Fluorescence Polarization Studies Research Articles

Steady-state, time-resolved fluorescence polarization behaviour and determination of dipole moments of coumarin laser dye

Steady-state and time-resolved fluorescence polarization studies have been carried out on coumarin 30 laser dye. The rotational reorientation of this probe has been measured in butanol at higher values of viscosity varying temperature. It is found that coumarin 30 rotates faster in butanol than in n-octanenitrile. The ground state ( μ g) and the excited state ( μ e) dipole moments are studied at room temperature in various solvents. These have been measured using absorption and fluorescence spectral band shift in solvent with varying dielectric constant ( ε) and refractive index ( n). It is observed that the dipole moment of excited state is higher than ground state dipole moment.

Phospholipid Interactions Protect the Milk Allergen α-Lactalbumin from Proteolysis during in Vitro Digestion

Interactions with food components may alter the resistance of food proteins to digestion, a property thought to play an important role in determining allergenic properties. The kinetics of breakdown of the bovine milk allergen alpha-lactalbumin during in vitro gastrointestinal digestion was found to be altered by interactions with physiologically relevant levels of phosphatidylcholine (PC), a surfactant that is abundant both in milk and is actively secreted by the stomach. Breakdown during gastric digestion was slowed in the presence of PC and accompanied by small alterations in the profile of resulting peptides, with little effect being observed during subsequent duodenal digestion. alpha-Lactalbumin was found to unfold at gastric (acid) pH, giving a CD spectrum similar to that obtained for the partially folded state it is known to adopt at pH values below its isoelectric point. Fluorescence polarization studies performed at low pH indicated that this partially unfolded form of the protein was able to penetrate into the PC vesicles. These interactions are probably responsible for the slowing of gastric digestion by reducing the accessibility of the protein to pepsin. These findings show that interactions with other food components, such as lipids, may alter the rate of breakdown of food proteins in the gastrointestinal tract. It underlines the importance of the food matrix in affecting patterns of food allergen digestion and hence presentation to the immune system and that in vitro digestion systems used for assessing digestibility of allergens must take account of surfactants.

Absorption enhancement, mechanistic and toxicity studies of medium chain fatty acids, cyclodextrins and bile salts as peroral absorption enhancers

The objective of the present investigation was to evaluate an oral ‘drug delivery’ approach, which involves co-administration of absorption enhancers (AEs). The representative low permeable hydrophilic (biopharmaceutic classification system (BCS) Class III) drugs used in the study comprised of cefotaxime sodium and ceftazidime pentahydrate, whereas low permeable lipophilic (BCS Class IV) drugs include cyclosporin A and lovastatin. AEs from three different chemical classes, namely, medium chain fatty acids (sodium caprylate and caprate), cyclodextrins (β-cyclodextrin, hydroxypropyl β-cyclodextrin) and bile salts (sodium cholate and deoxycholate) were evaluated for absorption enhancement efficacy, mechanism of action and toxicity using in vitro everted intestinal sac model. These AEs were found to enhance intestinal permeability of drugs from 2- to 27-fold. Light microscopy studies of intestinal sac incubated with AEs for 120 min revealed morphological changes in absorptive mucosa and rank order of toxicity were cyclodextrins > bile salts ≅ medium chain fatty acids. Fluorescence polarization studies indicated that brush bordered membrane vesicles labeled with lipophilic (DPH, 12AS) and hydrophilic dyes (ANS), when treated with AEs exhibited concentration and time dependent decrease in fluorescence polarization. Total protein released in presence of AEs was more than control but considerably less than EDTA (0.58% w/v), which is known to cause toxic release of proteins from cell. Overall, AEs were found to significantly enhance drug permeability by decreasing lipid membrane fluidity and/or interacting with hydrophilic domains of membrane, and has the potential to improve oral delivery.

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein–dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.

Interaction of the Macrolide Antibiotic Azithromycin with Lipid Bilayers: Effect on Membrane Organization, Fluidity, and Permeability

To investigate the effect of a macrolide antibiotic, azithromycin, on the molecular organization of DPPC:DOPC, DPPE:DOPC, SM:DOPC, and SM:Chol:DOPC lipid vesicles as well as the effect of azithromycin on membrane fluidity and permeability. The molecular organization of model membranes was characterized by atomic force microscopy (AFM), and the amount of azithromycin bound to lipid membranes was determined by equilibrium dialysis. The membrane fluidity and permeability were analyzed using fluorescence polarization studies and release of calcein-entrapped liposomes, respectively. In situ AFM images revealed that azithromycin leads to the erosion and disappearance of DPPC and DPPE gel domains, whereas no effect was noted on SM and SM:cholesterol domains. Although azithromycin did not alter the permeability of DPPC:DOPC, DPPE:DOPC, SM:DOPC, and SM:Chol:DOPC lipid vesicles, it increased the fluidity at the hydrophilic/hydrophobic interface in DPPC:DOPC and DPPE:DOPC models. This effect may be responsible for the ability of azithromycin to erode the DPPC and DPPE gel domains, as observed by AFM. This study shows the interest of both AFM and biophysical methods to characterize the drug-membrane interactions.

A fluorescence polarization study of the O2+ A2Πu → X2Πg and the b4Σg− → a4Πu transitions in the excitation range 17–25 eV

The fluorescence yields and polarizations of the O2+ A2Πu → X2Πg and the b4Σg− → a4Πu transitions have been measured between their thresholds and 25 eV using synchrotron radiation. The O2+ ions may be formed either through direct photoionization or through excitation into a Rydberg state followed by autoionization. The alignment of the resulting (A2Πu or b4Σg− state) ion has been determined by measuring the degree of linear polarization of the fluorescence emitted in the subsequent decay. The fluorescence yields and polarizations display prominent structure associated with autoionizing Rydberg states belonging to series converging onto the b4Σg−, B2Σg− or the c4Σu− ionization thresholds. The structure in the fluorescence polarization data has enabled the symmetry of the super-excited states to be deduced. Although the interpretation of this symmetry information has been straight-forward in regard to the Rydberg series converging onto the c4Σu− limit, the situation is less clear for the Rydberg series converging onto the b4Σg− or the B2Σg− limits. The measured polarizations have been compared with calculated values and, even for the A2Πu → X2Πg transition where the theoretical model incorporates resonant processes, the agreement is not particularly good.

Protein self-association in crowded protein solutions: a time-resolved fluorescence polarization study.

The self-association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8-anilinonaphthalene-1-sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50-200 mg/mL range, using picosecond-resolved fluorescence anisotropy methods. It was found that apoMb molecules self-associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time-resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.

A mechanism for the proapoptotic activity of ursodeoxycholic acid: effects on Bcl-2 conformation.

Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x(L) antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x(L). Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers.

Apo-Calmodulin Binds with its C-terminal Domain to the N-Methyl-d-aspartate Receptor NR1 C0 Region

Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, ∼44% of phospholipids are left insoluble at 4 °C in bovine hippocampal membranes. Cholesterol depletion using methyl-β-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.

Changes in biophysical parameters of plasma membranes influence cisplatin resistance of sensitive and resistant epidermal carcinoma cells

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more “fluid” plasma membranes than the sensitive cells. Because of this observed difference in membrane “fluidity,” we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.

A vibrationally resolved fluorescence polarization study of theCO+ A 2Π → X 2Σ+ transition in the 16.5–20 eV excitation range

Synchrotron radiation has been used to formCO+ inthe A 2Π state, either through direct photoionization or through excitation into a Rydberg statefollowed by autoionization. The alignment of the resulting ion has been determined bymeasuring the degree of linear polarization of the fluorescence emitted in the or 1 transitions. These vibrationally resolved polarization data, together with thecorresponding fluorescence yields, have been recorded in the 16.5–20 eV excitation range. Inthis energy range the fluorescence yields exhibit prominent structure due to autoionizingRydberg states belonging to series converging onto various vibrational levels of either theA 2Π orthe B 2Σ+ ionization thresholds. Resonant excitation and decay processes produce substantial deviationsin the fluorescence polarization and these changes have been used to deduce the symmetryof the excited state. The vibrationally resolved data are particularly useful in this respectbecause the observed variations in the polarization can be related to the branching ratiobetween a specific vibrational level in the Rydberg state and a particular level in theA 2Π state. This information has allowed the assignments of the Rydberg series converging onto theB 2Σ+ ionization limit to be verified. In addition, the measured polarization has been comparedwith predictions derived from a theoretical approach in which only direct photoionization istaken into account.

Perturbation of Membrane Microarchitecture Significantly Affects the Temperature Dependence of Cytochrome B5 Reduction in Liver Microsomes: Stopped-Flow Kinetic Analysis of the Initial Burst

The temperature dependence of the initial burst in the NADH-dependent reduction of rat liver microsomal cytochrome b5 in situ in the membrane was examined by means of stopped-flow spectrophotometry. The rate of cytochromeb5 reduction increased only moderately with increasing temperature, in contrast to the larger increase in a pure, delipidated reconstituted system in free solution. Treatment with a low concentration (0.05%) of sodium deoxycholate, which perturbs the membrane integrity without causing disassembly, abolished the characteristically weak temperature dependence of cytochrome b5 reduction observed in intact microsomes. Despite the striking difference in the behavior of intact and deoxycholate-treated microsomes as regards temperature dependence, the microviscosity in these preparations was found to be essentially the same by means of fluorescence polarization studies. The microsomal NADH-ferricyanide reductase and NADH-cyto-chrome c reductase activities with a water-soluble electron acceptor showed a usual linear dependency on temperature, even in deoxycholate-treated microsomes. It is concluded that the microenvironment (membrane structure) of the microsomal enzyme system strongly influences the electron transfer from the reductase to cytochrome b5. A model explaining the results is proposed and discussed.

Studies on the Binding Mode of Pinacyanol Chloride to Nucleic Acids

AbstractThe interaction of pinacyanol chloride (PC) with nucleic acids has been investigated by a series of experiments. Extensive hypochromism, appreciable peak shifts, isosbestic points and new peaks of the product of binding to nucleic acids in the spectra were observed. They showed that the interaction between PC and nucleic adds occurred. The results from absorption spectra of DNA, DNA melting, electrophoresis and fluorescence polarization studies have indicated that PC binds to DNA in nonintercalative way. Consistent with the nonintercalation, the studies of fluorescence titration and absorption titration specified that the binding of PC to nucleic acids occurred by an outside stacking binding, in which nucleic acids served for acting templates. The fact that the new absorption peaks of bound PC at ca. 485 nm are just close to the absorption bands of H‐aggregate of PC at high concentrations without DNA further supports the outside stacking binding mode. In addition, other evidence indicated that the interaction between PC and nucleic acids is not purely electrostatic.

Thermodynamic and Structural Studies of Triton X-100 Micelles in Ethylene Glycol−Water Mixed Solvents

Micellar properties of p-tert-octyl-phenoxy (9.5) polyethylene ether (Triton X-100) in aqueous mixtures of ethylene glycol (EG) were determined using such techniques as surface tension, static and dynamic light scattering, and fluorescence spectroscopy. Thermodynamics of micellization was obtained from the temperature dependence of critical micelle concentration values. The differences in the Gibbs energies of micellization of Triton X-100 between water and binary solvent systems were calculated to evaluate the influence of cosolvent on the micellization process. From this study, it can be concluded that the structure-breaking ability of EG and its interaction with the oxyethylene groups of the surfactant are dominating factors in the micellization process. Thermodynamics of adsorption of the solution−air interface was also evaluated. It was found that the surface activity of the surfactant decreases slightly with increasing concentration of EG at a given temperature. By a combination of static and dynamic light scattering measurements, a reduction of the micelle size was observed, mainly due to a decrease of the micellar aggregation number, whereas the micellar solvation was not substantially modified in magnitude with EG addition. However, the change of the surface area per headgroup of the surfactant suggested an alteration in the nature of its solvation layer, produced probably by a certain participation of cosolvent in the micellar solvation layer. This point was corroborated from the fluorescence polarization studies of several luminescent probes, including coumarin 6, merocyanine 540, and rhodamine B. These experiments revealed a slight increase of the micellar microviscosity. Finally, the proposed mechanism was also supported by the increase observed in the cloud point of Triton X-100, induced by the EG addition.

Organization and Orientation of a Triblock Copolymer Poly(ethylene glycol)-b-poly(p-phenylene ethynylene)-b-poly(ethylene glycol) and Its Blends in Thin Films

Molecular organization and orientation in thin films (∼100 nm) of a triblock copolymer, PPEPEG, composed of a rigid, π-conjugated middle block poly(phenyleneethynylene), PPE, and two poly(ethylene glycol) end blocks, PEG, were studied by fluorescence, secondary ion mass spectroscopy (SIMS), and atomic force microscopy (AFM). Fluorescence polarization studies of the triblock copolymer and its blends with poly(methyl methacrylate) (PMMA) led to the conclusion that the triblock copolymer molecules were oriented perpendicularly to the silicon substrate surface. Orientation of molecules in the vertical direction was confirmed by measurement of the oxygen depth profile of the film with the use of SIMS. Copolymer molecules in large fraction of PMMA (∼2 wt % in PMMA) were molecularly dispersed to give rise to high fluorescence emission intensity at a wavelength characteristic of nonaggregated chains. The reinforced action of this rigid polymer for PMMA was obtained. The secant modulus of the 2 wt % blend in a tensile test was 5.7 times the value of PMMA.

Abnormalities in lipid composition of brush-border membranes isolated from renal cortex of spontaneously hypertensive rats

The lipid composition and fluidity in brush-border membrane vesicles (BBMV) isolated from renal cortex of spontaneously hypertensive rats (SHR) and their normotensive control, Wistar Kyoto (WKY) rats have been studied. The activity of Na +-dependent D-glucose transport has been also determined. A significant increase in total phospholipids and free cholesterol were observed in renal BBMVs from SHR, which led to a decrease in the ratio of phospholipid-to-free cholesterol in these hypertensive rats. A reduction in the content of phosphatidylcholine and an increase in the percentage of sphingomyelin were observed in SHR. As a consequence, a diminished ratio of phosphatidylcholine-to-sphingomyelin was present in kidneys from SHR. The content of phosphatidylserine and phosphatidylinositol was reduced in SHR. However, the levels of phosphatidylethanolamine, cardiolipin, and lysophosphatidylethanolamine were increased in hypertensive animals. An increase in the level of saturated fatty acids, together with a decrease in the level of unsaturated fatty acids was responsible for the lower ratio of unsaturated-to-saturated fatty acids found in kidneys from SHR. The ratio of linoleic-to-arachidonic acid was increased in SHR when compared to WKY rats, as a result of the observed decrease in the content of arachidonic acid in hypertensive rats. Studies of diphenylhexatriene fluorescence polarization indicated no changes in the fluidity between BBMVs from both experimental rats, which did not correlate to the observed changes in the lipid composition of these membranes. These modifications in brush-border membrane lipid composition were accompanied by changes in the Na +-dependent d-glucose transport through renal BBMVs from SHR.

The aggregation behavior of collagen in aqueous solution and its property of stabilizing liposomes in vitro

The aim of this study is to acquire the relationship between the aggregation behavior of collagen in aqueous solution and its property of stabilizing liposomes in vitro. Firstly, we used an intramolecular charge transfer compound 3-methoxy-4′- N, N-dimethylamino flavone derivative (DMMF) as a fluorescence probe to detect collagen association in aqueous solution. Results showed that, when collagen concentration was increased to 0.5 mg/ml or above, the fluorescence intensity of DMMF at 465 nm increased dramatically. This indicated that collagen could associate in aqueous solution. The subsequent fluorescence polarization study on the same collagen solutions also proved that the aggregation behavior existed in the aqueous solution of collagen. Secondly, to evaluate the in vitro stability of small unilamellar vesicles (SUV), made from soybean phosphatidylcholine (PC) and coated with collagen, 5(6)-carboxyfluorescein was used as a fluorescence probe to determine the change of their permeability. We found that the coating with a collagen/PC weight ratio of 2:1, significantly decreased the permeability of liposomal membranes. The likely weight ratio of collagen and PC was mainly dependent on the critical aggregation concentration of collagen in aqueous solution. In addition, the fluorescence polarization method was also used to study the influence of a coating with collagen at the above ratio on the fluidity of liposomal membranes, employing DMMF as a fluorescence probe. The fluidity of liposomal membranes coated with collagen showed no obvious change, indicating that SUV coated with collagen at an appropriate ratio, could significantly improve the in vitro stability of liposomal membranes without disturbing their fluidity. It is thus clear that collagen self-association behavior is closely correlated to its coating liposomes to improve their stability.

Fluorescence polarization studies of different forms of angiotensin-converting enzyme.

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied using fluorescence polarization. The dissociation constants Kd of the enzyme-inhibitor complexes in 50 mM Hepes-buffer (pH 7.5) containing 150 mM NaCl and 1 microM ZnCl2 at 37 degrees C were (2.3 +/- 0.4).10(-8), (2.1 +/- 0.3).10(-8), and (2.1 +/- 0.2).10(-8) M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 +/- 0.2).10(-3) M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 +/- 0.01) sec(-1) and (0.026 +/- 0.001) sec(-1) that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 +/- 0.001) and (0.041 +/- 0.001) sec(-1) for the N-domain and for testicular ACE, respectively.

Analysis of the interaction between the HIV-inactivating protein cyanovirin-N and soluble forms of the envelope glycoproteins gp120 and gp41.

The novel virucidal protein cyanovirin-N (CV-N) binds with equally high affinity to soluble forms of either H9 cell-produced or recombinant glycosylated HIV-1 gp120 (sgp120) or gp160 (sgp160). Fluorescence polarization studies showed that CV-N is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41) (as well as SIV glycoprotein 32), albeit with considerably lower affinity than the sgp120/CV-N interaction. Pretreatment of CV-N with either sgp120 or sgp41 abrogated the neutralizing activity of CV-N against intact, infectious HIV-1 virions. Isothermal calorimetry and optical biosensor binding studies showed that CV-N bound to recombinant sgp120 with a K(d) value ranging from 2 to 45 nM and to sgp41 with a K(d) value of 606 nM; furthermore, they indicated an approximate 5:1 stoichiometry for CV-N binding to sgp120 and a 1:1 stoichiometry for CV-N binding to sgp41. Circular dichroism studies additionally illuminated the binding of CV-N with both sgp120 and sgp41, providing the first direct evidence that conformational changes are a consequence of CV-N interactions with both HIV-1 envelope glycoproteins.