon dots nanocomposite and graphene

In this work, a new nanomaterial of thiol functional ferrocene derivative (Fc-SH) stabilized Au NPs/carbon dots nanocomposite (Au/C NC) coupling with graphene modified glassy carbon electrode (Fc-S-Au/C NC/graphene/GCE) was fabricated to serve as a quadruplet detection platform for ultrasensitive and simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and acetaminophen (AC). The Au/C NC was synthesized by adding HAuCl₄ into carbon nanodots solution without using any additional reductant and stabilizing agent. Then the Fc-SH was utilized as the protective and capping agent to modify the Au/C NC. Transmission electron microscopy (TEM), UV–Vis, Fourier-transform infrared (FT-IR), scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) were adopted to characterize the morphology and electrochemical properties of the materials and the electrodes. The Fc-S-Au/C NC/graphene/GCE exhibits a synergistic catalytic and amplification effects towards oxidation of AA, DA, UA and AC owing to the existence of the nanomaterial and electron mediator. When simultaneous detection of AA, DA, UA and AC, the oxidation peak potentials of the four compounds on the electrode can be well separated and the peak currents were linearly dependent on their concentrations. The quadruplet detection platform shows excellent linear range and ultrasensitive response to the four components, the detection limits were estimated to be as low as 1.00, 0.05, 0.12 and 0.10 μM (S/N = 3), and the modified electrode exhibits excellent stability and reproducibility. The proposed electrode has been successfully applied to detect of these four analytes in real samples with satisfactory results.     

Mass spectrometric analysis of the S-layer proteins from Clostridium difficile demonstrates the absence of glycosylation

Like many other bacterial cell surfaces, the cell wall of Clostridium difficile is also encapsulated by a proteinaceous paracrystalline layer, the surface (S)-layer. In many bacterial species, the S-layer proteins (SLPs) have been shown to be glycosylated, whereas in other species glycosylation is absent. Unusually, the S-layer of C. difficile is composed of two distinct proteins, the high-molecular weight (HMW) and low-molecular-weight (LMW) SLPs. Previous investigations have reported that one or both of these SLPs are glycosylated, though no definitive study has been conducted. We have used a variety of mass spectrometric approaches to analyse SLPs from a number of strains of C. difficile for the presence of associated glycans. Analysis of intact SLPs by matrix assisted laser desorption/ionisation time of flight (MALDI-ToF) mass spectrometry demonstrated that the observed molecular masses matched the predicted masses of the LMW and HMW SLPs. Furthermore, analysis of Cyanogen bromide (CNBr) and tryptic peptides displayed no evidence of post-translational modification. In the first in-depth study of its kind, we unequivocally demonstrate that the S-layer proteins from the C. difficile strains investigated are not glycosylated.     

Cation and anion diversity in [M(dithiooxalate)2]2- salts: structure robustness in crystal synthesis

A series of crystalline salts based on the [M(dto)2]2- (dto = 1,2-dithiooxalate, M = Ni, Pt, Cu) dianion with hydrogen-bond donor cations have been synthesised following a molecular tectonics approach. The chelating M(dto)[dot dot dot]HN supramolecular synthon has been exploited in a systematic study of its robustness. The effects of competition between hydrogen-bond acceptors, of the shape and functionality of the cations and of varying the metal in the anion are discussed. The preparation and structural characterisation of the new crystalline phases [4,4'-H(2)bipy][Pt(dto)2] (2), [HNC5H4CO2H-4]2[Pt(dto)2] (5), [HNC5H4CO2H-3]2[Pt(dto)2] (6), [HNC5H4CH2CO2H-4]2[Ni(dto)2] (7), [HNC(5)H(4)CH(2)CO(2)H-3]2[Ni(dto)2] (8), [HNC5H4CONH2-4]2[Ni(dto)2] (9), [HNC5H4CHNOH-4]2[Ni(dto)2] (10), [HNC5H4CHNOH-3]2[Ni(dto)2] (11), [4,4'-H2bipip][Ni(dto)2] (12), [H2NC5H9CO2H-4]2[Pt(dto)2] (12), [H2NC5H9CO2H-4]2[Cu(dto)2] (14), [H2NC5H9CO2H-3]2[Ni(dto)2][H2O]2 (15), [H2NC5H9CO2H-3]2[Pt(dto)2][H2O]2 (16), [H2NC5H9CO2H-3]2[Cu(dto)2][H2O]2 (17), [H(Me)NC5H9CO2H-4]2[Ni(dto)2][H2O]2 (18) is reported. The charge-assisted NH[dot dot dot]dto synthon is formed in each of compounds 1-20, and is apparently much more robust than the conventional synthons used (such as the carboxylic acid dimer), which have a much lower rate of occurrence. The NH[dot dot dot]dto synthon may be generalised to 3- and 4-pyridinium species and 3- and 4-piperidinium derivatives. In the latter cases branching of the hydrogen-bond networks through the NH2 groups arises. The robustness of the NH...dto synthon allows structures of the form [NH cation]2[M(dto)2] to be regarded as being formed by the packing of neutral supermolecules. Cases of isomorphism (as in 16-18) and latent polymorphism (e.g. in 4 and 6) are noted.     

Characterization of wheat gluten proteins by hplc and MALDI TOF mass spectrometry

We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the ω-gliadin fractions (F1–4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5–8), 59 in the α- and β-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9–31) and 5 peaks in the γ-gliadin fractions (F32–35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.     

Hyaluronic acid‐dependent protection against alkali‐burned human corneal cells

Hyaluronic acid (HA) is a high‐molecular‐weight glycosaminoglycan and extracellular matrix component that promotes cell proliferation. This study aimed to evaluate the effects of HA on alkali‐injured human corneal epithelial cells in vitro, and to elucidate the mechanisms by which HA mediates corneal cell protection. A human corneal epithelial cell line (HCE‐2) was treated with sodium hydroxide before incubation with low‐molecular‐weight HA (LMW‐HA, 127 kDa) or high‐molecular‐weight HA (HMW‐HA, 1525 kDa). A global proteomic analysis was then performed. Our data indicated that HA treatment protects corneal epithelial cells from alkali injury, and that the molecular weight of HA is a crucial factor in determining its effects. Only HMW‐HA reduced NaOH‐induced cytotoxic effects in corneal cells significantly and increased their migratory and wound healing ability. Results from 2D‐DIGE and MALDI‐TOF/TOF MS analyses indicated that HMW‐HA modulates biosynthetic pathways, cell migration, cell outgrowth, and protein degradation to stimulate wound healing and prevent cell death. To our knowledge, our study is the first to report the possible mechanisms by which HMW‐HA promotes repair in alkali‐injured human corneal epithelial cells.     

Phase transfer of CdS nanocrystals mediated by heptamine β-cyclodextrin

A fundamental and systematic study on the fabrication of a supramolecularly assembled nanostructure of an organic ligand-capped CdS nanocrystal (NC) and multiple heptamine β-cyclodextrin ((NH(2))(7)βCD) molecules in aqueous solution has been here reported. The functionalization process of presynthesized hydrophobic CdS NCs by means of (NH(2))(7)βCD has been extensively investigated by using different spectroscopic and structural techniques, as a function of different experimental parameters, such as the composition and the concentration of CD, the concentration of CdS NCs, the nature of the NC surface capping ligand (oleic acid and octylamine), and the organic solvent. The formation of a complex based on the direct coordination of the (NH(2))(7)βCD amine groups at the NC surface has been demonstrated and found responsible for the CdS NC phase transfer process. The amine functional group in (NH(2))(7)βCD and the appropriate combination of pristine capping agent coordinating the NC surface and a suitable solvent have been found decisive for the success of the CdS NC phase transfer process. Furthermore, a layer-by-layer assembly experiment has indicated that the obtained (NH(2))(7)βCD functionalized CdS NCs are still able to perform the host-guest chemistry. Thus, they offer a model of a nanoparticle-based material with molecular receptors, useful for bio applications.     

磷酰化组氨酸特异性抗体的制备与应用

以4-碘吡唑和亚磷酸二乙酯为初始原料,经3步反应合成了新型的磷酰化组氨酸结构类似物,并以其为半抗原制备了磷酰化组氨酸特异性抗体(Anti-pHis).采用酶联免疫吸附(ELISA)、斑点印迹(Dot blot)和免疫印迹(Western blot)实验等方法对Anti-pHis的特异性进行了检测.结果表明,Anti-pHis对组氨酸磷酰化蛋白表现出优异的亲和力和选择性识别能力,并可应用于微生物细胞裂解液中组氨酸磷酰化蛋白的检测.     

基于RNA杂交的马铃薯纺锤块茎类病毒检测芯片

报道了一种检测植物类病毒RNA的新方法——RNA杂交芯片技术, 即将cDNA芯片技术与RNA斑点杂交技术相结合, 将马铃薯样品的总RNA直接固定在玻片上, 用荧光标记制备检测马铃薯纺锤块茎类病毒(PSTVd)的特异探针, 探针与芯片杂交后分析杂交信号以确定相应的样品有无PSTVd侵染. 参照膜杂交的方法, 确定了RNA芯片的制备条件, 并用以检测了马铃薯样品的PSTVd侵染情况, 检测结果与RT-PCR结果相符, 阳性产物经克隆测序证实为PSTVd.     

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.