Uses of metal-ion buffers

One of the major applications of pM buffers is in maintaining concentrations of necessary metal ions in biological nutrient media at essentially constant levels. As free metal ions are removed from the system, perhaps by hydrolysis or by incorporation into metalloenzymes, they are replenished by the reversible dissociation from a reservoir of metal complex. Among the first complexing agents used in this way were citrate and tartrate ions, but more recently aminopoly-carboxylic acids such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and nitrilotriacetic acid (NTA) have become the chelating agents 

This metal-buffering is important when hydrolysis or insolubility limits the attainable free metal ion concentration. Thus, in toxicity studies of lead ion, the presence of phosphate and chloride ions in the physiological media and the very small solubility products of Pb3(P04)2 (lO ), PbHP04 (10 ) and PbCl2 severely limit the possible concentration of free lead ion. It is not feasible to add directly to the medium a soluble lead salt, such as the nitrate, in sufficiently small amounts that precipitation can be avoided but it is practicable to calculate the lowest value of pPb that would not result in precipitation and to use chelating agents to maintain this level in the system. 

Maintenance of constant pM levels may also be important in controlling the catalytic action of metal ions in industrial chemical processes. A distinction is made between metal-ion buffering and the masking of metal ions. The latter topic, discussed elsewhere (Perrin, 1970), is concerned with the use of chelating agents to diminish metal ion concentrations below levels at which they exert specified chemical or biological effects. 

A promising apphcation of metal-ion buffers is in the standardization of ion-selective electrodes. 

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N. Ishibashi, T. Imato, and K. Tsukiji, Potentiometric and Spectrophotometric Flow-Injection Determinations of Metal Ions with Use of Metal Ion Buffers. Anal. Chim. Acta, 190 (1986) 185. 

In experimental work, the need often arises to maintain a predetermined concentration of a given metal ion, a concentration that is not allowed to rise above this level nor fall below it. This situation is met by the use of metal ion buffers which maintain a steady pM just as hydrogen ion buffers maintain a steady pH. With their help, free metal ions are replenished (as they are removed by the reaction) from a reservoir of bound metal complex. The first complexing agents to be used for this purpose were citrate and tartrate ions, but much more application has been found for ethylenediaminetetra-acetic acid (EDTA) (cf. 11.27), diethylenetriaminepenta-acetic acid (DTPA), and nitrilotriacetic acid (NTA). The necessary calculations will be found in Perrin and Dempsey (1974). 

Chemical and biological reactions often depend critically on the presence of low concentrations of certain free metal ions. Concentrations less than 10 M are difficult to maintain in the presence of adventitious complexing agents, hydrolytic equilibria, adsorption and, possibly, contamination. Many problems can be overcome by the use of metal-ion buffers which provide a controlled source of free metal ions in a manner similar to the regulation of hydrogen ion concentration by pH buffers. 

Mosher, R. A., The use of metal ion-supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis, Electrophoresis, 11, 765, 1990. 

Another approach for changing selectivity in MEKC is the use of metal ions. In particular, the MEKC separation of oligonucleotides is improved by the addition of Mg(II), Cu(II), and Zn(II) ions. Metal ions are electrostatically attracted to the surface of a negatively charged micelle where they can be selectively complexed with analytes. Separation is due to differences in the distribution of solutes between the buffer and the metal-micelle surfaces. Retention is proportional to the complexation constant... 

Although most of the chelating agents used in metal-ion buffers are also capable of serving as pH buffers, their values do not usually lie in the required pH range. For example the pATa values of NTA, tartaric acid and citric acid are too remote from pH 7 for them to be used as pH buffers in near-physiological media. Similarly A-hydroxyethyl-ethylenediamine triacetic acid (pATa 5.33), EDTA (pATj 6.13) or trimethylenediamine tetraacetic add (pATa 7.91) are of only limited use for this purpose. 

Table 7.2 pK Values of some ligands useful as metal-ion buffers (at 25°Cand/=0.15)... 

If measurements are to be carried out at low activities (for example in studying complexation equilibria), standard solutions cannot be prepared by simple dilution to the required value because the activities would irreproducibly vary as a result of adsorption effects, hydrolysis and other side reactions. Then it is useful to use well-defined complexation reactions to maintain the required metal activity value [14, 50, 132]. EDTA and related compounds are very well suited for this purpose, because they form stable 1 1 complexes with metal ions, whose dissociation can be controlled by varying the pH of the solution. Such systems are often termed metal-ion buffers [50] (cf. also p. 77) and permit adjustment of metal ion activities down to about 10 ° m. (Strictly speaking, these systems are defined in terms of the concentration, but from the point of view of the experimental precision, the difference between the concentration and activity at this level is unimportant.)... 

Cd ion-selective electrodes The usual version of the Cd " ISE contains a membrane of a sintered or pressed mixture of CdS and Ag2 S [121, 325,408]. Membranes from sintered Ag2S, CuS and CdS mixtures [157] have also been proposed, similarly as forPb ISEs. CdS precipitate in a polyethylene matrix [250] or a CdS-Ag2S precipitate mixture in a silicone rubber matrix [153] can also be used for Cd ISEs. Cd ISEs can be calibrated using a metal diethylenetriamine buffer [66]. Similar substances interfere in the response of the Cd ISE as for the Hg, Ag and Cu electrodes. 

The equilibrium constant of an enzyme-catalyzed reaction can depend greatly on reaction conditions. Because most substrates, products, and effectors are ionic species, the concentration and activity of each species is usually pH-dependent. This is particularly true for nucleotide-dependent enzymes which utilize substrates having pi a values near the pH value of the reaction. For example, both ATP" and HATP may be the nucleotide substrate for a phosphotransferase, albeit with different values. Thus, the equilibrium constant with ATP may be significantly different than that of HATP . In addition, most phosphotransferases do not utilize free nucleotides as the substrate but use the metal ion complexes. Both ATP" and HATP have different stability constants for Mg +. If the buffer (or any other constituent of the reaction mixture) also binds the metal ion, the buffer (or that other constituent) can also alter the observed equilibrium constant . ... 

The stability of solutions of the aminochromes is influenced by many factors such as the nature of the solvent, the pH of the medium, the nature of the buffer used (if any), the presence or absence of dissolved oxygen in the solution, the presence of traces of metal ions, traces of oxidizable organic matter, etc. In general, solutions of the aminochromes with a 3-hydroxyl group [e.g. adrenochrome (1)] are more stable than those without [e.g. epinochrome (27)].106 126,127 On the other hand, solutions of adrenochrome methyl and ethyl ethers were slightly more stable than those of adrenochrome.108... 

A Ca2+ ion-selective electrode was calibrated in metal ion buffers whose ionic strength was fixed at 0.50 M. Using the following electrode readings, write an equation for the response of the electrode to Ca2+ and Mg2+. 

Dithizone is used for analytical extractions, for colorimetric determinations of metal ions, and for removing traces of metals from aqueous buffers. 

It is essential that all traces of DTT should be removed from the RIP after the reduction step. The presence of even very low concentrations of DTT at this stage will release S-pyridyl groups from the antibody in preference to the formation of conjugate. For this reason, the G-25 column should be rigorously cleaned before use. EDTA in the column buffer inhibits disulfide bond formation, catalyzed by trace amounts of metal ions. 

As Mamiya and Gorin (11) note, Sumner occasionally had difficulty in preparing crystalline urease from some jack bean meals and this is a technical problem that has received attention in each recent improved method of preparation (11-13). Modification of Sumner s extraction procedure by including / -mercaptoethanol (11, 14) to diminish aggregation and ethylenediaminetetraacetate (EDTA) (13,15) to maintain a low concentration of metal ions has been helpful. The procedure used in this laboratory with consistent success over a period of 4 years, and involving several students, is essentially that of Mamiya and Gorin (11) but with EDTA included in the buffers. [There is an error in Mamiya and Gorin (11). The concentration of acetone used was 32%, not 36%.]... 

The use of chemiluminescence reactions for the detection of metal ions by liquid chromatography was recently reported [59,60]. The detectors made use of the chemiluminescence produced in the reaction between luminol and hydrogen peroxide which is catalyzed by transition metals. The column effluent was mixed with the reagents in order to yield the chemiluminescence. The reaction was fast and was carried out at room temperature. By varying the pH of the buffer, selectivity towards certain metals was also achieved. For example, at pH 10-11 nickel could be analyzed but lead and aluminium were inactive at pH 13-14, the converse was true [59]. Aminco-Bowman has marketed a liquid chromatographic system in which amino acids and amines are analyzed by means of the fluorescence produced on reaction with the reagent fluorescamine. Fluorescamine does not fluoresce, but it does react with primary amino groups to produce fluorescent derivatives. The reaction is instantaneous and may be carried out at room temperature, usually at pH 9. This detection system promises to be far more sensitive than the ninhydrin detection system and is much more easily adapted to HPLC. 

Determination of metal ions (Cr3+, Co2+, Cu2+) has been achieved using the metal-catalyzed luminol-peroxide system. The EOF delivery of the CL reagent (H202 pH 11.7) in the aqueous system can be achieved using a side channel (with luminol present in the separation buffer with a pH of 6.0) [168]. 

Figure 5.4 shows the separation of histidine-containing dipeptides by CZE using a buffer containing zinc ions.7 Copper ions or zinc ions in acetate or phosphate buffers (pH 2.5) were used, with good results. In the absence of metal ions, little resolution was evident, but as the metal ion concentra-... 

A very interesting application of affinity chromatography to the purification of halophilic enzymes was reported by Sundquist and Fahey (1988). These authors have purified the enzymes bis-y-glu-tamylcysteine reductase and dihydrolipoamide dehydrogenase from H. halohium using immobilized metal ion affinity chromatography in high-salt buffers. 

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