Dialysis experiments

FIGURE 5A.2 A dialysis experiment. The solution of macromolecules to be dialyzed is placed in a semipermeable membrane bag, and the bag is immersed in a bathing solution. A magnetic stirrer gently mixes the solution to facilitate equilibrium of diffusible solutes between the dialysate and the solution contained in the bag. 

Dialysis experiments and DNA thermal denaturation studies of bisimidazoletetrachloroirida-te(III), (250), suggest poor binding of (250) to DNA, with no formation of interstrand crosslinks.422... 

In a dialysis experiment, a dialysis bag containing the dissolved humic materials is placed in a solution of a pollutant (preferably radiolabeled). The dialysis tubing is chosen so the pollutant is free to diffuse through the bag while the humic materials are retained inside the bag. The solution is shaken at constant temperature until it comes to an equilibrium point. At equilibrium, the pollutant inside the dialysis bag consists of two fractions that truly dissolved and the bound to the humic materials. The concentration of pollutant on the outside of the dialysis bag consists only of the free, truly dissolved fraction. Any increase of the pollutant concentration on the inside of the dialysis bag is due to binding by dissolved humic materials. A series of dialysis experiments, therefore, can measure the bound fraction concentration as a function of the free concentration. 

We have made some preliminary comparisons of the methods used in this research.7 In general the dialysis experiments give the most reliable results. These experiments can not, however, be run with all compounds. 

To illustrate the principles of an equilibrium dialysis experiment, we will describe the binding of [ H] acetylcholine to the nicotinic acetylcholine receptor (nAChR) in native membranes from Torpedo electro-plax. The data obtained from such an experiment are shown in Figure 10-7 where they are compared with data obtained from a centrifugation assay described below (Protocol 4.2). 

Next, the RBDCL was screened against the TAMRA-labeled DNA sequences. As seen in Fig. 3.11, only one pool of resin showed significant fluorescence. This pool contained the monomer Cys-Ser-Ser-Quin, and as such the homodisulfide (Cys-Ser-Ser-Quin) was selected as the best binder. Equilibrium dialysis experiments confirmed that (Cys-Ser-Ser-Quin) bound the target DNA Sequence 2 with a dissociation constant of 2.8 tM. While it is certainly true that identification of amplified compounds from large solution-phase DCLs is possible, given sufficiently... 

The equilibrium dialysis experiment revealed that histidine-substituted salicylamide was selected as an RNA ligand. Subsequent binding analysis by UV titrations and Job plot revealed the histidine-substituted salicylamide Cu + complex bound the target RNA hairpin with an apparent dissociation constant of 150 nM. This binding constant likely reflects more complex binding processes than a simple 1 1 interaction, as the observed binding curve saturates well below the concentration of the histidine-substituted salicylamide, and thus the actual affinity of the complex for targeted RNA is probably lower. Importantly, however, titrations with the... 

A quantitative measure of interaction between polymer and monomer in solution can be expressed by a value of preferential solvation. Preferential solvation can be calculated from the measurements of refractive index increments in dialysis experiments. This experiment can be illustrated as in Figure 3.1. 

Kl can be calculated from dialysis experiments. However, it was demonstrated that there is a large error in the calculation of Kl from the refractive index increments. 

Ryan et al. (2001) studied the effects of hydroxyapatite addition to soils impacted by Pb from smelter operations. Dialysis experiment were conducted where the soil and hydroxyapatite solids were placed in separate dialysis bags suspended in 0.01 M NaN03. Chloropyromor-phite formed on the dialysis membrane containing the soil. The dissolution of solid-phase soil Pb was the rate-limiting step for pyromorphite formation. EXAFS showed that after the 240 day incubation the hydroxyapatite treatment caused a change in the average, local molecular bonding environment of soil Pb. 

You can demonstrate the size of colloidal particles with a dialysis experiment in which two solutions are separated by a semipermeable membrane that has pores with diameters of 1—5 nm.3 Small molecules diffuse through these pores, but large molecules (such as proteins or colloids) cannot. (Collecting biological samples by microdialysis was discussed at the opening of Chapter 25.)... 

Dialysis experiments" have shown that Ca2 , Mg2 , Ba2 , and Sr2 form soluble chelates in aqueous alkaline solution with D-galactose, D-glucose, D-fructose, D-arabinose, D-ribose, maltose, and lactose. The absence of any precipitation of alkaline-earth metal hydroxide when an aqueous solution containing D-fructose and an alkaline-earth metal salt is made alkaline... 

Peptide nitriles are reversible inhibitors of cysteine proteases. 1,2 Peptide nitrile reacts with the active site thiol group to form an imidothioate, a dead-end product that does not undergo hydrolysis to an amide.134 This imidothioate derivative has been detected by NMR spectroscopic studies.P 5 The inhibition of papain, a cysteine protease, by a peptide nitrile proved to be reversible in a dialysis experiment. 3 Peptide nitriles are weaker inhibitors of cysteine proteases than the corresponding aldehydes. 61 Most peptide nitriles show poor inhibition toward serine proteases, however those nitriles with proper peptide sequences are potent inhibitors of serine proteases. 7-9 ... 

Figure 13.3 Illustration of a Donnan dialysis experiment to separate and concentrate uranyl nitrate, U02(N03)2, after Wallace [5]...

The biocatalyst a-chymotrypsin s ability to hydrolyze 20 is inhibited in the presence of copolymer 19a loaded with 0.2 mol% of the triphenyl carbinol units. 47b Photoirradiation of 19a results in heterolytic bond cleavage and the formation of the cationic copolymer 19b. In this polymer structure, the biocatalyzed hydrolysis of 20 is activated (V = 1.0 pM min-1). The polymer-induced photostimulated activation and deactivation of a-chymotrypsin in the different membrane environments correlates with the permeability and transport properties of the substrate 20 through the different structures of the polymer membranes.1471 Flow dialysis experiments showed that the polymer states 17a, 18a, and 19a are nonpermeable to 20, and hence the biocata-lytic functions of the immobilized enzyme are blocked. The polymer structures 17b,... 

The key concept of the analysis developed here is the interaction coefficient, which we will use to assess the net interactions (favorable or unfavorable) taking place between ions and an RNA. We first introduce interaction coefficients by describing the way they might be measured in an equilibrium dialysis experiment, and give an overview of their significance. These parameters are defined in more formal thermodynamic terms in Section 2.2 and are subsequently used to derive formulas useful in the interpretation of experimental data. 

The two ion gradients that develop in the dialysis experiment are conveniently quantitated in terms of interaction coefficients. A histogram (Fig. 21. IB) diagrams the ion concentrations in a similar experiment as cartooned in Fig. 21.1 A. The total number of KC1 ion pairs that migrates across the membrane as equilibrium is established, when normalized by the number of RNA molecules present, becomes the interaction coefficient rkci- Another way to find the same number is to count the total number of KC1 ion pairs on each side of the membrane at equilibrium... 

In the definitions of T, two variables in addition to the ion chemical potential must also be specified as constant. In an equilibrium dialysis experiment, these are temperature and the chemical potential of water. This partial derivative is known as the Donnan coefficient. (Note that the hydrostatic pressure is higher in the RNA-containing solution.) In making connections between T and the Gibbs free energy, it is more convenient if temperature... 

At this point, we have defined an ideal reference state for the RNA in which there are no net interactions with ions, and introduced the RNA activity coefficient as a factor that assesses the deviation of the RNA from ideal behavior due to its interactions with all the ions in solution. No assumptions have been made about the nature of the ion interactions anions and cations, long- and short-range interactions all contribute. The ion interaction coefficients (Eqs. (21.4a) and (21.4b)) also reflect the ion—RNA interactions that create concentration differences in a dialysis experiment, and there is an intimate relationship between activity coefficients (y) and interaction coefficients (F), as developed below. This relationship will be extremely useful y comes from the chemical potential and gives access to free energies and other thermodynamic functions, while F is directly accessible by both experiment and computation (see Pappu et al., this volume, 111.20). 

In Eq. (65), the prime indicates the salt concentration in the reference chamber of the Donnan dialysis experiment with T and P held fixed. 

Serge Timasheff made the next major contribution to this analysis (Timasheff, 1992, 1998). Through a series of elegant and extremely difficult dialysis experiments, he showed that stabi-... 

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