Protein extraction by Winsor‐III microemulsion systems

Proteins (bovine serum albumin (BSA), α‐chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L^?1 aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol‐OT, or AOT, and a 1,3‐dioxolane (or cyclic ketal) alkyl ethoxylate, CK‐2,13‐E_5.6), producing a three‐phase (Winsor‐III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5–13 g L^?1) and small in volume (12–20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W‐III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK‐2,13‐E₃, or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α‐chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α‐chymotrypsin being >90% of its original value. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α₁-adrenergic receptors induced by agonists. α₁-receptors were studied on DDT₁ MF-2 smooth muscle cells (DDT₁-MF-2 cells) by specific [³H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [³H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [³H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT₁-MF-2 cells preequilibrated with [³H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α₁-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT₁-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [³H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α₁-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

High and low affinity binding of folate to proteins in serum of pregnant women

Binding of [³H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37°C). A Scatchard analysis revealed the presence of high-affinity (K_ass = 10¹⁰M^?1, N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (M_r ≈ 30 000–35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/ 30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (M_r > 200 000) protein, probably α₂-macroglobulin.High-affinity binding which displayed positive cooperatively was saturated at folate concentrations above 10^?10 M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N¹⁰-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10^?10–10^?7 M. Dissociation of folate was rapid.     

Determination of hydrophobicity of myelinic,synaptosomal, and mitochondrial surfaces in the rat brain

The hydrophobicity of myelinic, synaptosomal and mitochondrial surfaces in the rat brain was measured using the nonionic surfactant, C₁₈H₃₇O(CH₂CH₂O)₁₃H. This method is based on the adsorption of the hydrophobic alkyl group of the surfactant by the hydrophobic sites on the surfaces. Each preparations was mixed with an excess of the surfactant and the surfactant remaining in the supernatants was determined spectrophotometrically by measuring the absorbance of tetrabromophenolphthalein ethylester at 690 nm. The greatest amount was adsorbed by myelin, followed by synaptosomes and mitochondria. The hydrophobicity is shown to be a reflection of the surface lipids. This method showed good reproducibility and was useful for the quantitative determination of hydrophobicity.     

Specific binding of [H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [³H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 °C. Scatchard analysis revealed two classes of binding sites. The apparent K_d for high-affinity receptors is 2.14 × 10⁻⁸ M with 1.48 × 10⁶ sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Calcium binding to placental plasma membranes as measured by rate of diffusion in a flow dialysis system

The Ca²⁺-binding properties of placental plasma membranes were studied using a flow dialysis system.Ca²⁺-binding was not detectable at pH 4.0, but increased at higher pH values to a maximum binding at pH 11.0.Two types of Ca²⁺-binding sites were identified: high-affinity sites with dissociation constant K_s = 3.1 · 10^{−5 M} and a capacity of 26 nmoles per mg protein; low-affinity sites with K_s = 1.1 · 10^{−3 M} and a capacity of 266 nmoles per mg protein.The affinities of Mg²⁺ and Sr²⁺ for the high-affinity sites were 10-fold lower than that of Ca²⁺, and for the low-affinity sites were 4- and 8-fold lower, respectively.The placental plasma membranes contain sites for Ca²⁺ with capacity, specificity and affinity within the range reported for other membranes involved in an active transport of Ca²⁺ (mitochondria, sarcoplasmic reticulum, cardiac microsomes). The presence of high-affinity Ca²⁺ sites as well as Ca²⁺-ATPase implicates these membranes in Ca²⁺ transport from the maternal to the fetal circulation.     

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K_a) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average K_a values of 1.3 (±0.2) × 10⁵ and 3.5 (±3.0) × 10² M⁻¹ for acetohexamide and values of 8.7 (±0.6) × 10⁴ and 8.1 (±1.7) × 10³ M⁻¹ for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K_a values of 1.3 (±0.1) × 10⁵ and 4.3 (±0.3) × 10⁴ M⁻¹, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K_a values of 5.5 (±0.2) × 10⁴ and 5.3 (±0.2) × 10⁴ M⁻¹, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions.     

Thyroxine binding to human serum albumin immobilized on sepharose and effects of nonprotein albumin-binding plasma constituents

¹²⁵I-thyroxine (¹²⁵I-T₄) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied.¹²⁵I-T₄ binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19±0.53×10^–6 M and 2.69±0.78×10^–5 M, respectively. The number of binding sites of the high and the low affinity sites were 1.28±0.46 mol/mol and 23.5±9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of¹²⁵I-T₄ to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T₄ binding via reduction of the binding capacity. Unlabeled T₄ showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T₄ binding to HSA-Sepharose.     

Interaction of uncouplers with the mitochondrial membrane: a high-affinity binding site

2-Nitro-4-azidocarbonylcyanide phenylhydrazone (N₃CCP), a potent water-soluble uncoupler at pH 6–8, was used to determine the nature of binding of the uncoupler to the mitochondrial membrane. Equilibrium binding studies with N₃CCP showed that isolated pigeon heart mitochondria contain 1.6 ± 0.3 high-affinity binding sites per cytochrome a. Several different types of chemical uncouplers were also found to bind to the same high-affinity site as evidenced by their observed competition with N₃CCP. The potassium ionophore valinomycin and the respiratory inhibitor antimycin A did not affect uncoupler binding to the high-affinity sites nor did active respiration of the mitochondria. The number of high-affinity binding sites was essentially unchanged by extraction of 80% of the mitochondrial phospholipids. The ability of the uncouplers to bind to the high-affinity binding sites is proportional to the uncoupler activities. These data support the idea that the high-affinity binding sites of mitochondria are protein(s) which are involved in the coupling reactions of oxidative phosphorylation and that uncoupler bound at these sites is responsible for the uncoupling activity.     

Acute influences on the two GDP-binding sites in brown-adipose-tissue mitochondria

Scatchard analysis of³H-guanosine diphosphate (GDP) binding to rat brown-adipose-tissue mitochondria demonstrated that binding to the high- and low-affinity sites (K_d=0.05 and 2.0 M) was abolished by denaturation at 100°C but non-specific binding remained constant (0.2% of free-GDP). Prior incubation of mitochondria at 37°C reduced binding to the high-affinity site, but this could be reversed by incubating samples at 0°C. Addition of palmitic acid (5–40 nmole/mg of mitochondrial protein) did not affect GDP-binding, but similar concentrations of palmitoyl CoA caused a slight reduction in the number of high-affinity sites and a significant decrease in the number of lower-affinity sites. Acute treatments known to stimulate thermogenesis in vivo (a single meal, cold exposure, or noradrenaline injection 40–80 min before sacrifice) all increased binding to both binding sites, and tended to raise the dissociation constants, whereas injection of 2-deoxy-D-glucose, which depresses metabolic rate in the rat, decreased dissociation constants of both sites and the maximum number of high-affinity sites. These data indicate that both GDP-binding sites respond rapidly to acute thermogenic stimuli, possibly due to conformational changes in the mitochondrial inner membrane, and that palmitoyl CoA may influence mitochondrial proton conductance via an association with purine nucleotide binding sites.     

Further studies on the specific interaction of S-100 protein with synaptosomal particulates.

Abstract— The specific interaction of S-100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca²⁺ -dependent. Two affinities affect the interaction (K_ins= 7.04 × 10^?9 M. 1.28 × 10¹² binding sites/ mg protein; K_ins2= 3.91 × 10^?7M, 2.96 × 10¹³ binding sites/mg protein). The half-saturation time is about 5.5 min at 37°C. The half-life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one-third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S-100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane-bound fraction of S-100.     

1H, 13C and 15N backbone and side-chain chemical shift assignment of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

SH2 domains are interaction modules uniquely dedicated to recognize phosphotyrosine sites, playing a central role in for instance the activation of tyrosine kinases or phosphatases. Here we report the ¹H, ¹⁵N and ¹³C backbone and side-chain chemical shift assignments of the SH2 domain of the human protein tyrosine kinase Fyn, both in its free state and bound to a high-affinity phosphotyrosine peptide corresponding to a specific sequence in the hamster middle-T antigen. The BMRB accession numbers are 17,368 and 17,369, respectively.     

Improved high-affinity binding of 3H-apomorphine with a subcellular membrane fraction of mammalian brain tissue

The binding of low concentrations of [³H](?)apomorphine to preparations of calf and rat forebrain tissue was evaluated. Fractionation of crude homogenates to prepare a membrane fraction (P₄) of striatal or caudate homogenates increased the proportion of saturable to total binding from 33% to over 80%, and increased the apparent density of binding sites from 94 to 681 fmol/mg protein. Binding in calf caudate P₄ tissue was protein-dependent and optimal at pH = 7.0 to 7.5, and T = 20 to 25°C; at higher temperatures tissue binding sites appeared to degrade. The half-time of association and dissociation at 22°C were, respectively, 14.0 and 18.5 min; equilibration was complete in 60 min. Kinetic characteristics of high-affinity binding obtained from association and dissociation constants and from saturation isotherms were similar (K_d = 2.1 to 3.4 nM). The pharmacology of competition for ³H-APO suggests selectivity for dopamine-agonist interactions. These results indicate that the P₄ membrane preparation may be useful for the evaluation of dopamine-agonist binding sites or “receptors.”     

Past vegetation changes in the Brazilian Pantanal arboreal–grassy savanna ecotone by using carbon isotopes in the soil organic matter

Measurements of the organic carbon inventory, its stable isotopic composition and radiocarbon content were used to deduce vegetation history from two soil profiles in arboreal and grassy savanna ecotones in the Brazilian Pantanal. The Pantanal is a large floodplain area with grass-dominated lowlands subject to seasonal flooding, and arboreal savanna uplands which are only rarely flooded. Organic carbon inventories were lower in the grassy savanna site than in the upland arboreal savanna site, with carbon decreasing exponentially with depth from the surface in both profiles. Changes in ¹³C of soil organic matter (SOM) with depth differed markedly between the two sites. Differences in surface SOM ¹³C values reflect the change from C₃ to C₄ plants between the sites, as confirmed by measurements of ¹³C of vegetation and the soil surface along a transect between the upland closed-canopy forest and lowland grassy savanna. Changes of ¹³C in SOM with depth at both sites are larger than the 3–4 per mil increases expected from fractionation associated with organic matter decomposition. We interpret these as recording past changes in the relative abundance of C₃ and C₄ plants at these sites. Mass balances with ¹⁴C and ¹³C suggest that past vegetational changes from C₃ to C₄ plants in the grassy savanna, and in the deeper part of the arboreal savanna, occurred between 4600 and 11 400 BP, when major climatic changes were also observed in several places of the South American Continent. The change from C₄ to C₃, observed only in the upper part of the arboreal savanna, was much more recent (1400 BP), and was probably caused by a local change in the flooding regime.