Cooperative specific adsorption phenomena in biology

The importance of cooperative specific adsorption for the interpretation of biological processes is pointed out by the agreement of experimental data with a cooperative specific adsorption isotherm which has been derived on the basis of statistical mechanics as well as stochastically. An approach is described to account for physiological excitation on the basis of cooperative specific adsorption of potassium and sodium after a switch in the adsorption preference of the sites for those ions. To derive the accompanying bioelectric phenomena, the vacant negative adsorbing sites and the changes of occupancy of the interstitial sites have to be taken into account. This leads to the kinetics of cooperative phenomena in which each state variable can assume at least three values. For this purpose a master-equation has to be solved or a Monte Carlo method used. It is suggested that the calcium concentration be considered as a control variable when catastrophe theory is applied.     

A source of error in equilibrium dialysis

Equilibrium dialysis is often used to study the binding of steroids to proteins. With this technique it is customary to determine the percent bound and unbound steroid in the sample, the affinity constant for the steroid-protein binding reaction, and the concentration of binding sites on the protein. Investigators have used many different ratios of dialysis buffer to sample volumes in their experiments assuming that the equilibrium in the post-dialysis sample was the same as existed before dialysis. Chemical equilibrium expressions for the system before and after dialysis indicate that during dialysis the concentration of steroid in the sample decreases resulting in a new equilibrium in which the percent bound and unbound are different from the original sample. The magnitude of the difference between the pre- and post-dialysis systems is proportional to the ratio of dialysis buffer to sample volumes. Accurate values for the affinity constant and binding site can be obtained only if this change in the equilibrium is considered.Experimental verification of the application of these principles was made in an equilibrium dialysis study of testosterone-albumin binding.     

Preparation of plasma samples for amino acid analysis by equilibrium dialysis.

A claim that losses of free amino acids in plasma can occur on deproteinisation with sulphosalicylic acid has been confirmed in studies with synthetic amino acid mixtures. The use of equilibrium dialysis is proposed as a simple, quick, and effective means of preparing protein-free extracts of plasma amino acids from plasma. Dialysis against citrate buffer ensures that the amino acid-containing extract is in a form suitable for direct application to an amino acid analyser. Apart from tryptophan, the amino acid concentrations of the dialysate appear stable at 0° for up to 10 weeks.     

Binding of calcium to glycosaminoglycans: an equilibrium dialysis study

Binding of calcium to the glycosaminoglycans (GAGs) heparin, chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA) has been studied by equilibrium dialysis using exclusion of sulfate to correct for Gibbs-Donnan effects. Calcium binding occurs to all of these GAG species, suggesting that both sulfate and carboxylate groups are involved in cation binding. For all GAGs, the binding stoichiometry is consistent with a calcium-binding "site" consisting of two anionic groups. The order of calcium binding affinities is heparin greater than CS greater than KS greater than HA, and is critically dependent upon charge density; heparin binds calcium with 10-fold higher affinity than CS. The mode of calcium binding to GAGs is consistent with a recently proposed mechanism of growth plate calcification which states that cartilage proteoglycan functions as a reservoir of calcium for calcification of epiphyseal cartilage.     

Enzyme adsorption in porous supports: local thermodynamic equilibrium model

Enzyme adsorption from a finite bath (batch adsorption) onto porous spherical supports is investigated both experimentally and theoretically using beta-galactosidase and Duolite ion-exchange resin as a model system. Efficient numerical techniques are presented that have been used in conjunction with a parameter estimation routine to evaluate adsorption isotherm constants. Results show that even for adsorption processes lasting almost 10 h, the majority of the enzyme is confined to the outer half of the support and, for high initial enzyme concentrations in the bath, this loading takes place as a slowly moving front. Information on the enzyme distribution has practical importance in the design of immobilized enzyme reactors that in previous works have almost always been analyzed assuming a uniform catalyst distribution.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

The analysis of fatty acid binding to protein using a modified equilibrium dialysis method: detailed analysis of chromophore-fatty acid-protein interactions

In this paper we extend our previous analysis of fatty acid-chromophore-protein interactions using a modified equilibrium dialysis method described previously. A more rigorous mathematical treatment is combined with a micro-dialysis method using a maximum volume of dialyzate of between 250 microliters and 400 microliters to examine the suitability of different chromophores (mepacrine, quinine, chloroquine, chlorpromazine, methylene blue, rhodamine 6G, 6-carboxyfluorescein) for studying the binding of fatty acid to protein. The macro- and micro-methods of dialysis are compared, and the binding of fatty acid to bovine serum albumin and beta-lactoglobulin discussed as examples of the method. Problems associated with propagated errors in the measurements and obtaining the number of binding sites and the binding constants from curve-fitting are also considered.     

An evaluation of ways of using equilibrium dialysis to quantify the binding of ligand to macromolecule.

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Utilization of conical equilibrium dialysis cells to shorten equilibration time

Equilibrium dialysis is commonly used to characterize the binding properties of macromolecules; however, it is not always preferred because of the lengthy time required to reach equilibrium. Consequently, other methods have been developed. This paper compares the disadvantages of various methods for obtaining binding data and shows that utilization of a conically shaped chamber can accelerate the process of equilibrium dialysis. For cylindrical and conical cells of different dimensions a linear relationship was found for the time of equilibration versus the ratio of volume to surface area. Minimizing the volume to surface area ratio by using a conical chamber shortens the time of equilibration by more than half and facilitates sampling of the chambers' contents.     

Application of thin film dialysis to the determination of equilibrium constants

A theoretical basis for thin-film dialysis involving binding between a ligand and nondialyzing species is presented. A general differential equation that applies to the case of equivalent, noninteracting sites is derived relating [A]_F, [A]_T, [P]_T, and K. Numerical solutions to this equation are used to develop a series of escape curves corresponding to specific values of the parameters [P]_T, [A]_i, K, and k₀. A general method for determining an equilibrium binding constant from thin-film dialysis data is given. A comparison of thin-film dialysis results predicted by this theory with literature data shows essential agreement.     

Comparison of serum free thyroxine measurements by chemiluminescence and equilibrium dialysis

A study of 239 patients compared free thyroxine (FT4) measurements made by equilibrium dialysis (ED) with measurements made using the Magic Lite FT4 chemiluminescence (Cl) immunoassay (Ciba Corning Immunodiagnostics). Patient groups: 41 normals; 27 hyperthyroid; 29 hypothyroid; 37 sick euthyroid; 10 chronic renal failure (CRF) and 25 pregnant patients; 13 oestrogen; 10 heparin; 12 salicylate; and 9 dilantin-treated patients; 3 lipaemic; 5 haemolysed; 6 hyperbilirubinaemic patients; 6 low thyroid binding protein (TBP) and 6 high TBP level patients. The two assays gave comparable results in most groups. Both assays tended to give elevated values in heparinized patients but FT4–ED results were more obviously affected. Pregnant patients and women on oral oestrogen had higher mean values with FT4–ED. In both assays the sick euthyroid and CRF patients had mean FT4 values similar to healthy euthyroid patients; the range of values in sick euthyroid and CRF patients was similar in both assays but wider than in healthy euthyroid patients. A supplemental study of 81 unselected acutely ill patients using FT4–Cl alone confirmed the wider range of values to be anticipated in sick euthyroid patients.     

Effects of charged groups in haptens on adsorption equilibrium of hapten antibody

Antisera against charged (p-azobenzoate and p-azoben zenesulfonate) and uncharged (dinitrophenyl) haptenic groups were produced in rabbits, and the equilibrium characteristics of hapten-antibody were measured by use of immunoadsorbents. The antibody to the uncharged hapten formed a stable binding with the hapten to the changes in ionic strength and pH. On the other hand, the antibodies to the charged haptens showed affinities sensitive to the changes in pH and ionic strength. Therefore, the effect of the pK(a) of ionizable haptens on the pH dependence of the hapten-antibody binding was studied by comparing the interactions between a series of para-substituted benzoic acids and the anti-p-azobenzoate antibody. The pH dependence of the interactions was strongly affected by the pK(a) of ionizable groups in haptens. Furthermore, the equilibrium characteristics of anti-p-aminobenzoyl dipeptides were compared. The characteristics of interactions were affected by the features of amino acid residues.     

Substrate and water adsorption phenomena in a gas/solid enzymatic reactor

The adsorption of water and substrates to dry deposited alcohol dehydrogenase from Lactobacillus brevis (LBADH) is studied in a continuous enzymatic gas/solid reactor. This work is aiming at obtaining a deeper and more thorough understanding of the enzyme microenvironment in the gas/solid system of acetophenone reduction with concomitant oxidation of 2-propanol. Extensive water adsorption studies showed that the effect of sucrose in the enzyme preparation on the water adsorption isotherm is significant for water activities exceeding 0.5 and reaches a factor 2 with respect to bead mass at water activity of 0.9. Significant hysteresis during water desorption is identified, resulting in up to 0.6 mg_water/mg_protein, for lyophilized enzyme preparation and up to 10 mg_water/mg_protein for deposited enzyme preparation. The adsorption of the substrates is quantified here for the first time. Whereas the adsorption of the main substrate, acetophenone, may reach a significant level of up to 6 mg/mg_protein, at an acetophenone activity of 0.32, the secondary substrate, 2-propanol is not adsorbed at a detectable degree. The presence of water leads to a decrease of the adsorbed amount of acetophenone, by approximately 25%, at a water activity of 0.54.     

True and apparent temperature dependence of protein adsorption equilibrium in reversed-phase HPLC

The adsorption behavior of bovine insulin on a C(8)-bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed-phase HPLC. Changes in the molar volume of insulin (deltaV(m)) upon adsorption were derived from the pressure dependence of the isothermal retention factor (k'). The values of deltaV(m) were found to be practically independent of the temperature between 25 and 50 degrees C at -96 mL/mol and to increase with increasing temperature, up to -108 mL/mol reached at 50 degrees C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln(k'). In the first series the average column pressure was kept constant. The second series involved measurements of ln(k') under constant mobile-phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln(k') and the temperature, but the values obtained for ln k' were higher in the first than in the second case. The relative difference in ln(k'), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, T(c) approximately equals 53 degrees C, at which ln(k') is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain.     

A simple, inexpensive, and precise microcell for the exchange dialysis and equilibrium dialysis of small samples

A simple equilibrium dialysis cell may be quickly prepared from common, inexpensive microcentrifuge tubes. The resulting cell is easy to use and precise enough for quantitative dialysis studies of small samples (<50 μl). In addition, by using a portion of the cell, exchange dialysis of small samples can easily be done.     

Failure of equilibrium dialysis to show selective monosaccharide binding by erythrocyte membranes

Summary Equilibrium dialysis has been reported to show stereoselective binding of the preferred sugar transport substrate,D-glucose, by NaI protein extracts of human erythrocyte membranes. However, we were unable to show any detectable differential binding ofD-glucose (as compared with the poorly transported analogue,L-sorbose) with NaI protein extracts. The basis for this decided dissonance is not clear. Extracts with nonionic detergents, various alcohols, and pyridine were also used, but the results with these were also negative. Our data indicate either that the transport sites are not thus extractable in a functional condition, or that only a very small number of binding sites (less than 100,000) are involved with the sugar translocation; and that this method cannot serve to measure the site population unless a far greater concentration of the binding material can be achieved than has so far been possible.