Kinetic analysis of the interactions of complement receptor 2 (CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220.

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.

A biosensor assay for studying ligand-membrane receptor interactions: binding of antibodies and HIV-1 Env to chemokine receptors.

The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor, which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env-coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced, stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env-receptor binding constants to viral tropism and pathogenesis.

High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. METHODS: Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. RESULTS: All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. CONCLUSIONS: The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation, despite removal of circulating Abs and adequate immunosuppression.

Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1.

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1-specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a K(d) = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (k(on) and k(off), respectively) were determined, and they yielded a dissociation constant (K(d)) of 3.2x10(-9) mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1-mediated cell-extracellular matrix and cell-cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury. (Blood. 2000;95:3788-3795)

A molecular basis for functional peptide mimicry of a carbohydrate antigen.

Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti-peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody-binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes.

Exploring biomolecular recognition using optical biosensors.

Understanding the basic forces that determine molecular recognition helps to elucidate mechanisms of biological processes and facilitates discovery of innovative biotechnological methods and materials for therapeutics, diagnostics, and separation science. The ability to measure interaction properties of biological macromolecules quantitatively across a wide range of affinity, size, and purity is a growing need of studies aimed at characterizing biomolecular interactions and the structural elements that drive them. Optical biosensors have provided an increasingly impactful technology for such biomolecular interaction analyses. These biosensors record the binding and dissociation of macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface coated with ligand molecule into an optical signal. Interactions of analytes and ligands can be analyzed at a microscale and without the need to label either interactant. Sensors enable the detection of bimolecular interaction as well as multimolecular assembly. Most notably, the method is quantitative and kinetic, enabling determination of both steady-state and dynamic parameters of interaction. This article describes the basic methodology of optical biosensors and presents several examples of its use to investigate such biomolecular systems as cytokine growth factor-receptor recognition, coagulation factor assembly, and virus-cell docking.

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Randomization of the receptor alpha chain recruitment epitope reveals a functional interleukin-5 with charge depletion in the CD loop.

We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization. Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain. Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage. (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues. This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized. From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90. Characterization of this variant expressed as a soluble protein in E. coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop. Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment.

Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein.

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.

Tissue factor regulates plasminogen binding and activation.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Construction and binding kinetics of a soluble granulocyte-macrophage colony-stimulating factor receptor alpha-chain-Fc fusion protein.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha). We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha. The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 x 10(-2)/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.

Copper-zinc superoxide dismutase activity in red blood cells and serum in demented patients and in aging.

The activity of the enzyme copper-zinc superoxide dismutase (Cu-Zn SOD) has been investigated in serum and red blood cells (RBC) homogenate obtained from demented patients with associated vascular lesions (VD), demented patients with probable Alzheimer's disease (DAT) and healthy controls (CG) of the same age. The increase in SOD activity was statistically significant (P < 0.01) in RBCs homogenate of DAT and VD patients, when compared to controls, but no differences appear between the two diseases groups. Additionally, a statistically significant increase in SOD activity (P < 0.01) in DAT patients above 70 years as compared to those 50-70 years old, and a relation between SOD and age were found. No changes in SOD activity with age in healthy controls nor in vascular dementia group were detected. A statistically significant increase in Circulating SOD activity (P < 0.01) was observed in vascular patients compared to controls. The observed increase in DAT Circulating SOD activity (against CG) was not significant. The increased levels of Cu-Zn SOD, probably represent a general alteration of the oxidative processes characteristic of these dementias and suggest that the enzyme might be used as a marker.

Dissociation of the crotoxin complex promoted by acetylcholine.

Neuromuscular blockage at the presynaptic level is the main biological effect exerted by crotoxin, the major toxin from Crotalus durissus terrificus venom. This effect requires the dissociation of this complex at the target membrane in spite of its tightness and stability under physiological conditions of pH, ionic composition and temperature. Complex dissociation should be determined by a specific set of physicochemical conditions prevailing at the neuromuscular junction. In this regard, we have studied the effect of acetylcholine on the stability of the crotoxin complex, since this effector is present at relatively high concentrations near the presynaptic membrane of the neuromuscular junction. Evidences arising from spectrofluorometric measurements, changes in enzymatic activity and crotoxin B inactivation by b-bromophenacyl bromide indicate that acetylcholine is indeed able to promote complex dissociation at neutral pH values, apparently by titration of 2-3 carboxylate groups in crotoxin A. This effect may, at least in part, contribute in determining the target specificity of this toxin.

Dissociation of the crotoxin complex promoted by acetylcholine.

Neuromuscular blockage at the presynaptic level is the main biological effect exerted by crotoxin, the major toxin from Crotalus durissus terrificus venom. This effect requires the dissociation of this complex at the target membrane in spite of its tightness and stability under physiological conditions of pH, ionic composition and temperature. Complex dissociation should be determined by a specific set of physicochemical conditions prevailing at the neuromuscular junction. In this regard, we have studied the effect of acetylcholine on the stability of the crotoxin complex, since this effector is present at relatively high concentrations near the presynaptic membrane of the neuromuscular junction. Evidences arising from spectrofluorometric measurements, changes in enzymatic activity and crotoxin B inactivation by b-bromophenacyl bromide indicate that acetylcholine is indeed able to promote complex dissociation at neutral pH values, apparently by titration of 2-3 carboxylate groups in crotoxin A. This effect may, at least in part, contribute in determining the target specificity of this toxin.

The mechanism of inhibition of phospholipase activity of crotoxin B by crotoxin A.

In the crotoxin complex isolated from Crotalus durissus terrificus venom, the component A inhibits the phospholipase A2 activity of crotoxin B only when the substrate is in the aggregated form, preventing the interaction of the enzyme with lecithin--water interfaces. In contrast, with similar rates of hydrolysis of dihexanoyllecithin monomers, the activity of the crotoxin complex is lower than that of crotoxin B when the substrate is aggregated into micelles. Crotoxin B readily hydrolyses dimyristoyllecithin vesicles, the rate being modulated by the physical state of the phospholipid, suggesting that the enzyme is tightly bound to the interface. With the crotoxin complex the rate of vesicle hydrolysis is much slower (about 1/10 that of crotoxin B) and is little affected by the physical state of the lecithin. Direct binding experiments demonstrate that, in contrast to crotoxin B, the crotoxin complex is unable to interact with lecithin--water interfaces. Together with the free accessibility of the enzyme active site in the crotoxin complex, this evidence suggests that a specific area on the enzyme surface, different from the active site and shielded by crotoxin A in the complex, is responsible for the interaction of crotoxin B with lipid--water interfaces.

[Resistance of Crotalus durissus terrificus and Bothrops neuwiedii to the neurotoxicity of massive quantities of Crotalid venom]. / Resistencia de Crotalus durissus terrificus y Bothrops neuwiedii a la neurotoxicidad de cantidades masivas de veneno crotálico.

The antitoxic potency of crude Crotalus durissus terrificus serum against crotalic venom is similar to that of a standard horse anticrotalic serum in protecting mice against 4 LD50, while the potency of Bothrops neuwiedii serum is 20% of the latter. Failure to form precipitin lines in immunodiffusion tests suggests that the antitoxic factors present in the sera from both species are not immunoglobulins. It is, therefore, probable that crotoxin is not neutralized by an antigen-antibody reaction, but rather by formation of inactive complexes with specific serum components. Resistance to the venom is not reciprocal, since specimens of C. d. terrificus die after the injection of similar amounts of B. neuwiedii venom, which are tolerated by the homologous species.

Accessibility of the active site of crotoxin B in the crotoxin complex.

Basic phospholipases A and the crotoxin complex isolated from Crotalus durissus terrificus venom exhibited similar initial reaction rates, time course and degree of hydrolysis of synthetic short chain lecithins in the monomeric state. Although monomeric lecithins seem to promote dissociation of crotoxin up to a certain extent, this cannot explain the high activity observed with the complex. The crotoxin complex is able to bind the non-hydrolyzable analog D-diheptanoyllecithin, as demonstrated by equilibrium gel-filtration, with a dissociation constant of 0.12 mM. This value is similar to the dissociation constant of the crotoxin B-D-diheptanoyllecithin complex (about 0.13 mM), estimated from the protection against enzyme inactivation by p-bromophenacyl bromide, which further supports the free accessibility of the substrate to the enzyme active site in the crotoxin complex. The lack of enzyme inactivation when crotoxin is treated with p-bromophenacyl bromide may be interpreted in terms of the specific requirements of the reagent to react with the enzyme rather than protection of the active site. Crotoxin B inhibition by complex formation with crotoxin A, which is not apparent on monomeric substrates, seems not to involve the active site of the enzyme.