An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.     

1H NMR study of effects of synergistic anion and metal ion binding on pH titration of the histidinyl side-chain residues of the half-molecules of ovotransferrin

Separation of ovotransferrin into C-terminal (OTf/2C) and N-terminal (OTf/2N) half-molecules has made possible the resolution of all expected histidinyl C(2)H resonances by proton nuclear magnetic resonance at 250 MHz. The chemical shift of many of the resonances decreases with increasing pH, allowing construction of titration curves, whereas a few resonances fail to titrate. On formation of the GaIIIOTf/2(C2O4) ternary complexes, two of the low-field C(2)H resonances in each half-molecule fail to titrate. This behavior implicates the imidazole groups giving rise to these resonances as ligands to the bound metal ion. A third C(2)H resonance in each half-molecule undergoes a marked reduction in pK'a on formation of the ternary complex. The imidazole group displaying this resonance is implicated in a proton-relay scheme involved in binding the synergistic anion, oxalate, and a water of hydration on the bound metal ion. The titration curves for the various imidazole resonances have been fit to a four-parameter equation involving estimation of the pK'a, the limiting chemical shift values, and a Hill constant n. Hill constants of less than 1 can be rationalized by correcting the titration curve for the charge Z on the protein as a function of pH and the work function w. The titration curve for the imidazole group in OTf/2C involved in the proton-relay scheme shows a value for n greater than 1, which suggests positive cooperativity in the titration of this residue. The basis for this behavior cannot be rationalized at this time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD were studied by potentiometric and spectroscopic (UV-Vis, CD and EPR) techniques. The results reveal that both ligands have effective metal binding sites, but the tripeptide is a much stronger complexing agent than the tetrapeptide. The formation of a macrochelate via the coordination of the imidazolyl residues is suggested in the copper(II)-Ac-HisValHis-NH2 system in the acidic pH range, while a 4N complex predominates at physiological pH. The interaction of Ac-HisValHis-NH2 with zinc(II) results in the formation of a precipitate indicating polynuclear complex formation. Both copper(II)-Ac-HisValHis-NH2 and copper(II)-HisValHis systems exhibit catalytic activity toward the dismutation of superoxide anion at physiological pH, but the saturated coordination sphere of the metal ions in both systems results in low reactivity as compared to the native enzyme.     

Kinetics of iron and copper catalysis of ascorbate oxidation.

The effects of oxidant, pH and ligands on iron- and copper-catalyzed ascorbate oxidation have been examined. The formation of the catalyst-substrate complex is affected by pH, whereas oxidant affects its breakdown. With copper-ion catalysis, ligands inhibit competitively. With iron catalysis, on the other hand, for a series of aminopolycarboxylic ligands at neutral pH, formation of catalyst-substrate is favored by ligands which form more stable iron complexes. Decreased rates caused by changes in metal environment (ligand or pH) may result for competing activities (e.g., catalase activity competing with peroxidase activity). Evidence for a ternary complex (catalyst-substrate-oxidant) is presented.     

Interactions of insulin-mimetic zinc(II) complexes with cell constituents: glutathione and ATP

Ternary complex formation of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 3-hydroxy-1,2-dimethyl-pyridinone with (O,O), 2-picolinic acid and 6-methylpicolinic acid with (N,O) and the tridentate 2,6-dipicolinic acid with (O,N,O) coordination modes was studied in aqueous solutions by pH-potentiometry and spectroscopic (UV, CD, ESI-MS) methods in the presence of critical cell constituents such as l-glutathione reduced (GSH) and adenosine 5′-triphosphate (ATP). Results showed that formation of the ternary complexes was hindered in the case of 2,6-dipicolinic acid, especially with ATP, while it was favoured with the bidentate ligands in the physiological pH range. Driving force of the formation of mixed-ligand species was found to be a more enhanced coordination of GSH and ATP as second ligands in the ternary complexes than in their binary ones due to steric and electrostatic reasons. The mitochondrial dehydrogenase activity of the zinc(II) complexes, as an indirect indicator for the glucose intake, was measured on Mono Mac and 3T3-L1 adipocyte cell lines. The activity of the complexes up to ∼10-100 μM concentration was in the range of the effect of 0.75-1.5 μM insulin, while at higher concentration it was broken down due to the sensitivity of the cells to toxicity of the complexes.     

Affinity labels for the anion-binding site in ovotransferrin

Bromopyruvate, a known alkylating agent, has previously been reported to function as an affinity label for the anion-binding site in the iron-binding protein ovotransferrin [Patch, M.G., & Carrano, C. J. (1982) Biochim. Biophys. Acta 700, 217-220]. However, the present results indicate that hydroxypyruvate also functions in an almost identical manner, which implies that alkylation of a susceptible nucleophile cannot be the mechanism responsible for the covalent attachment of the anion. Model complexes and amino acid analysis of labeled ovotransferrin suggest that initial Schiff base formation, followed by reduction of the imine bond between the affinity anion and a lysine within the locus of the anion-binding site, accounts for the irreversible labeling. As expected, the covalently attached anions render the iron in the ovotransferrin-iron-anion ternary complex much more resistant to loss at low pH. It is proposed that the covalently labeled protein be used to test the hypothesis that iron removal from transferrin occurs by protonation and loss of the anion in low-pH lysosomal vesicles.     

Transport of Cu(II) from an albumin mimic peptide, GlyGlyHisGly, to histidine and penicillamine

Cu in blood has been believed to transport into cell via albumin and some amino acids. To shed light on the Cu transport process we studied the reaction of the Cu(II)-peptide with the amino acid by absorption and CD spectra. Albumin mimic peptides GlyGly-L-HisGly (GGHG) and penta-Gly(G5) formed stable 4N coordinated Cu(II) complexes, but in the reaction with histidine (His) and penicillamine (Pes) the ternary Cu(II) complex formations were observed different by the kinetic study. Cu(II)-G5 complexes reacted with Pes to form the ternary complex Cu(H(-1)G5)(Pes(-)) which was subsequently transformed to the binary complex Cu(Pes(-))(2). In the system with GGHG the Cu(II) was also transported from GGHG to Pes, but the ternary Cu(H(-1)GGHG)(Pes(-)) complex as the intermediate was detected a trace. The ternary complex would be spontaneously transformed to Cu(Pes(-))(2) upon forming, because the rate constant of the ternary complex formation k(1+)= approximately 2M(-1)s(-1) was less than k(2+)= approximately 5 x 10(2)M(-1)s(-1) for the Cu(Pes(-))(2) formation at physiological pH. In the Cu(II)-GGHG-His system the ternary Cu(H(-1)GGHG)(His) complex was also hardly identified because the formation constant K(1) and k(1+) were very small and the equilibrium existed between Cu(H(-2)GGHG) and Cu(His)(2) and its overall equilibrium constant beta(2) for Cu(His)(2) was very small to be 1.00+/-0.05 M(-1) at pH 9.0. These results indicated that the ternary complex is formed in the Cu transport process from the albumin to the amino acid, but His imidazole nitrogen in the fourth-binding site of Cu(II) strongly resists the replacement by the incoming ligand.     

Speciation studies of ternary complexes of some essential divalent metal ions with L-histidine and L-glutamic acid in DMSO-water mixtures

Abstract

Chemical speciation of ternary complexes of Ca(II), Mg(II) and Zn(II) ions with L-histidine as the primary ligand (L) and L-glutamic acid as the secondary ligand (X) has been studied pH metrically in the concentration range of 0.0-60.0% v/v DMSO-water mixtures maintaining an ionic strength of 0.16 mol L^-1 using sodium chloride at 303.0 K. Titrations were carried out in different relative concentrations (M:L:X = 1.0:2.5:2.5, 1.0:2.5:5.0, 1.0:5.0:2.5) of metal (M) to L-histidine to L-glutamic acid with sodium hydroxide. Stability constants of ternary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The predominant species detected for Ca(II), Mg(II) and Zn(II) are ML₂XH₂, MLXH₂ and MLX₂. Extra stability of ternary complexes compared to their binary complexes was explained to be due to electrostatic interactions of the side chains of ligands, charge neutralisation, chelate effect, stacking interactions and hydrogen bonding. The species distribution with pH at different compositions of DMSO and the plausible equilibria for the formation of species are discussed.     

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 50-70-fold lower than that observed for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, the binding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn-BSPAP. F and FeZn-BSPAP.PO(4).F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.     

Copper(II) complexes with β-cyclodextrin-homocarnosine conjugates and their antioxidant activity

Copper(II) complexes of the β-cyclodextrin (β-CD) functionalized with homocarnosine (HC) in the primary (CDHC6) and secondary rim (CDHC3) were characterized by means of different spectroscopic techniques such as UV-Vis absorption, circular dichroism, electron paramagnetic resonance and electron-spray mass spectrometry. Taken together, all the spectroscopic parameters indicate the formation of different copper(II) complex species at various pH values. In the CDHC3 copper(II) complex species, a direct involvement of the secondary hydroxyl group 2 of functionalized β-CD’s ring has been pointed out.The antioxidant activity of the copper(II) complexes of the two derivatives was determined through pulse radiolysis measurements. The results obtained provide direct evidence for a high catalytic activity of both complexes towards the dismutation of the superoxide anion radical. It is also demonstrated that the complex formation is not detrimental to the excellent scavenger activity exhibited by the ligands alone towards hydroxyl radicals. These copper complexes then represent very intriguing antioxidant agents against well known toxic reactive oxygen species.     

The influence of inorganic anions on the formation and stability of Fe3+-transferrin-anion complexes

Harris (Biochemistry 24 (1985) 7412) reports that inorganic anions bind to human apotransferrin in such a way as to perturb the ultraviolet spectrum. The locus of binding is thought to involve the specific metal/anion-binding sites since no perturbation is observed with Fe3+-transferrin-CO3(2-). Paradoxically, we were unable to demonstrate the formation of Fe3+-transferrin-inorganic anion complexes despite the presence of high concentrations of SO4(2-), H2PO4-, Cl-, ClO4- or NO3-. Similar results were found for human lactoferrin. Electron paramagnetic resonance spectroscopy and visible spectrophotometry were used to monitor the results. An attempt to form the H2PO4- complex by displacement of glycine from Fe3+-transferrin-glycine resulted only in the disruption of the ternary complex. A series of inorganic anions varied in their ability to release iron from Fe3+-transferrin-CO3(2-) at pH 5.5, the approximate pH of endosomes where iron release takes place within cells. The order of effectiveness was H2P2O7(2-) much greater than H2PO4- greater than SO4(2-) greater than NO3- greater than Cl- greater than ClO4-. The rate of iron removal from Fe3+-transferrin-CO3(2-) at pH 5.5 by a 4-fold excess of pyrophosphate was greatly enhanced by physiological NaCl concentration. Iron removal was complete within 10 min, the approximate time for iron release from Fe3+-transferrin-CO3(2-) in developing erythroid cells. Thus, inorganic anions may have a significant effect on the release of iron under physiological conditions despite the fact that such inorganic anions cannot act as synergistic anions. The results are discussed in relation to a special role for the carboxylate group in allowing ternary complex formation.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Alizarin complexone-lanthanide(III)-fluoride system: Revised speciation and the origin of the analytical signal

Potentiometric titrations of alizarin complexone (ALC) in the presence of La(III), Eu(III) and Y(III) in 50% vol. DMSO reveal formation of mostly binuclear complexes with monoprotonated (LH) and completely deprotonated (L) forms of the ligand of the type M₂L₂H₂, M₂L₂H and M₂L₂. Only for La(III) a single mononuclear complex LaLH is observed in acidic media. Titrations of the same mixtures with added fluoride show that with Eu(III) and Y(III), fluoride forms ternary complexes without shifting the distribution profile of complexes with differently protonated forms of bound ALC, but with La(III) a strong shift in distribution in favor of complexes with the deprotonated ligand occurs, which explains the appearance of blue colored species in acidic medium giving rise to the analytical signal employed for quantitative determination of fluoride. Detailed analysis of the spectral properties of La(III)-ALC-fluoride system on the basis of the established species distribution diagrams demonstrate the following factors to be involved: (1) fluoride-induced conversion of LaLH to La₂L₂HF around pH 3; (2) anomalously high affinity of La(III)-ALC complexes to fluoride and (3) some sort of mutual effect of fluoride and ALC ligands in ternary La(III) complexes, probably involving change in the metal coordination number, leading to enhanced absorptivity of La₂L₂HF as compared to La₂L₂H.     

Simultaneous binding of two proteins to opposite sides of a single transfer RNA

Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase--tRNA--Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery.     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new macrocyclic tetraamine, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane):synthesis, cation reporter properties, and recognition of 1-methylthymine by its zinc(II) complex

A new functional macrocyclic ligand, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane), has been synthesized and isolated as its trihydrochloric acid salt (L·3HCl). The protonation constants (log K_n) for three secondary nitrogens of L were determined by potentiometric pH titration to be 10.10, 7.33 and <2 with I = 0.10 (NaNO₃) at 25°C. The 2,4-dinitrophenylaniline chromophore was proven to be a good reporter signaling proton- and metal-binding events in the macrocyclic cavity. The UV absorption band (λ_max 370 nm, 8200) of the 2,4-dinitrophenylaniline moiety at pH ≥ 9 becomes quenched as pH is lowered (to pH 3.1, where the major species is L·2H⁺), due to the strong protonation effect extended to the aniline moiety within the macrocyclic cavity. This is in sharp contrast to the pH-independent UV absorption (λ_max 390 nm, 14 000) of a reference compound, N,N-diethyl-2,4-dinitroaniline. The UV absorption band of L is shifted to lower wavelengths with Zn²⁺ (λ_max 320 nm), Cd²⁺ (λ_max 316 nm) and Pb²⁺ (λ_max 317 nm), while it almost disappears with Cu²⁺ and Ni²⁺. The 1:1 Zn²⁺ and Cu²⁺ complexes with L were isolated and characterized. The Zn²⁺ complex recognizes 1-methylthymine anion (MT⁻) in aqueous solution at physiological pH to yield a stable ternary complex ZnL-MT⁻. The X-ray crystal structure of ZnL-MT⁻ showed that Zn²⁺ is four-coordinate with three secondary nitrogens of L and the deprotonated imide anion that is cofacial to the 2,4-dinitrophenyl ring.     

The complex formation of copper(II) with GHL and HSA

The formation constants for complexes of copper(II) with GHL have been determined by means of pH titrations and ESR spectroscopy in aqueous solutions. GHL has an extremely high affinity for copper(II) and forms very stable 1:1 complexes and a comparatively weak 1:2 complex. The ? amino group of GHL seems not to be involved in complex formation as can be deducted from both equilibrium constants and ESR spectroscopy. The ternary system copper(II)-GHL-HSA was investigated by ESR spectroscopy and optical absorption spectroscopy in aqueous solution at physiological pH (7.4). At equimolar concentrations, copper(II), HSA and GHL form a ternary complex.     

Acid-base and metal ion-binding properties of flavin mononucleotide (FMN). Is a ‘dielectric’ effect responsible for the increased complex stability?

Due to contradictions in the literature we have redetermined the acid-base properties of riboflavin (=RiFl; vitamin B₂), i.e. 7,8-dimethyl-10-ribityl-isoalloxazine, and of flavin mononucleotide (FMN²⁻), also known as riboflavin 5′-phosphate, via potentiometric pH titrations (I = 0.1 M, NaNO₃; 25 °C). In contrast to various claims, the isoalloxazine ring cannot be protonated at pH > 1, a result in agreement with an early study (pK_a = −0.2; L. Michaelis, M.P. Schubert and C.V. Smythe, J. Biol. Chem., 116 (1936) 587–607); deprotonation of the ring system occurs in both compounds with pK_a 10. The pK_a value of 0.7 determined for the deprotonation of H₂(FMN) must be attributed to the release of the first proton from the fully protonated phosphate group; its second proton is released with pK_a = 6.18 in agreement with the acidity constants of various other monoprotonated monophosphate esters. The stability constants of the 1:1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Cd²⁺ (---M²⁺) and FMN²⁻ were determined by potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO₃; 25 °C). The log stability constants of all these M(FMN) complexes are about 0.2 log units higher than expected from the basicity of the phosphate group. This slight stability increase cannot be attributed to the formation of a seven-membered chelate involving the ribit-hydroxy group at C-4′ as the stability constants for the M²⁺ 1:1 complexes of glycerol 1-phosphate (G1P²⁻) demonstrate: G1P²⁻ contains the same structural unit which would also allow in this case the formation of the mentioned seven-membered chelate; however, the stability of the M(G1P) complexes is solely determined by the basicity of the phosphate group. Hence, in agreement with earlier conclusions (J. Bidwell, J. Thomas and J. Stuehr, J. Am. Chem. Soc., 108 (1986) 820–825) regarding Ni(FMN) one must conclude that the slight stability increase of the M(FMN) complexes has to be attributed to the isoalloxazine ring. The equality of the stability increase of the complexes for all the mentioned ten metal ions precludes its attribution to an interaction with an N site and makes a specific interaction with an O site also somewhat unlikely. In addition, carbonyl oxygens appear as not very favorable for the formation of macrochelates by a further interaction with already phosphate-coordinated metal ions. Therefore, we propose that the slight but significant stability increase originates from M(FMN) species (with a formation degree of about 30%) in which the hydrophobic flavin residue is close to the metal ion, thereby lowering the ‘effective’ dielectric constant in the microenvironment of the metal ion and thus indirectly promoting the −PO₃²⁻/M²⁺ interaction.     

Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes

Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-550 s(-1)) is a common step that precedes binding of all inhibitors. At all pH values studied, the rate constants for proton release or uptake matched those for formation of ternary complexes, and the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) was observed for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transient data for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of the enzyme-NAD+ complex is coupled to the conformational change and controls the formation of ternary complexes.     

Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding

Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)