Charles W. Carr

Studies on the binding of small ions in protein solutions with the use of membrane electrodes. IV. The binding of calcium ions in solutions of various proteins.

Abstract The binding of sodium and potassium with 14 different proteins has been determined with collodion membrane electrodes. Many of these proteins do not show any appreciable interaction with these ions at pH 7.5. However, pepsin, β-lactoglobulin, zein, and three plasma protein fractions show a significant binding of both sodium and potassium at pH 7.5.

Ion-binding studies of ribonucleic acid and Escherichia coli ribosomes

Abstract The binding of calcium and magnesium ions to RNA from various sources has been studied by equilibrium dialysis. At concentrations of free ion which produce maximum binding (greater than 5 m m ), the binding is the same for both ions ( Mg 2+ P = 0.50 ± 0.04 at pH 6.0 ). This value remains constant at 0.50 over the pH range 6.0 to 9.6. Below pH 6.0, the maximum binding decreases, and above pH 9.5 it increases, which is consistent with what would be expected from the acid-base titration studies of others. Ion-binding studies with Escherichia coli ribosomes show that there is no difference between the 30 s and 50 s ribosomes in binding of magnesium ions. For both types of ribosomes the ratio, Mg P , reaches a maximum value of 0.50 at 10 m m free Mg2+. This behavior is almost identical with that observed for RNA alone, which indicates that magnesium ion-binding occurs only at phosphate groups and that all these groups are available as binding sites for small cations. Apparently these ribosomes are organized in such a way that the phosphate groups are not directly involved in the formation of the RNA-protein complex, and all of these groups are available for Mg2+ binding at the level of free Mg2+ concentration at which in vitro protein biosynthesis takes place at its maximum rate. Competitive binding studies show that Mg2+ can be displaced by other cations such as Na+, Ca2+, spermidine3+ spermine4+, and protaminen+. It is suggested that such competitive binding may be involved in the cellular control mechanism of protein biosynthesis.

Studies on the binding of small ions in protein solutions with the use of membrane electrodes. I. The binding of the chloride ion and other inorganic anions in solutions of serum albumin

Abstract When the protamine-collodion membrane electrode is used to study the binding of chloride and thiocyanate to bovine serum albumin, the results are found to be in agreement with the experimental results of other investigators. The binding of bromide, nitrate, sulfate, iodide, and perchlorate ions has also been measured.

Studies on the binding of small ions in protein solutions with the use of membrane electrodes. II. The binding of calcium ions in solutions of bovine serum albumin.

The sulfonated polystyrene-collodion membrane is used to study the binding of calcium to bovine serum albumin in solutions of CaCl2. A maximum of eight calcium ions is bound per albumin molecule at pH 7.4; increasing pH favors increased binding and vice versa. The binding of chloride is the same as in the presence of NaCl.

Studies on the structure and function of lysozyme: I. The effect of pH and cation concentration on lysozyme activity

Abstract A study has been made of the effect of salts on the assay for lysozyme (mucopeptide N -acetylmuramylhydrolase, EC 3.2.1.17) in which the decrease in turbidity of a suspension of lyophilized Micrococcus lysodeikticus is measured. Determinations were carried out in the presence of several types of salts in the concentration range of 5–500 mM and the pH range 5–10. Lysozyme is inactive in distilled water, is activated by low concentrations of salt and is inhibited by high concentrations of salt. Analysis of the data shows that the activation at low salt concentration is most closely correlated with a non-specific ionic strength effect and that the inhibition at high salt concentration is most closely correlated with cationic concentration and charge. At a given ionic strength, polyvalent cations are stronger inhibitors than monovalent cations. The effect of cations changes with pH such that the optimal cation concentration decreases with increasing pH. For example, 50 mM monovalent cation is optimal at pH 7.0, but at pH 9.0 it is strongly inhibitory. Thus, the activity of lysozyme cannot be adequately represented by a pH-activity curve, but it is necessary to establish an activity-pH cation profile.

The Binding of Calcium in Mixtures of Phospholipids.

Summary The binding of calcium in phos-pholipid mixtures is very dependent on the pH, it being a quantitative reflection of the types of dissociable groups in the phospho-lipids present. As the pH increases from 6.5 to 8.5, there is a considerable increase in Ca-binding if triphosphoinositide, phosphatidyl-serine, or phosphatidylethanolamine is present. This effect may be of some consequence in certain physiological processes.

Competitive Binding of Calcium and Magnesium with Serum Albumin.

Summary A study has been made of the binding of calcium and magnesium to serum albumin when both are present at the same time. It is shown that the ratio of the concentrations of bound calcium and bound magnesium is equal to the ratio of the concentrations of the ionized calcium and ionized magnesium.

The binding of calcium with deoxyribonucleic acid and deoxyribonucleic acid-protein complexes

Abstract The binding of Ca 2+ to highly polymerized DNA in the presence of several different small cations has been studied by the technique of equilibrium dialysis. In the neutral range of pH, the cations Na + , diamine 2+ , La 2+ , Th 4+ , spermidine 3+ and sperimine 4+ compete with Ca 2+ for the phosphate binding sites. The competitive effect for a given ion is almost entirely a function of the magnitude of the cationic charge. The effect of pH on the binding of Ca 2+ also shows the electrostatic nature of the binding process because the binding curve is closely correlated with the H + titration curve in the pH range 2.0–11.0. In studies with DNA-protein complexes, competition between Ca 2+ and cationic proteins is also shown. When the Ca 2+ concentration is below 5 mM, cationic proteins such as protamine and histones will block DNA binding sites for Ca 2+ in direct proportion to the concentration of cationic groups present. When the concentration of Ca 2+ is greater than 20 mM, Ca 2+ is bound to its full-capacity to DNA, indicating a complete displacement of protein from the binding sites.

APPLICATIONS OF MEMBRANE ELECTRODES

Membrane electrodes have been used almost exclusively for the determination of ionic activities in solution. In biological work, the studies have been concerned with these determinations in solutions of the biological polyelectrolvtes because of the extensive interest in the interaction of small ions with such macromolecules. There have been many different experimental approaches to the study of ionic interactions with biological macromolecules, each of which has its advantages and disadvantages. Membrane electrodes are particularly useful because they yield data which in the ideal case are the most direct, and their development has played a considerable role in the understanding of small ion interactions with macromolecules. By far the largest contribution has been with the use of the glass electrode for measuring hydrogen ion binding with more modest contributions being made in studies of many other small ions. Because the glass electrode for the measurement of hydrogen ion activity is SO well known and so widely used and because it is being discussed by others at this conference, the present report will be restricted to applications of membrane electrodes involving ions other than hydrogen. Two general types of electrodes will be considered: (a) ion exchanger electrodes with high permeability and low selectivity for the critical ion-6 and (b) glass electrodes with low permeability and relatively good selectivity for the critical i ~ n . ~ ~ The clay membrane electrodes originally developed by Marshall and coworkers10 will not be included in this report because they have not been used to any extent in studies of biological systems. Although the principal use of membrane electrodes has been for the measurement of ionic activities, their use as indicator electrodes in titration reactions and their use in certain model systems will be discussed briefly.

Studies on the binding of small ions in protein solutions with the use of membrane electrodes. VII. The binding of the alkali-metal ions in solutions of phosphoproteins.

Abstract The binding of the alkali ions with phosvitin, casein, and a phosphopeptide of casein has been studied as a function of pH and cation concentration. The maximum binding of sodium at pH 7.5 is 175 moles/ 10 5 g., 18 moles/10 5 g., and 100 moles/10 5 g. for phosvitin, casein, and the phosphopeptide, respectively; lithium binding is slightly stronger, and potassium binding is slightly weaker. It is suggested that the localization of negatively charged groups within the casein molecule is responsible for its binding of the alkali ions.