potassium-thiocyanate and potassium-fluoride

Protein solubility was measured using the crystalline precipitate of a recombinant therapeutic antibody, in monovalent salt solutions containing KF, KCl, and KSCN (up to ∼ 0.7 M) at different pH conditions. For all three anions, the antibody solubility demonstrated complex behavior, both monotonic and nonmonotonic, with dependence on pH and salt concentration. At pH 7.1, close to the isoelectric point (pI) of 7.2, a typical salting-in behavior was observed with the salting-in constants of 12.7, 8.0, and 2.8 M for KSCN, KCl, and KF, respectively, suggesting that the anions follow the order of SCN(-) > Cl(-) > F(-) for increasing antibody solubility. Nonmonotonic behavior, as described by an initial solubility decrease followed by a solubility increase with ionic strength, was observed at pH 5.3, far below its pI. The effectiveness of the anion for reducing the solubility followed the order of SCN(-) > Cl(-) > F(-) . After the solubility reached the minimum, the anion's effectiveness for raising the antibody solubility was in agreement with that at pH 7.1. The mechanisms for the above phenomena are discussed based upon specific binding of the anions to the antibody surface. The mechanistic view of anion binding and its charge neutralization effect at pH 5.3 was supported by the results from the effective charge and zeta-potential measurements.

Topics: Anions; Chromatography, High Pressure Liquid; Fluorides; Immunoglobulin G; Osmolar Concentration; Potassium Chloride; Potassium Compounds; Recombinant Proteins; Salts; Solubility; Thiocyanates

Liquid-liquid phase separation was studied for a monoclonal antibody in the monovalent salt solutions of KF, KCl, and KSCN under different pH conditions. A modified Carnahan-Starling hard-sphere model was utilized to fit the experimental data, establish the liquid-liquid coexistence curve, and determine antibody-antibody interactions in the form of T(c) (critical temperature) under the different solution conditions. The liquid-liquid phase separation revealed the complex relationships between antibody-antibody interactions and different solution conditions, such as pH, ionic strength, and the type of anion. At pH 7.1, close to the pI of the antibody, a decrease of T(c) versus ionic strength was observed at low salt conditions, suggesting that the protein-protein interactions became less attractive. At a pH value below the pI of the antibody, a nonmonotonic relationship of T(c) versus ionic strength was apparent: initially as the ionic strength increased, protein-protein interactions became more attractive with the effectiveness of the anions following the inverse Hofmeister series; then the interactions became less attractive following the direct Hofmeister series. This nonmonotonic relationship may be explained by combining the charge neutralization by the anions, perhaps with the ion-correlation force for polarizable anions, and their preferential interactions with the antibody.

Topics: Anions; Antibodies, Monoclonal; Fluorides; Hydrogen-Ion Concentration; Isoelectric Point; Models, Chemical; Osmolar Concentration; Phase Transition; Potassium Chloride; Potassium Compounds; Temperature; Thiocyanates

It was established that nitrite in the presence of chloride, bromide, and thiocyanate decreases the rate of hydrogen peroxide decomposition by catalase. The decrease was recorded by the permanganatometric method and by a method of dynamic calorimetry. Nitrite was not destroyed in the course of the reaction and the total value of heat produced in the process was not changed by its presence. These facts suggest that nitrite induces inhibition of catalase with no change in the essence of the enzymatic process. Even micromolar nitrite concentrations induced a considerable decrease in catalase activity. However, in the absence of chloride, bromide, and thiocyanate inhibition was not observed. In contrast, fluoride protected catalase from nitrite inhibition in the presence of the above-mentioned halides and pseudohalide. As hydrogen peroxide is a necessary factor for triggering a number of important toxic effects of nitrite, the latter increases its toxicity by inhibiting catalase. This was shown by the example of nitrite-induced hemoglobin oxidation. The naturally existing gradient of chloride and other anion concentrations between intra- and extracellular media appears to be the most important mechanism of cell protection from inhibition of intracellular catalase by nitrite. Possible mechanisms of this inhibition are discussed.

Topics: Animals; Bromides; Catalase; Cattle; Enzyme Inhibitors; Fluorides; Hemoglobins; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Liver; Nitrites; Oxidation-Reduction; Potassium Compounds; Sodium Chloride; Thermodynamics; Thiocyanates