Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes.

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (ß-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers. Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria. In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible. Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to ß defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.

Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures.

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study.

The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.

The interaction of an anti-lipid antibody (TEPC 15) with a model biomembrane system (monolayer).

The interaction which occurs between an anti-lipid antibody (TEPC 15) and two phospholipids, phosphatidylcholine and phosphatidylethanolamine, when they are arranged in a lipid monolayer system has been studied. It is shown that the antibody is stabilised under the influence of a high lateral pressure when it is mixed with a lipid monolayer and that the behaviour of the antibody depends upon the lipid used. Measurements of the surface pressure and surface potential parameters of the lipid monolayers indicate that the antibody interacts differently with phosphatidylcholine compared with phosphatidylethanolamine. The antibody also exhibits a different interaction when it is pretreated with phosphorylcholine prior to being spread with a phosphatidylcholine monolayer. The interaction of the antibody with phosphatidylcholine-cholesterol monolayers has also been studied.

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers.

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.

Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study.

Laurdan (6-dodecanoyl-2-dimethylamine-naphthalene) is a fluorescent membrane probe of recent characterization. It was shown that this probe discriminates between phase transitions, phase fluctuations and the coexistence of phase domains in phospholipid multilamellar aggregates. We measured the excitation and emission generalized polarization (GP(ex) and GP(em)) of Laurdan in aggregates of complex glycosphingolipids in their pure form and in mixtures with dipalmitoylphosphatidylcholine (DPPC). Our results show that Laurdan detects the broad main phase transition temperature of the neutral ceramide-tetrasaccharide Gg(4)Cer (asialo-G(M1)) and shows a value of GP(ex) in between that of DPPC and that of ganglioside G(M1). In contrast, Laurdan was unable to detect the thermotropic phase transition of G(M1). The probe also appears to be unable to detect phase coexistence in both types of pure glycolipid aggregates. Deconvolution of the excess heat capacity vs. temperature curves of pure Gg(4)Cer and DPPC/Gg(4)Cer mixtures indicates that the thermograms are composed by different transition components. For these cases, Laurdan detects only the high cooperativity component of the transition of the mixture. The peculiar behaviour of Laurdan in aggregates containing complex glycosphingolipids may result from the inherent topological features of the interface that are conferred by the bulky and highly hydrated polar head group of these lipids.

Molecular parameters of gangliosides in monolayers: comparative evaluation of suitable purification procedures.

The molecular area, collapse pressure, and surface potential of gangliosides obtained by different methods were systematically compared in monolayers at the air-water interface. Different values of these parameters are obtained depending on the purification procedure employed for the isolation of pure gangliosides. This is due to impurities (such as peptidaceous material) that remain in different amounts in the various preparations and that modify the ganglioside surface behavior. Routine purity checking by HPTLC analysis of gangliosides usually fails to reveal these impurities. On the other hand, even if the monolayer technique cannot identify the nature or amount of contaminants, it is extremely sensitive to reveal alterations of the surface molecular parameters caused by relatively small amounts of other components coextracted with the ganglioside or adventitiously introduced with the solvents or subphases employed. This is a serious problem for the obtention of correct and reproducible values of such important parameters as the molecular area of gangliosides, their electrostatic potential in oriented interfaces, and their interactions with other lipids and proteins. A procedure leading to consistent molecular parameters that remain reproducible after several repurification cycles is to perform an alkaline treatment on previously purified gangliosides species with NaOH, this is followed by dialysis against bidistilled water, rechromatography on DEAE-Sephadex A25, silicic acid or Iatrobeads, and Sephadex LH-20 columns; repurified gangliosides are stored in chloroform-methanol-0.01 M NaOH (60:30:4.5).

The effect of phospholipase A(2) immobilization upon calcium interaction: a kinetic study.

In this work we studied the effect of Ca(2+) on the ability of immobilized PLA(2) to hydrolyze phospholipid substrates either in aggregate or monomeric forms. We use a kinetic methodology for the determination of dissociation constants of soluble and immobilized PLA(2)-Ca(2+) complexes. This approach allows us to obtain the values of the dissociation constants of enzyme-Ca(2+) (K(x)) and enzyme-Ca(2+)-substrate (K'(x)) complexes from the kinetic data obtained at different substrate and Ca(2+) concentrations. Results using mixed micelles of phospholipid-Triton X-100 showed that, in most cases, productive complexes were destabilized by immobilization of PLA(2). However, a correct analysis of the interaction must be independent of the classical modes of PLA(2) action toward lipid surfaces. Thus, a substrate in monomeric form was also employed to analyze the effect of immobilization on hydrolysis rate in the absence of interfacial activation. Kinetic data showed that the immobilization affected severely the mode of PLA(2) action. The kinetic data also suggested that immobilization promoted conformational alterations in the Ca(2+)-binding site, destabilizing the productive complex enzyme-Ca(2+)-phospholipid.

Extremely high thermal stability of streptavidin and avidin upon biotin binding.

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.

The post-translational incorporation of arginine into a beta-amyloid peptide increases the probability of alpha-helix formation.

The beta-amyloid peptide (beta AP1-40) inhibited the in vitro post-translational incorporation of [14C]arginine at the N-terminus of brain soluble proteins and was labelled by the incorporation of [14C]arginine. Addition of arginine at the N-terminal position of beta AP1-40 is predicted to increase the probability of an alpha-helix structure being formed on the first residues with a higher hydrophilic characteristic, increasing the possibility of these residues being exposed to the aqueous environment. Unmodified beta AP1-40 has a low alpha-helix content and a higher probability of beta-turn formation. Accumulation of beta AP1-40 in Alzheimer's disease may therefore be due to a reduced arginylation reaction and consequently to a decrease in its normal degradation by the ubiquitin pathway.

Interaction of alpha-MSH and substance P with interfaces containing gangliosides.

In the present work we studied the interaction of alpha-MSH and substance P neuropeptides with gangliosides using lipid monolayers, fluorescence spectroscopy, and differential scanning calorimetry techniques. The positively charged weak amphiphilic neuropeptides did not show surface activity in the range of concentrations tested (0.1-0.3 muM), but they were preferentially able to penetrate monolayers formed by acidic lipids, showing the best interaction with the more complex gangliosides. The general order of interaction found for both peptides is GTIh > GDIa = GMI > DLPA > sulphatide. Neither neuropeptide interacted with phosphatidylcholine monolayers above 10 mN.m-1. The binding of alpha-MSH to GMI micelles followed by changes in the fluorescence of its tryptophan residue takes place with an increase in the hydrophobic environment of the neuropeptide. An apparent dissociation constant of 13 muM was estimated for this process. Similar result was found with GMI:DMPC vesicles (1:10 molar ratio). The thermotropic profile of GMI micelles is modified in the presence of the neuropeptides. The calorimetric enthalpy of GMI transition increased 21% and 37% in the presence of alpha-MSH and substance P, respectively. Both neuropeptides induced the same increment in the transition temperature Tm from 19 to 20.5 degrees C. The basic physicochemical studies herein indicated that both positively charged neuropeptides, alpha-MSH and substance P, interact with interfaces containing gangliosides in a mainly electrostatic form, whereas the hydrophobic interaction seems to play a secondary role.

Synergic action of gangliosides on alpha-MSH-induced cyclic AMP levels in rat brain slices.

Gangliosides are particularly enriched in neuron cell membranes and they were postulated to be involved in the modulation of membrane-mediated transduction of information. In this study we explore the possibility that the increase in cAMP tissular levels induced by alpha-MSH may be modulated by the action of exogenously added gangliosides. We measured the level of cAMP in both tissues and medium in response to the alpha-MSH in slices previously incubated with total bovine brain gangliosides (TBG). When slices were exposed to TBG, the effect of alpha-MSH on inducing an increase in the content of cAMP was practically twice compared to the changes induced by alpha-MSH or TBG alone. We conclude that the presence of gangliosides may facilitate the alpha-MSH interaction with its receptor, increasing the cAMP levels in slices containing the CP and Acc nuclei.

Anti-inflammatory effect of gangliosides in the rat hindpaw edema test.

The influence of total brain gangliosides on acute inflammation was investigated using the rat hind paw edema test. Total gangliosides (10 micrograms/paw) inhibited the edema produced by the injection of bee venom phospholipase A2 (5 micrograms/paw) when the lipids were co-injected or injected 15 min before the phospholipase A2. Sulphatide (10 micrograms/paw) did not inhibit the edema but potentiated it. Gangliosides (40 micrograms/paw) inhibited the edema induced by carrageenin 1% when they were injected 1 h before the agent. However, gangliosides (up to 200 micrograms/paw) failed to inhibit the dextran-induced edema. The edema test was also used to investigate the effect of gangliosides on the production of mediators of inflammation by peritoneal adherent macrophages. Gangliosides inhibited the production of mediators of inflammation only when they were incubated with these cells before the stimulation with phospholipase A2 or carrageenin. Gangliosides did not inhibit the production of mediators of inflammation when arachidonic acid was added to the cells. These results suggest that the anti-inflammatory effect observed with gangliosides is mediated by inhibition at or before endogenous phospholipase activity.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.

A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.

A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties.

Although 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase-transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well-resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN dimethylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDAN's red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground-state interactions on LAURDAN's emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.

Gangliosides raise the intracellular Ca2+ level in different cell types.

Total gangliosides from bovine brain at micromolar concentration induce intracellular Ca2+ increments in a temperature, time and dose dependent manner when assayed with suspensions of rat macrophages, rat and chicken neurons, human erythrocytes and liposomes, loaded with the fluorescent Ca2+ indicator FURA 2. The effect was independent on the endogenous ganglioside composition of the cells and in the case of neurons it was also independent on the differentiation state. Gangliosides do not induce the release of Ca2+ from inner stores. These findings indicate that the reported inhibition of arachidonic acid release (Bressler, J., et al., (1994) Life Sci., 54, 49-60) and anti-inflammatory properties of gangliosides (Correa, S.G. et al., (1991) Eur. J. Pharmacol. 199, 93-98) are not due to impairments of Ca2+ flux. The results also suggest the possibility that the well-known neurotrophic effect produced by gangliosides on undifferentiated neurons in culture may be due to subtoxic cytosolic Ca2+ increments.

Effect of gangliosides on Trypanosoma cruzi infection in mice.

Albino Swiss male mice were inoculated with Trypanosoma cruzi, Tulahuen strain trypomastigotes, and separated into three groups: control, without treatment; control, treated with Nifurtimox 25 mg/day; and experimental, treated with total brain gangliosides 1 mg/day, intramuscular. The treatment was started immediately after infection and maintained for 4 weeks. Parasitemia was determined twice a week and histopathological analyses of hearts were performed. The parasitemia was significantly lowered by the ganglioside treatment. All untreated mice died by day 14 post infection. Survival at day 30 was 96% for mice in the experimental group. Hearts from untreated animals showed acute chagasic myocarditis, while those from mice treated with gangliosides presented only minor mononuclear infiltration. The effect of gangliosides is probably due to interference of parasite penetration into the host cells.

Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin.

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.