A new radiochemical method to investigate ion binding with polyelectrolytes

A new method for investigating the binding of ions with polyelectrolytes has been developed. This method, based on Donnan equilibrium and an isotope exchange between the electrolyte and polyelectrolyte, can distinguish territorial from specific binding of ions and can determine fractions of ions bound with the polyion. This method can determine ion binding with polyelectrolytes in a wide range of polyelectrolyte concentrations in multicomponent solutions. The method was tested with radioactive tracers 22Na+, 36Cl- and heparin sodium salt. The influence of the ionic strength on the Na+ binding with heparin was investigated at 310 K. In the limit of zero ionic strength, all Na+ ions are bound to heparin, but only 45% of them are exchangeable. Thus Na+ ions can be bound both territorially and specifically. The fraction of bound ions decreases rapidly with increasing ionic strength. The fraction of the specifically bound ions becomes negligible when the ionic strength exceeds 0.01 M, whereas the fraction of territorially bound ions can be neglected at ionic strengths higher than 0.45 M.     

Studies of calcium ion binding to poly A

Ultraviolet differential spectra of poly A we studied in the presence of Ca2+ ions with 10(-3)M Na+ in the solution. At concentrations lower than 10(-3)M Ca2+, the ions bind to phosphate groups of the single helical polymer, thus increasing its degree of helicity. At higher concentrations, the ions start binding to the bases of poly A, producing aggregates whose effective radius, as found with an electric microscope, is not more than 10(2) A. These particles stack to form aggregates of an order-of-magnitude higher size. The mutual orientation of bases in the poly A aggregates is of a high degree of order. The calculation of concentration dependences of Ca2+-poly A binding constants shows that this process is cooperative.     

Hydroxide ion binding to methemerythrin. An investigation by resonance Raman and difference spectroscopy

The pH dependence for the interconversion of the acid and base forms of methemerythrin from Themiste dyscritum was investigated by difference spectroscopy. A new technique was designed to be able to study mixtures without knowledge of extinction coefficients or exact protein concentrations. The resultant pKa value of 8.4 proved that T. dyscritum hemerythrin crystals used for previous X-ray crystallographic studies at pH less than or equal to 6.5 were in the acid form. Since this material contains a 5-coordinate iron atom with no evidence of a ligated water molecule, it is more appropriately referred to as methemerythrin than aquomethemerythrin. The presence of an iron-bound hydroxide in the base form of methemerythrin was verified by resonance Raman spectroscopy for both T. dyscritum and Phascolopsis gouldii. At pH greater than 9, the protein from either species exhibited a new feature at 490 cm-1 that shifted to 518 cm-1 in D2O and was assigned to a coupled Fe-OH stretching and O-H bending vibration. Thus, hydroxomethemerythrin is the correct designation for the base form of the protein. The other resonance-enhanced vibration, the Fe-O-Fe symmetric stretch, was observed at 506 cm-1 in hydroxomethemerythrin and at 511 cm-1 in methemerythrin and was unaffected by deuteration. Addition of perchlorate to methemerythrin had no effect on the Raman spectrum, despite its known role in stabilizing the met form relative to the hydroxomet form.     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

Mg and Ca binding to heparin in the presence of added univalent salt

Activities of Mg and Ca in solutions of heparin with added sodium or potassium chloride have been determined by means of a previously described dye spectrophotometric method. The polyion concentration, C_p (expressed as mol univalent anionic charges/I) is 0.001 in all cases, solution total ionic strengths are 0.005, 0.0075, 0.01, 0.02 and 0.04 mol/l. Divalent metal ion concentrations are varied between 0 and 1.0 C_p. The results for the metal ion activities are expressed in the form of binding isotherms, θ₂ vs. C₂/C_p (θ₂=C_2b/C_p; C_2b=bound divalent metal ion concentration) and Scatchard plots, K₂ vs. θ₂/(C₂-C_2b, at different ionic strengths. Results are correlated with a theoretical treatment of the mixed counterion system, called the ‘two variable theory’, recently developed by Manning. The ionic strength dependence of θ₂ and K₂ is found to agree with the prediction of the two variable theory. Mg interaction with heparin appears to be independent of the nature of the charged groups on the polyion and is in very good agreement with the two variable theory. Ca binding was found to be considerably higher than Mg binding and is in excess of theoretical predictions, suggesting a localized or specific interaction of Ca with heparin.     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

Surface potential effects on metal ion binding to phosphatidylcholine membranes 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles

31P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln3+ or Ca2+ shifted the 31P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca2+ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb3+ and Ca2+ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

The calcium ion binding site in bovine chymotrypsin A

The effect of Gd³⁺ on the nuclear magnetic resonance (nmr) relaxation rates, T_1m^?1 and T_2m^?1, of inhibitor protons in metal-inhibitor-α-chymotrypsin ternary complexes has been measured. The Solomon-Bloembergen equations were used to calculate the distance from the methyl protons of p-toluamidine (a competitive inhibitor) to the Gd³⁺ binding site which is 9.2 ± 0.5 Å. Calcium ion and gadolinium ion compete for the same binding site on α-chymotrypsin. Distances from the specificity pocket of α-chymotrypsin to the metal binding site have been measured by fluorescence energy transfer experiments. By observing energy transfer between proflavine and Nd³⁺, Pr³⁺, or Ho³⁺, we have been able to calculate a distance of approximately 10 Å between the two chromophores. This agrees well with the data obtained by nmr techniques and also gives nearly identical values to those obtained for trypsin (Darnall, D., Abbott, F., Gomez, J. E., and Birnbaum, E. R., Biochemistry15, 5017, 1976). This is consistent with the calcium ion binding sites being composed of the same residues in both trypsin and α-chymotrypsin.     

On the nature of calcium ion binding between phosphatidylserine lamellae

Ca2+ binding between phosphatidylserine (PS) lamellae gives rise to a phase with the composition Ca(PS)2. When aqueous Ca2+, hydrated PS, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant. At Ca2+ concentrations below this critical value, no binding of Ca2+ to PS is detected. Above this value, Ca2+ binds to PS to form Ca(PS)2. The invariant Ca2+ concentration is 0.14 microM for palmitoyloleoylphosphatidylserine (POPS) and 3.0 microM for dioleoylphosphatidylserine (DOPS). For the mixed acyl chain PS derived from bovine brain (BBPS) this Ca2+ concentration ranges from 0.25 to 0.7 microM. The observed phase behavior is described by the phase rule for the three-component system of water, Ca2+, and PS, with temperature and pressure constant. In order for Ca2+ to bind between PS lamellae to form the Ca(PS)2 phase, the aqueous Ca2+ concentration must be supersaturated. The equilibrium Ca2+ concentration is determined by dissolving Ca(PS)2 by use of Ca2+ chelators.     

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine

The effects of calcium ion on phosphatidyl L-serine (PS) have been studied with PS membranes containing a lecithin spin label (L¹). The calcium ion makes the ESR spectra of the L¹ in PS membranes broadened owing to the intermolecular spin-spin exchange interactions. The results indicate that the calcium ion binds to PS molecules to form rapidly rigid calcium ion-bound PS aggregates, the lecithin molecules being thereby separated from the host PS bilayers to form clusters. The magnesium ion is ineffective for the aggregation and exerts a quite different effect only at much higher concentrations.     

Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro

The effect of calcium ion on 3,5,3'-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, the binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM calcium, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.     

An investigation on calcium taurodeoxycholate micellar aggregates

Abstract

The formation of micellar aggregates in the presence of calcium(II) ions in solutions containing sodium and taurodeoxycholate ions and their composition at 25°C and in 0.5 mol dm^?3 N(CH₃)₄Cl as constant ionic medium was studied. The study was carried out by means of two different procedures. In the first one, solid calcium oxalate was equilibrated with taurodeoxycholate, sodium and hydrogen ions and the free concentration of sodium and hydrogen ions was determined. After filtration, the calcium(II) (by atomic absorption spectrophotometry) and oxalate concentration were also determined. In the second approach, hydrogen and sodium ions free concentrations were obtained by electromotive force measurements carried out in solutions containing taurodeoxycholate. The results of both procedures could be explained by assuming the presence of aggregates of different composition with the participation of sodium, calcium(II) and taurodeoxycholate ions, depending on the concentration of the reagents. Protonated species were even present in appreciable concentrations. All the found species have taurodeoxycholate aggregation numbers in multiples of three. A mechanism for the micellar aggregates containing calcium and sodium is proposed. Sodium taurodeoxycholate in the presence of calcium(II) forms larger aggregates than does taurocholate in the presence of calcium(II); the building block of the former is a trimer whereas the latter system has lower aggregation numbers and its building block is a dimer or an octamer.     

A chlorotetracycline fluorescent probe--an indicator of calcium ion binding with calcium-binding proteins

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.