Non-electrostatic Binding Research Articles

Capacitive Deionization Systems for Water Desalination Applications: Role of the Electrosorption Resistance and Non-Electrostatic Binding in the Porous Electrodes

The unprecedented global water scarcity and shortage of fresh water have drawn attention to an abundant yet overlooked water resource: Brackish ground water. Although the common desalination methods are energy intensive for low salinity water streams, Capacitive Deionization (CDI) offers a highly efficient and economical alternative for desalinating brackish water. In CDI, an electrically charged porous material is employed to remove ionic species from aqueous solutions. CDI includes different multi-scale transport phenomena and requires robust theories to accurately model its performance. In this work, we introduce a dynamic two-dimensional model which couples the diffusion and advection in the bulk solution with the diffusion and electrosorption in the porous material. The significance of this model lies in the incorporation of the transport theories with the adsorption/desorption resistances and non-constant chemical attraction forces in the micropores. A lab-scale CDI unit is employed to validate the proposed model at different transport time scales and electrochemical techniques are used to characterize these systems. Efficiency of the studied cases is also evaluated using the proposed inclusive figure-of-merits, where both numerical and experimental data present a trade-off between adsorption and energetic performance of the systems with low and high mass Péclet number. The results obtained here highlight the significant role of the rate limiting resistance for the ionic species transferring from macropores into the micropores in the porous electrode, especially at low initial concentrations.

Effects of electrostatic interactions on ligand dissociation kinetics.

We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.

Genomic RNA Binding Promotes Retroviral Gag Protein Interactions in an Assembly-Competent Conformation Leading to Selective Genome Packaging

In HIV-1 infected cells, full-length viral genomic RNA (gRNA) is selectively packaged by the HIV-1 Gag protein despite a vast excess of spliced viral and host RNAs. The mechanism of this selective packaging is incompletely understood but a region of gRNA known as “Psi” mediates specific Gag interaction. HIV-1 Gag binds Psi and non-Psi RNA with similar affinity under physiological salt concentration (∼150 mM NaCl), but the salt dependence of these two binding events differs dramatically (Webb, JA, et al, RNA 2013). HIV-1 Gag binds Psi RNA with a strong non-electrostatic binding component and a small effective charge (Zeff ∼ 5). In contrast, HIV-1 Gag binds non-Psi RNA with a very weak non-electrostatic binding component and a larger effective charge (Zeff ∼ 9). In this work, we use salt-titration binding assays to study the effect of various Gag and Psi RNA mutations on the Psi and non-Psi binding interactions of HIV-1 and Rous sarcoma virus Gag proteins. Our findings are consistent with a model in which Gag binding to cognate Psi RNA shifts the equilibrium from a non-assembling to an assembling Gag state, providing an advantage for nucleation of assembly only on gRNA.

DNA-binding studies of valrubicin as a chemotherapy drug using spectroscopy and electrochemical techniques

In this study, the molecular interactions between valrubicin, an anticancer drug, and fish sperm DNA have been studied in phosphate buffer solution (pH 7.4) using UV–Vis spectrophotometry and cyclic voltammetry techniques. Valrubicin intercalated into double stranded DNA under a weak displacement reaction with methylene blue (MB) molecule in a competitive reaction. The binding constant (kb) of valrubicin-DNA was determined as 1.75×103L/mol by spectrophotometric titration. The value of non-electrostatic binding constant (kt0) was almost constant at different ionic strengths while the ratio of kt0/kb increased from 4.51% to 23.77%. These results indicate that valrubicin binds to ds-DNA via electrostatic and intercalation modes. Thermodynamic parameters including ΔH0, ΔS0 and ΔG0 for valrubicin-DNA interaction were determined as −25.21×103kJ/mol, 1.55×102kJ/mol K and −22.03kJ/mol, respectively. Cyclic voltammetry study shows a pair of redox peaks for valrubicin at 0.45V and 0.36V (vs. Ag/AgCl). The peak currents decreased and peak positions shifted to positive direction in the presence of DNA, showing intercalation mechanism due to the variation in formal potential.

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain.

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA coating agent, called C8-BSso7d, which binds to and coats with high specificity and affinity, individual DNA molecules as well as folded origami nanostructures. C8-BSso7d coats and protects without condensing, collapsing or destroying the spatial structure of the underlying DNA template. C8-BSso7d combines the specific nonelectrostatic DNA binding affinity of an archeal-derived DNA binding domain (Sso7d, 7 kDa) with a long hydrophilic random coil polypeptide (C8, 73 kDa), which provides colloidal stability (solubility) through formation of polymer brushes around the DNA templates. C8-BSso7d is produced recombinantly in yeast and has a precise (but engineerable) amino acid sequence of precise length. Using electrophoresis, AFM, and fluorescence microscopy we demonstrate protein coat formation with stiffening of one-dimensional templates (linear dsDNA, supercoiled dsDNA and circular ssDNA), as well as coat formation without any structural distortion or disruption of two-dimensional DNA origami template. Combining the programmability of DNA with the nonperturbing precise coating capability of the engineered protein C8-BSso7d holds promise for future applications such as the creation of DNA-protein hybrid networks, or the efficient transfection of individual DNA nanostructures into cells.

Study of Ribavirin–Nucleic Acids Interaction

The UV–Vis absorption spectra of ribavirin in the absence and presence of calf thymus DNA are presented and discussed in this paper. The molecular structure of ribavirin was investigated by the semiempirical AM1 method, which triggered two polymorphic modifications of the antiviral drug also reported in the literature. Our experimental results point out two types of the binding. The first type involves a non-electrostatic (internal) binding, consisting of the intercalation of drug between the nitrogenous bases of nucleic acid. The second type (an external binding) involves the drug binding to the nucleic acid grooves. In addition, the binding constant of the second process has an order of magnitude greater than the binding constant for the first process, calculated by the Benesi–Hildebrand, Scott, and Scatchard methods, which supposes a 1:1 binding ratio. Also, the interactions of two polymorphic modifications of ribavirin (V1 and V2) with nucleic acids by the molecular mechanic and semiempirical AM1 methods were analyzed. The experimental data pointed out that in the ribavirin–nucleic acid complexes, the 1,2,4-triazole-3-carboxamide chromophore is intercalated between the bases of the nucleic acid sequences, the carboxamidic group is set outside of the nucleic acid sequence toward the major groove, and the 2-hydroxymethyl-tetrahydrofuran-3,4-diol fragment is located in the minor groove. In order to stress the sequence specificity of ribavirin, different models of the nucleic acid sequences containing adenine (A), thymine (T), cytosine (C), and guanine (G) in AAAAAA, TTTTTT, CCCCCC, GGGGGG, ATATAT, CGCGCG, ATCGAT, and CGATCG were used. The theoretical results outline the differences in the contributions of the electrostatic and van der Waals interactions to the total binding energy and the preference of ribavirin for the binding at the sequences of nucleic acids containing adenine and thymine bases.

Impregnation and Swelling of Wood with Salts: Ion Specific Kinetics and Thermodynamics Effects

The effects of salt impregnation are studied within wood cell walls, occurring upon soaking from concentrated salt solutions. Osmotic deswelling is in some cases followed by ion specific swelling linked to Hofmeister effects. Taking into account microstructure, this study models the free energy changes associated with the ions and water uptake at molecular, colloidal, and macroscopic mechanical scales, to show that slow swelling until osmotic equilibrium originates from charge separation of the salt diffused into wood cell wall material. Kinetic effects as well as mechanical effects linked to transfer of species and swelling of the interstitial matrix between cellulose crystals are discussed. Predictions by minimal models taking into account nonelectrostatic ion complexation allow to estimate the order of magnitude of the nonelectrostatic binding free energy of adsorbed chaotropic anions and complexed divalent cations to be 8 and 10 kJ mole–1, respectively.

Fluorescence anisotropy-based salt-titration approach to characterize protein-nucleic acid interactions.

Many proteins bind nucleic acids (NA) via cationic residues that interact electrostatically with the anionic phosphate backbone of RNA or DNA. These electrostatic interactions are often insensitive to NA sequence and structure, but confer strong salt dependence to the binding interactions. In contrast, salt-independent non-electrostatic contacts reflect more specific binding interactions. Proteins with multiple cationic NA-binding domains connected by flexible linkers, such as the HIV-1 Gag polyprotein, may bind different NA molecules in distinct ways. For example, Gag binding to the Psi-packaging signal of the HIV-1 RNA genome optimizes the specific non-electrostatic binding component of this protein-RNA interaction. In contrast, Gag binding to a non-psi RNA optimizes the electrostatic interactions at the expense of specific contacts. Here, we describe a fluorescence anisotropy-based salt-titration approach that allows complete characterization of both electrostatic and non-electrostatic binding components for any protein-NA complex in a quantitative manner within a single assay.

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC.

DNA binding properties and cytotoxic activities of two trinuclear ruthenium(II) polypyridyl complexes

The interactions between two trinuclear Ru(II) complexes and calf thymus DNA(CT DNA) were studied via absorption spectroscopy, reverse salt titrations, binding stoichiometry, DNA melting experiments, as well as viscosity measurement. The results indicate that complexes 1 and 2 bind to DNA via the interaction of the planar π-delocalized system of the complexes with intrinsic binding constants of 4.18×105 and 3.85×106 L/mol, respectively, and non-electrostatic binding free energy makes a predominant contribution to the binding free energy. The in vitro cytotoxic activity of complexes 1 and 2 was evaluated by the MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]_method. Complex 1 shows higher anticancer potency than complex 2 against four tumor cell lines. Further mechanism study indicates that complexes 1 and 2 can cause cell cycle arrest in the G2/M phase.

Computing the Electrophoretic Mobility of Large Spherical Colloids by Combining Explicit Ion Simulations with the Standard Electrokinetic Model

The electrophoretic mobility of large spherical colloids in different salt solutions of varying valency and concentration is studied via a combination approach of numerically solving the standard electrokinetic model with a ζ potential that has been obtained from explicit ion simulations of the restricted primitive model, thus going beyond the standard mean-field treatment. We compare our theoretical mobility curves to two distinct sets of experimental results and obtain good agreement for monovalent and divalent salt solutions. For the case of the trivalent La(3+) salt, the experimentally obtained mobility reversal at high ionic strengths can be obtained only by adding an additional attractive interaction of 4k(B)T to the potential between the colloid and La(3+), hinting at the presence of a nonelectrostatic binding term for this ion. It is also shown that, contrary to intuition, charge inversion does not necessarily result in mobility reversal.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

Antimicrobial and nuclease activity of mixed polypyridyl ruthenium(II) complexes

Abstract The ruthenium(II) complexes with 2,9-dimethyl-1,10-phenanthroline (dmphen) and terpyridine derivatives have been synthesized and screened for their antimicrobial potency. The interaction of these complexes with Herring Sperm DNA was investigated by thermal denaturation, viscosity measurements, gel electrophoresis and spectrophotometric methods. The results indicate that the complexes bound to DNA via partial intercalative mode. The salt-dependent binding of these complexes has been determined by UV–Vis spectrophotometric titration. The contribution of the non-electrostatic binding free energy (ΔG t o ) to the total binding free energy change (ΔG o ) is found to be ~ 88% at [Na + ] = 0.075 M. The large value suggests that the stabilization of the DNA binding is mostly due to the contribution of non-electrostatic process.

ChemInform Abstract: Conformational Regulation of Amyloid β‐Peptide by Lipid Membranes and Metal Ions

AbstractReview: 28 refs.

脂質膜と金属イオンによるアミロイドβペプチドのコンホメーション制御

Conformational transition of monomeric amyloid beta-peptide (Abeta) to a self-associated beta-sheet structure is considered to be an initial step in the development of Alzheimer's disease. Several lines of evidence suggest that physiologically abundant lipid membranes and metal ions are involved in this step. We have demonstrated that Abeta binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the liquid crystalline phase by using fluorescence and circular dichroism spectroscopy. The membrane-bound Abeta molecule takes alpha-helical or beta-sheet structure depending on the temperature. Tightly packed phosphatidylcholine membranes appear to serve as a platform for non-electrostatic binding and self-association of Abeta. We have also examined Zn(II) and Cu(II) binding modes of Abeta by Raman spectroscopy. The Raman spectra demonstrate that three histidine residues in the N-terminal region of Abeta provide primary metal binding sites. Zn(II) binds to the N(tau) atom of histidine and the peptide aggregates through intermolecular His-Zn-His bridges. In contrast, Cu(II) ion is chelated by the N(pi) atom of histidine and deprotonated main-chain amide nitrogens to form a soluble complex. Our findings on the conformational regulation of Abeta may help in better understanding the molecular basis for the development of Alzheimer's disease.

Non-electrostatic binding and self-association of amyloid β-peptide on the surface of tightly packed phosphatidylcholine membranes

Self-association of amyloid β-peptide (Aβ) is considered to be an initial step in the development of Alzheimer’s disease and is known to be promoted by negatively charged lipid membranes. We have examined the possibility of non-electrostatic Aβ–membrane interaction by using neutral phosphatidylcholine lipids. Fluorescence and circular dichroism spectra have clearly shown that Aβ binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the ripple gel or liquid crystalline phase, indicating the importance of the tight lipid packing characteristic of the lamellar gel phase. The Aβ–membrane binding occurs at both low and high salt concentrations, ensuring the non-electrostatic nature of the interaction. The membrane-bound Aβ molecule takes a monomeric α-helical or self-associated β-sheet structure depending on the temperature, peptide/lipid ratio, and salt concentration. The flat surface of tightly packed phosphatidylcholine membranes appears to serve as a platform for non-electrostatic binding and self-association of Aβ.

DNA Binding of Iron(II)-Phenanthroline Complexes: Effect of Methyl Substitution on Thermodynamic Parameters

The influence of methyl substitution on the thermodynamic parameters for the binding of [Fe(DMP)3]2+ and [Fe(TMP)3]2+ (DMP = 4,7-dimethyl-1,10-phenanthroline, TMP = 3,4,7,8-tetramethyl- 1,10-phenanthroline) to calf thymus DNA (ct-DNA) has been studied by determining their equilibrium binding constants (Kb) at various salt concentrations and temperatures. Kb of the iron(II) complexes to ct-DNA decreases with the salt concentration in the solution, suggesting considerable electrostatic interaction in the ct-DNA binding of the iron(II) complexes. In contrast, Kb of the DNA binding increases with temperature, indicating that the DNA binding reaction of the complex is endothermic and entropically driven. The evaluation of the non-electrostatic binding constant (K0 t) based on polyelectrolyte theory has revealed that the K0 t portions of the total binding constant (Kb) are relatively large and reach 46.4% for [Fe(DMP)3]2+ at [Na+] = 0.075 M and 43.9% for [Fe(TMP)3]2+ at [Na+] = 0.100 M. The contribution of non-electrostatic binding free energy (ΔG0 t ) to total binding free energy change (ΔG0) is extremely large, i. e. > 90% for both iron(II) complexes at [Na+] = 0.05 M, suggesting that the stabilization of the DNA binding is mainly contributed from the non-electrostatic process. The effect of methyl substitution on electrostatic (ΔG0 pe) and non-electrostatic (ΔG0 t ) binding free energy changes has been systematically evaluated using the quantity of ΔΔG0 pe and ΔΔG0 t relative to that of the parent iron(II) complex, [Fe(phen)3]2+. The results indicate that the substitution of hydrogen atoms in the phen ligand by methyl groups decreases slightly the electrostatic binding free energy changes, but tremendously increases the non-electrostatic ones to yield net binding free energy changes which are more favorable for the ct-DNA binding.

Salt-dependent binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline and dipyrido[3,2- a:2′,3′- c]phenazine to calf thymus DNA

The salt-dependent binding of racemic iron(II) mixed-ligand complex containing 1,10-phenanthroline (phen) and dipyrido[3,2- a:2′,3′- c]phenazine (dppz), [Fe(phen) 2(dppz)] 2+ to calf thymus DNA (ct-DNA) has been characterized by UV–VIS spectrophotometric titration. The equilibrium binding constant ( K b) of the iron(II) complex to ct-DNA decreases with the salt concentration in the solution. The slope, SK = ( δlog K b / δlog [Na 2+]) has been found to be 0.49, suggesting that, in addition to intercalation, considerable electrostatic interaction is also involved in the ct-DNA binding of [Fe(phen) 2(dppz)] 2+. The calculation of non-electrostatic binding constant ( K t o) based on polyelectrolyte theory has revealed that the non-electrostatic contribution to the total binding constant ( K b) increases significantly with the increase in [Na +] and reaches 36% at 0.1 M NaCl. On the other hand, the contribution of the non-electrostatic binding free energy (Δ G t o) to the total binding free energy change (Δ G o) is considerably large, i.e. 87% at [Na +] = 0.1 M, suggesting that the stabilization of the DNA binding is mostly due to the contribution of non-electrostatic process. Moreover, the effect of specific ligand substitutions on Δ G o has been rigorously evaluated using the quantity ΔΔ G t o, i.e. the difference in Δ G t o relative to that of the parent iron(II) complex, [Fe(phen) 3] 2+, indicating that each substitution of phen by dip and dppz contributes 7.5 and 17.5 kJ mol − 1 , respectively to more favorable ct-DNA binding.

Ruthenium(ii) complex of 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline: synthesis, spectrophotometric pH titrations and DNA interaction

A Ru(II) complex of [(bpy)2Ru(aip)](ClO4)2·H2O (bpy = 2,2′-bipyridine, aip = 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline) was newly synthesized. The ground- and excited-state acid–base properties of the complex were studied by UV–visible and emission spectrophotometric pH titrations. The excited-state ionization constants of pKa1* = 2.57, pKa2* = 8.47 are comparable to and 1.3 orders of magnitude greater than the ground-state ones, respectively. The interaction of the complex with calf thymus DNA was investigated by UV–visible and emission spectroscopy, steady-state emission quenching by [Fe(CN)6]4–, competitive binding with ethidium bromide, reverse salt titrations, DNA melting experiments, as well as viscosity measurements. The results indicated that the complex bound to the DNA by interaction of the anthryl moiety of the complex with an intrinsic binding constant of the order of 104 M−1 in buffered 50 mM NaCl at room temperature, and non-electrostatic binding free energy made a predominant contribution to the binding free energy.

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

The interaction of purified alpha alpha cross-linked hemoglobin (alpha alpha Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-alpha) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of alpha alpha Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25 degrees to more than 50 degrees. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of alpha alpha Hb interaction. Thus, in the absence of alpha alpha Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of alpha alpha Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to alpha alphaHb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of alpha alpha Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.