The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

The interaction of europium(III) ion with nucleotides

Apparent composite stability constants have been determined at pH 7.0 and 8.0 for the interaction of Eu(III) with ADP and ATP in 0.1 M N-ethylmorpholine buffer at 20°C. The values were obtained using a competitive spectrophotometric technique with 8-hydroxyquinoline as the competing ligand and experiments were performed in the presence of relatively low concentration of europium so as to avoid precipitation of hydrolysed species of the metal ion. The data have been used, together with an assumed hydrolysis constant for europium of 10^?8 M, to calculate that the stability constant for the Eu-ADP⁰ complex is about 10⁶ M^?1. The results were not sufficiently accurate to determine the stability constant of the Eu(OH)-ADP^? complex.Values are also reported for the stability constants and molar extinction coefficients of the complexes formed by the reaction of europium and magnesium with 8-hydroxyquinoline.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

The influence of heavy water of low concentration on Spirogyra,Planaria and on enzyme action

Further experiments support the original suggestion of one of the authors (T. C. B.) that low concentrations of the heavy isotope of hydrogen in water will yield more significant physiological results than pure heavy water. The water employed had been slightly concentrated by commercial electrolysis and had a specific gravity of 1.00061 or 1 part in 2,000 of deuterium. Short lengths (5 to 50 cells) ofSpirogyra filaments lived longer in the isotope water (average 6.3 days for 355 cells) as compared to their longevity in ordinary distilled water (average 3.3 days for 322 cells). The experiments indicate that this effect was not due to differences in salt, CO₂, or O₂ content of the water samples.Planaria maintained their body size for longer periods in the isotope water. Fermentation by suspensions of commercial yeast, and digestion of starch by pancreatic amylase were inhibited only after a period of incubation of the enzyme in the heavy water; the degree of inhibition of the former reaction was, however, very nearly indentical for incubation periods of 16 and 166 hours indicating a stoichiometrical relationship between the isotope and the enzyme.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). I. Preparation and absorption properties in solution

Poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine) and poly(N^γ-acetoacetyl-L -diaminobutyric acid) form colored complexes with ferric ions in water/dioxane solutions. These complexes are soluble at pH values lower than 2.8 and show their maximum absorption at 257 nm in the uv and at 478 nm in the visible region; whereas the ferric complex of the model compound n-hexylacetoacetamide exhibits absorptions centered at 258 and 536 nm, respectively. It is shown that in the complex of the model compound one metal ion is bound per acetoacetamide group, while in the complexes of the three polymers two β-ketoamides side chains are bound per ferric ion under the same solvent, pH, concentration, and ionic strength conditions. The binding constants of ferric ions to the three polymers, and the formation constant of the ferric complex of the model compound are also evaluated.     

Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. I. The concentration of cations, anions, amino acids in, and pH of, the xylem sap

Sunflower plants (Helianthus annuus L.) were subjected to soil drying with their shoots either kept fully turgid using a Passioura-type pressure chamber or allowed to decrease in water potential. Whether the shoots were kept turgid or not, leaf conductance decreased below a certain soil water content. During the soil drying, xylem sap samples were taken from individual intact and transpiring plants. Xylem sap concentrations of nitrate and phosphate decreased with soil water content, whereas the concentrations of the other anions (SO₄² and Cl^?) remained unaltered. Calcium concentrations also decreased. Potassium, magnesium, manganese and sodium concentrations stayed constant during soil drying. In contrast, the pH, the buffering capacity at a pH below 5 and the cation/anion ratio increased after soil water content was lowered below a certain threshold. Amino acid concentration of the xylem sap increased with decreasing soil water content. The effect of changes in ion concentrations in the xylem sap on leaf conductance is discussed.     

Study of the interaction of polynucleotide chains with oligomers by means of chromatography. II. Effect of magnesium ions and noncomplementary bases on the stability of complexes

The interaction of the oligonucleotides ApA, ApApA, ApApC and ApApU with poly(U) and (Ip)₅I and (Ip)₆ with poly(C) has been studied by means of equilibrium gelfiltration through Sephadex.From sorption isotherms the free energies, energies and entropies of complexing have been computed for different concentrations of magnesium ions in the medium.The stoichiometric ratio of polymers to oligomers has been measured and found equal to 2 in the case of ApApA and ApApC. This shows that the cytidylic acid residue is included in the ternary complex. But in the case of ApApU the noncomplementary base is partly squeezed out of the complex.The stacking free energy of neighbouring oligomers has been found to be in the range 1000–3000 $\text{cal}\text{mole}$ depending on the conditions.The stoichimetric ratio has been found to be 1 in the case of poly(C): oligo(I), the stacking energy is equal to 1.2 $\text{kcal}\text{mole}$. The effect of magnesium is somewhat different in the case of double and triple helices and probably reflects the formation of coordination compounds with the nitrogen bases of nucleotides.     

On the application of polyelectrolyte limiting laws to the helix–coil transition of DNA. V. Ionic effects on renaturation kinetics

The bimolecular rate constant k₂ for the association of complementary polynucleotide strands has been observed to increase strongly with increasing ionic strength—in fact, proportional to its third or fourth power. This effect is here interpreted quantitatively by means of polyelectrolyte theory starting with the Wetmur–Davidson postulate of a pre-equilibrium between separated strands and aligned segments close to one another but unbonded. The correct form, a power dependence of k₂ on ionic strength, is predicted. Comparison of the theoretical exponent with data allows the conclusion that each of the two single-stranded segments in the aligned but unbonded configuration consists of about 13–16 nucletides (not to be confused with the much smaller number of bonded base pairs in the nucleus), and that this number, denoted by Q, is possibly correlated either with a minimum length for duplex stability or with the persistence length of a single polynucleotide strand. It is suggested that experimental determination of the dependence of Q on (G+C)-content may distinguish between these possibilities. It is also suggested that addition of sufficient amounts of divalent metal ions such as Mg²⁺, Ca²⁺, or Co²⁺ may reverse the dependence of k₂ on ionic strength; under these conditions, k₂ is predicted to decrease with about the first power of ionic strength. At fixed ionic strength, k₂ should increase with increasing concentration of divalent metal ion, and, in fact, the published observation that the formation of poly(A)·2 poly(U) from poly(A)·poly(U) and poly(U) is second order in Mg²⁺ concentration is here correctly predicted from a priori molecular considerations. Finally, published association rate data for oligonucleotides are discussed in the present theoretical context.     

Interaction of β‐cyclodextrin‐capped CdSe quantum dots with inorganic anions and cations

A facile method was developed for the preparation of water soluble β‐Cyclodextrin (β‐CD)‐modified CdSe quantum dots (QDs) (β‐CD‐QDs) by directly replacing the oleic acid ligands on the QDs surface with β‐CD in an alkaline aqueous solution. The as‐prepared QDs show good stability in aqueous solution for several months. Oxoanions, including phosphoric acid ion, sulphite acid ion and carbonic acid ion, affect the fluorescence of β‐CD‐QDs. Among them, H₂PO₄^– exhibited the largest quenching effect. For the polyprotic acids (HO)₃AO, the effect of acidic anions on the fluorescence of β‐CD‐QDs was in the order: monoanion (HO)₂AO₂^– > dianion (HO)AO₃^2– >> trianion AO₄^3–. After photoactivation for several days in the presence of anions at alkaline pH, the β‐CD‐QDs exhibited strong fluorescence emission. The effect of various heavy and transition metal ions on the fluorescence properties of the β‐CD‐QDs was investigated further. It was found that Ag⁺, Hg²⁺ and Co²⁺ have significant quenching effect on the fluorescence of the β‐CD‐QDs. The Stern–Volmer quenching constants increased in the order: Hg²⁺ < Co²⁺ <Ag⁺. The adsorption model of metal ions on β‐CD‐QDs was explored. Copyright © 2011 John Wiley & Sons, Ltd.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Study of complex formation with 2-hydroxyiminocarboxylates: specific metal binding ability of 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid

Complex formation properties of a novel water soluble thiazolyloxime 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid (H₃L¹) with Cu²⁺ and Ni²⁺ were investigated in solution by potentiometrical and spectral (UV-Vis, EPR, NMR) methods. All Cu²⁺ and most of Ni²⁺ complex species detected in solution were found to have square-planar MN₄ core with oxime and heterocyclic nitrogen atoms which was rationalized in terms of destabilizing effect of repulsive interaction between oxygen atom of carboxylic group and nitrogen atom of thiazole ring in N,O-coordinated ligand conformation. It has been found that stability of metal complexes in a series of oxime ligands is dependent upon basicity of nitrogen atom of oxime group. The thiazolyloxime forms less stable complexes with Cu²⁺ but stronger ones with Ni²⁺ ions when compared to parent 2-(hydroxyimino)propanoic acid. The lower stability obtained for Cu²⁺ complexes was elucidated in terms of negative inductive effect of the thiazole and nitrile substituents as well as an effect of intramolecular attractive interaction between thiazolyl sulfur and oxime oxygen atoms in thiazolyloxime. In the case of Ni²⁺ the complexes formed are square-planar and it is why thiazolyl ligand is more effective in metal ion binding than simple 2-(hydroxyimino)propanoic acid forming only octahedral species. The solid state structure of the Co³⁺ complex K₃[Co(HL¹)₃]·5.5H₂O (1) was studied by X-ray analysis. The thiazolyloxime ligand is coordinated to Co³⁺ via oxime nitrogen and carboxylate oxygen atoms forming five-membered chelate rings.     

Calcium,magnesium and zinc ion coordination equilibria of vincristine

The zinc ion coordination of vincristine was studied by polarography; the analogous calcium ion coordination process was studied potentiometrically by a calcium ion selective electrode. In both cases, complexes of 1:1 composition were formed. The formation constant of the calcium complex was found to be 1g K = 3.27 ± 0.1. On the basis of the substitution of zinc in its vincristine complex by calcium and magnesium ions respectively, the ratio of the corresponding stability constants could be estimated as K_Zn:K_Ca (and K_Zn:K_Mg) ∼ 10⁵−3 × 10⁴. The complex formation processes proved to be pH-independent in the pH range 3.4–5.5, indicating that the metal ions are coordinated by the unprotonated oxygen donor atoms of vincristine.     

The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid)

Studies of the binding of Ni²⁺ to adenylyl-3',5'-adenosine (ApA) at pH 6-0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 °C. and ionic strength μ = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (±0.6) M^?1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, k_f = 1430 M^?1 s^?1 and k_b = 665 s^?1 (25°C. μ = 0.5M). determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni²⁺ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni²⁺. Within the concentration range [Ni²⁺ = 1 × 10^?5 × 10^?3 M the data can be represented in the form of a linear Scatchard plot. i.e., the process can be described as the binding of Ni²⁺ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K = 8.2×10³ M^?1 (25°C μ = 0.1 M). In kinetic studies of the reaction of Ni²⁺ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.     

Heavy water and 15N labelling with NanoSIMS analysis reveals growth rate‐dependent metabolic heterogeneity in chemostats

To measure single‐cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and ¹⁵N ammonium in combination with multi‐isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single‐cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single‐cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (～0.1 atom % ²H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (～6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.     

Helix–random chain transitions of polyamino acids in organic acid–salt solutions

Polyamino acids which are soluble and helical in acetic acid and dichloroacetic acid (DCA) have been observed to undergo a helix to random chain transition upon the addition of lithium salts of strong acids. The transition can be reversed by diluting the salt. Apparently only lithium cations are able to bring about the polycarbobenzoxy-L -lysine (PCBL) transition in acetic acid, whereas the anions display a varying degree of effectiveness; ClO₄^? > Br^? > TSA^? > Cl^? > NO₃^?. The lithium salts of carboxylate anions such as OAc^? and TFA^? do not cause polymer unwinding in acetic acid. Neither do the acids, TSA, HCl, TFA, or DCA induce the transformation in acetic acid. Poly-L -alanine (PLA) in DCA unfolds as LiBr is added, but does not unfold in the presence of 0.5M (CH₃)₄NBr, 0.25M CsBr, or 0.32M HCl. These results are explained on the basis of a direct interaction of the lithium salt with the polymer amide groups to form an ion-pair complex. The extent to which the union of the ion pair can dissociate from the complex in the low dielectric constant, environment determines the degree of unfolding of the polymer. The anion dissociation equilibrium presumably therefore would lie in the same order as given above. Acids such as HCl and TSA are considered to substantially protonate and ion-pair with the polymer, but do not readily dissociate the anion partner from the complex, and therefore do not produce an unstable positively charged helical structure.     

Transition-state structure for a conformation change of ribonuclease

The rate constants k₁₂ⁿ for isomerization of the E₁H isomer (pK_a 8 in H₂O) of ribonuclease-A to the E₂H isomer (pK_a = 6.1 in H₂O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k₁₂ⁿ = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)²sec⁻¹ at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization.     

ELECTRON STAINS : I. Chemical Studies on the Interaction of DNA with Uranyl Salts

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO₂⁺⁺/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.     

Stability and solvation of alkali metal ion complexes with dibenzo-30-crown-10

Stability constants for the 1:1 complexes of dibenzo-30-crown-10 (DB30C10) with alkali metal ions have been determined at 25 °C in nitromethane and water by conductometry and capillary electrophoresis, respectively. Transfer activity coefficients of DB30C10 and its complexes from nitromethane to S (S = water, acetonitrile, propylene carbonate, methanol, and N,N-dimethylformamide) have been determined at 25 °C to evaluate the solvation properties. The stability constant in the poorly solvating solvent, nitromethane, decreases with increasing metal ion size, Na⁺ > K⁺ > Rb⁺ > Cs⁺, reflecting the intrinsic selectivity governed by electrostatic interaction between the metal ion and the ether oxygen atoms. It is also suggested that a part of the ether oxygen atoms does not bind to the metal ion in the Na(DB30C10)⁺ complex. The aqueous stability constant varies as Na⁺ ? K⁺ ≈ Rb⁺ ≈ Cs⁺; this selectivity pattern is similar to that in acetonitrile, propylene carbonate, and methanol. The complex stability in water is very low compared to that in the nonaqueous solvents, owing to hydrogen bonding of water to the oxygen atoms of the free crown ether. The transfer activity coefficient values show that DB30C10 shields all the metal ions effectively from the solvents and lead to the conclusion that the complexation selectivity in S receives a significant contribution from the solvation of the free metal ions. The Na(DB30C10)⁺ complex has specific interaction with water, causing much lower K⁺/Na⁺ selectivity in H₂O than in MeOH.     

Interaction of the antimalarial drug fluoroquine with DNA,tRNA, and poly(A): 19F-NMR chemical-shift and relaxation,optical absorption,and fluorescence studies

The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and ¹⁹F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (T_m) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the ¹⁹F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D₂O vs H₂O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. ¹⁹F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T₁), ¹⁹F{¹H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.     

Transport kinetics of hydrophobic ions in lipid bilayer membranes Charge-pulse relaxation studies

A modified version of the charge-pulse relaxation technique with improved time resolution was applied to the study of transport kinetics of hydrophobic ions (tetraphenylborate, dipicrylamine) through lipid bilayer membranes. Besides a better time resolution the charge-pulse method has the additional advantage that the perturbation of the membrane can be kept small (voltage amplitudes between 1 and 10 mV). The results of the analysis support the model proposed earlier, according to which the overall transport takes place in three consecutive steps, adsorption of the ion from water to the interface, translocation to the opposite interface, and desorption into the aqueous phase. The translocation rate constant K_i and the partition coefficient γ of the hydrophobic ion between water and the membrane were measured for lecithins with different mono-unsaturated fatty acid residues. Increasing the chain length of the fatty acid from C₁₆ to C₂₄ resulted in a decrease of k_i by a factor of about 9 in the case of tetraphenylborate and by a factor of about 17 in the case of dipicrylamine.