Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Hydroxyapatite chromatography of phage-display virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.     

Isolation of a novel agglutinin with complex carbohydrate binding specificity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji.

A hemagglutinin, with a molecular weight of 30,000 and expressing hemagglutinating activity which could not be inhibited by simple sugars and glycoproteins, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. The protein was adsorbed on CM-Sepharose even in 20 mM ammonium acetate (pH 5.5) containing 1 M NaCl and was desorbed by 20 mM ammonium bicarbonate (pH 9). The hemagglutinating activity was subsequently adsorbed on Mono S in 20 mM ammonium acetate (pH 5.5) and was desorbed by a linear gradient of 0.2-0.5 M NaCl in ammonium acetate buffer. The hemagglutinin exhibited a novel N-terminal sequence not found in any lectin and hemagglutinin reported so far. It was devoid of antifungal activity.     

红壤中镉在有机酸作用下的解吸行为

采用平衡批处理法,研究了3种有机酸及其两两混合液在序列pH值梯度下（pH 3.0～7.0）对华南山地红壤Cd解吸行为的影响.结果表明,草酸与苹果酸不利于Cd的解吸,反而促进了吸附,其中草酸只是在较高浓度(20 mmol·L^-1)且土壤溶液pH＞5.0时促进解吸.随着pH值升高,草s酸、苹果酸以及不含有机酸的对照溶液对红壤中Cd的解吸率都快速下降.柠檬酸在pH＜5.0时不利于Cd解吸;在pH＞5.0时显著促进Cd解吸,但两种浓度柠檬酸解吸特征有所不同,在低浓度(2 mmol·L^-1)下对镉的解吸率呈降低-升高-降低变化,在高浓度(20 mmol·L^-1)下呈降低-升高变化.在低pH条件下（pH 3.0、4.0）,苹果酸最有利于Cd的解吸,但3种酸对Cd解吸率差别不大,在较高pH条件下(pH 5.0～7.0),柠檬酸最有利于解吸,且解吸率大大高于草酸与苹果酸.有机酸混合没有明显的交互作用,对Cd的解吸率介于相应单独有机酸之间.     

How the surface functionalized nanoparticles affect conformation and activity of proteins: Exploring through protein-nanoparticle interactions

To understand the effect of counter ions (Na⁺) on the secondary conformation and functionality of the lysozyme, we have studied the interaction of lysozyme with counterion associated iron oxide nanoparticles (IONPs). The investigation was carried out at pH 7.4 and 9.0, with three different types of NPs, namely, bare IONPs, low molecular weight chitosan modified IONPs (LMWC-IONPs) and the counterion (Na⁺) associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs) and confirmed by using various spectroscopy techniques. The difference in UV–vis absorbance (ΔA) between native and STP-LMWC-IONPs interacted hen egg white lysozyme (HEWL) was greater than that between native and NPs interacted HEWL at pH 9.0 compared with pH 7.4. Furthermore, STP-LMWC-IONPs exhibited quenching effect on lysozyme fluorescence spectrum at pH 9.0 due to binding of Na⁺ counterions to the protein, confirming denaturation of the latter. After HEWL interaction with STP-LMWC-IONPs (pH 9.0), CD spectra revealed a conformational change in the secondary structure of HEWL. Also, counterion induced lysozyme inactivation, due to interaction with nanoparticles at pH 9.0, was confirmed by enzymatic activity assay involving lysis of Micrococcus lysodeikticus. In conclusion, pH 9.0 was observed to be a more favorable condition, compared to pH 7.4, for the strongest electrostatic interaction between lysozyme and NPs. We postulate that the counterions in nanoparticle surface-coating can ameliorate protein misfolding or unfolding and also prevent their aggregation and, therefore, can be considered as a powerful and potential therapeutic strategy to treat incurable neurodegenerative disorders.     

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.     

Silica nanoparticles based label-free aptamer hybridization for ATP detection using hoechst33258 as the signal reporter

In this work, we have developed a simple and sensitive method for ATP detection using silica nanoparticles (NPs) as the platform and hoechst33258 as the signal reporter. The ATP-binding aptamers hybridize with the probe DNA (DNA(p)) immobilized NPs to form the aptamer/DNA(p) duplex on the NPs surface. The conformational change of the aptamer leads to the decrease of the aptamer/DNA(p) duplex on the NPs due to the ATP-binding aptamer switches its structure from the aptamer/DNA(p) duplex to the aptamer/target complex in the presence of ATP. ATP detection can be easily realized by separating the silica nanoparticles and adding the hoechst33258 of intercalating to aptamer/DNA(p) (dsDNA). Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between ATP aptamer and ATP. The K(d) was estimated to be ～1mM from 0 to 4mM and a liner response was observed from 0 to 0.2mM with a detection limit of ～20μM. Compared with other methods, the carboxyl-modified silica nanoparticles (～60nm) prepared by the reverse microemulsion method can serve as a stable and sensitive sensor platform because of their smaller size and facile conjugation with amine-containing molecules. In addition, the high sensitivity and selectivity of hoechst33258 was employed for the ssDNA and dsDNA determination, which takes advantage of the label-free aptamer and lower cost.     

A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3

A xylanase-coding gene (xynAHJ3, 1,104 bp) was cloned from Lechevalieria sp. HJ3 harbored in a saline soil sampled from Heijing town, aka the "town of salt", on the famous "Silk Route of the South". The gene encodes a 367-residue polypeptide (XynAHJ3) with the highest identity of 74.0 % with the endoxylanase from Streptomyces thermocarboxydus HY-15. The coding sequence of the mature protein (without the predicted signal peptide from M1 to S22) of xynAHJ3 was expressed in Escherichia coli BL21 (DE3). The activity of the purified recombinant XynAHJ3 (rXynAHJ3) was apparently optimal at 70 °C and pH 6.0, retained greater than 55 % xylanase activity at a concentration of 0.2-2.0 M Na(+) and 26 % at 4.0 M Na(+) (pH 7.5 20 °C), and showed 110.2 and 44.2 % xylanase activities in the presence of 100 mM SDS (pH 6.0 37 °C) and 10 % ethanol (pH 5.0 37 °C), respectively. rXynAHJ3 activity was stable at 50 °C and pH 4.0-11.0 for more than 60 min, in trypsin or proteinase K at 20 °C for 24 h (pH 7.5), in 10 % ethanol (v/v) (pH 5.0) at 30 or 37 °C for 72 h, in 80 % ethanol (v/v) for 1 h, and in 0.6 or 3 M NaCl (20 °C, pH 7.5) for 72 h. Compared with the majority of xylanases with tolerance to ethanol, salt, SDS, or protease (K (m) values of 1.42-15.1 mg ml(-1)), rXynAHJ3 showed a low K (m) value (0.8 mg ml(-1)) and showed only limited amino acid sequence identity with those other xylanases (less than 47 %).     

Colorimetric determination of urinary adenosine using aptamer-modified gold nanoparticles

This paper describes a colorimetric sensing approach for the determination of adenosine triphosphate (ATP) using aptamer-modified gold nanoparticles (Apt-Au NPs). In the absence of the analytes, the color of the Apt-Au NPs solution changed from wine-red to purple as a result of salt-induced aggregation. Binding of the analytes to the Apt-Au NPs induced folding of the aptamers on the Au NP surfaces into four-stranded tetraplex structures (G-quartet) and/or an increase in charge density. As a result, the Apt-Au NPs solution was wine-red in color in the presence of the analytes under high salt conditions. For mixtures of ATP (20.0–100.0 nM), Apt-Au NPs (3.0 nM), 10.0% poly(ethylene glycol), 0.2 μM TOTO-3, 150.0 mM NaCl, 15.0 mM KCl, and 16.0 mM Tris–HCl (pH 7.4), a linear correlation (R² = 0.99) existed between the ratio of the extinctions of the Apt-Au NPs at 650 and 520 nm (Ex_650/520) and the concentration of ATP. The limit of detection for ATP was 10.0 nM. The practicality of this simple, sensitive, specific, and cost-effective approach was demonstrated through the determination of the concentration of adenosine in urine samples.     

Thermal stability studies of a globular protein in aqueous poly(ethylene glycol) by (1)H NMR

The reversible folding destabilization of hen lysozyme has been confirmed by a melting temperature (T(m)) decrease in aqueous poly(ethylene glycol) (PEG). The percent denatured, extracted from the histidine 15 C2H (H15 C2H) native and denatured peak areas from 500-MHz one-dimensional proton nuclear magnetic resonance (1D (1)H NMR) spectra in D(2)O, was analyzed through denaturation temperatures at 0% and 20% (w/w) PEG 1000. The lysozyme (3.5 mM) T(m) decreased by 4.2 degrees C and 7.1 degrees C in 20% (w/w) PEG 1000 at pH 3.8 and 3.0, respectively. The T(m) decreased with increasing lysozyme concentration. Additionally, the temperature-induced resonance migrations of 17 protons from 8 residues indicate that the native lysozyme structure undergoes temperature-induced conformational changes. The changes were essentially identical in both 0% and 20% (w/w) PEG 1000 at both pH 3.0 and 3.8. This small, local restructuring of the hydrophobic box region may be a manifestation of temperature-dependent solution hydrophobicity, whereas active-site cleft fluctuations may be due to the inherent active-site flexibility. The lysozyme structure in PEG at 35 degrees C was determined to be essentially native from the (1)H nuclear Overhauser effect spectroscopy (NOESY) fingerprint regions. Additionally, lysozyme chemical shifts, from 1D spectra, in PEG 200, 300, and 1000 at 35 degrees C and various concentrations were essentially identical, further confirming that the conformation remains native in various PEG solutions. (c) 1996 John Wiley & Sons, Inc.     

Selective adsorption/desorption of nucleic acids on submicron-sized polymeric particles

DNA can be removed or separated by the selective adsorption/desorption on positively charged submicronsized polymeric particles (SSPP). The selective adsorption of DNA, in the presence of protein, on positively charged SSPP was accomplished by increasing the concentration of potassium phosphate or sodium phosphate. The adsorption of DNA was not affected by the concentration of potassium phosphate or sodium phosphate up to 1.2M. On the other hand, the adsoprtion of a protein (bovine serum albumin) was completely impeded by 170mM potassium phosphate. DNA adsorbed on SSPP could be desorbed by increasing the concentration of NaCl or KCl, thus it can be recovered. DNA desorbed from SSPP when the concentration of NaCl or KC was higher than 0.6M. A complete desorption of DNA was achieved at the concentration of NaCl or KCl above 1.2M.     

Purification of human leukocyte elastase and cathepsin G by chromatography on immobilized elastin

Human leukocyte elastase and cathepsin G were isolated from purulent sputum by a simple procedure involving chromatography on elastin-agarose. Salt extracts of sputum were prepared, treated with DNase, and the precipitate which formed extracted and applied to a column of soluble elastin-Sepharose 4B. Contaminating protein was eluted with 50 mM Tris, 50 mM NaCl, pH 8.0 and then two column volumes of 50 mM acetate, 1.0 M NaCl, pH 5.0. The tightly bound elastase and cathepsin G together with a trypsin-like serine protease could finally be eluted with 50 mM acetate, 1.0 M NaCl, 20% DMSO, pH 5.0. Resolution of the proteases was accomplished by cation-exchange chromatography. Disc gel electrophoresis established the purity of elastase and cathepsin G and confirmed the existence of several isozymes for each.     

Heparin-binding properties of lactoferrin and lysozyme.

1. Binding of biotin-heparin to immobilized lactoferrin and lysozyme was optimum at pH 6.0, 100 mM NaCl. Complex interactions between NaCl and CaCl2 concentrations were observed for heparin binding to both proteins. 2. The metal ions Cu2+, Zn2+, Fe2+ and Fe3+ inhibited heparin binding, with half-maximal inhibition of binding to lactoferrin occurring between 600 microM and 1 mM and for lysozyme between 500 and 800 microM. 3. Binding of biotin-heparin to both proteins was inhibited to varying degrees by heparin, heparan sulfate, chondroitin sulfate A, dextran sulfate and DNA.     

Nonenzymatic amperometric response of glucose on a nanoporous gold film electrode fabricated by a rapid and simple electrochemical method

An enzyme-free amperometric method was established for glucose detection using a nanoporous gold film (NPGF) electrode prepared by a rapid one-step anodic potential step method within 5 min. The prepared NPGF had an extremely high roughness and was characterized by scanning electron microscopy (SEM) and cyclic voltammetry. Electrochemical responses of the as-prepared NPGF to glucose in 0.1M phosphate buffer solution (PBS, pH 7.4) with or without Cl(-) were discussed. In amperometric studies carried out at -0.15 V in the absence of Cl(-), the NPGF electrode exhibited a high sensitivity of 232 μA mM(-1)cm(-2) and gave a linear range from 1mM up to 14 mM with a detection limit of 53.2 μM (with a signal-to-noise ratio of 3). In addition, the oxidation of ascorbic acid (AA) and uric acid (UA) can be completely eliminated at such a low applied potential. On the other hand, the quantification of glucose in 0.1M PBS (pH 7.4) containing 0.1M NaCl offered an extended linear range from 10 μM to 11 mM with a sensitivity of 66.0 μA mM(-1)cm(-2) and a low detection limit of 8.7 μM (signal-to-noise ratio of 3) at a detection potential of 0.2V.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification of recombinant enhanced green fluorescent protein expressed in Escherichia coli with new immobilized metal ion affinity magnetic absorbents

A new immobilized metal ion affinity (IMA) adsorbent containing superparamagnetic nanoparticles and coated with hydrophilic resins are proposed here to improve the purification of His-tagged proteins. The magnetic chelating resin was prepared by radical polymerization of magnetite (Fe3O4), styrene, divinyl benzene (DVB) and glycidyl methacrylate-iminodiacetic acid (GMA-IDA) in ethanol/water medium. IDA is immobilized on magnetite as a ligand and pre-charged Cu2+, Zn2+ and Ni2+ as metal ions. To identify the GMA-IDA magnetic particles easily, we named these particles MPGI. The MPGI adsorbent was used to test their suitability for the direct recovery of an intracellular, polyhistidine-tagged protein, enhanced green fluorescent protein [EGFP-(His)(6)], from Escherichia coli lysates in a single step. Parameters influencing the purification efficiencies such as pH, ionic strength and imidazole concentration were optimized to achieve improved separation. The optimal selectively was observed in binding buffer (0.2M NaCl, 0.02M imidazole), washing buffer (0.4M NaCl, 0.03 M imidazole) and elution buffer (0.50M imidazole). The Cu2+-charged MPGI adsorbent had the highest yield and purification factor at 70.4% and 12.3, respectively. The calculated isotherm parameters (Q(m)=53.5 mg/g, K(d)=5.84 mg/mL and Q(m)/K(d)=9.2 mL/g) indicated that the MPGI adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution.     

Studies on the structure of isolated chromatin in three different solvents.

Properties of calf thymus chromatin, prepared by mild procedures, have been studied in various solvents. In 0.2 mM EDTA s-values ranged from 20 to 30 S and intrinsic viscosities from 5 to 24 dl/g. Dialysis against 0.15 M NaCl or 0.2 mM MgCl2 changed these values to 80 to 100 S and 0.2 to 5 dl/g, respectively, indicating an essentially more compact structure. In 0.2 mM EDTA X-ray scattering yielded a cross section diameter of 9 nm, which is associated with the tertiary structure of chromatin fiber (M/L = 21200 Dalton/nm). By dialysis against 0.15 M NaCl or 0.2 mM MgCl2 part of the material spontaneously formed quarterny structures (cross section diameters 25-29 nm). The rest of the material with cross section diameters less than 9 nm is supposed to be more strongly sheared tertiary structure which seems to be unable to form quarterny structure due to artificial conformational changes.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA.

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.