George Baum

Determination of acetylcholinesterase by an organic substrate selective electrode

Abstract A new electrochemical method is described for the determination of acetylcholinesterase activity. A liquid membrane electrode is used which has a high selectivity for acetylcholine over choline and various inorganic cations, thus permitting a continuous determination of the rate of change of substrate concentration in the presence of active enzyme. Enzyme activity ranging from 0.2 to 8 units was determined in 10 to 50 ml of saline buffered solutions. Consistent measurements of the rate of change in substrate concentration are obtained within one minute of introduction of the enzyme. The rate of hydrolysis can be monitored up to about 40% hydrolysis before the interference due to choline concentration becomes significant. The electrochemical procedure was correlated with the spectrophometric method of Hestrin.

Polymer membrane electrodes: Part I. A choline ester-selective electrode

Abstract An electroactive Polyvinylchloride membrane can be prepared which has a Nernstian response towards choline and choline esters. The membrane is prepared from a solution of acetylcholine tetra-p-chlorophenylborate in a phthalate ester which serves as the plasticizer for PVC. The membrane electrode can be used for the kinetic assay of acetylcholine esterase activity. A rate equation is derived which describes the response of the electrode towards simultaneous substrate disappearance and product formation during the enzyme-catalyzed hydrolysis of acetylcholine.

Polymer membrane electrodes: Part II. A potassium ion-selective membrane electrode

Abstract An ion-selective plasticized polymer membrane electrode of the ion-exchange type is described for potassium. The chemical nature of the plasticizer has a dominant influence on the selectivity of the electrode towards univalent cations. The selectivity ratio was examined over an extended time interval as a function of membrane thickness and plasticizer content. A simplified procedure for the determination of the selectivity ratio of the electrode in mixed electrolyte solutions is proposed and compared with several of the methods which have been described in the literature.

Stability, inhibition and reactivation of acetylcholinesterase covalently coupled to glass

Abstract Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was covalently coupled to a silanized fritted glass disc by a diazonium salt reaction and by a diimide condensation. The diimide-coupled adduct survived numerous assays and chemical studies for a period of 55 days at room temperature in contrast to the diazonium-coupled adduct which rapidly lost activity. Both adducts exhibited a lesser pronounced dependence of activity on pH than the free enzyme, reflecting the weak buffering action of the silanol-silane surface. The diimide adduct is inhibited by diethyl p -nitrophenyl phosphate and can be reactivated by either Toxigonin or alkaline phosphatase (orthophosphoric monoester phosphohydralase, EC 3.1.3.1).

Kinetic analysis of cholinesterases using a choline ester selective electrode.

Abstract A new electrochemical method for the determination of the cholinesterase activity of blood-derived fractions is presented. The rate of change of concentration of several choline esters in the presence of whole blood, serum and erythrocytes can be continuously monitored by an acetylcholine-selective electrode. Acetylcholine, butyrylcholine and acetyl β-methylcholine were employed as substrates. The assay of 0.05 units of enzyme activity can be readily conducted. The reproducibility of the procedure compares favorably to established colorimetric and pH-stat techniques.

Affinity chromatography of β-galactosidase on control-led-pore glass derivatives

Abstract Several controlled-pore glass (CPG) derivatives were examined as supports for the affinity chromatographic purification of the enzyme β-galactosidase. The competitive inhibitor p-aminophenyl-β- d -thiogalactopyranoside was coupled to an acid and a malonic acid derivative of 750-A alkylamine CPG of 80–120 mesh and to an azelaic acid derivative of 550-A alkylamine CPG of 40–80 mesh. The latter derivative exhibited particularly good load capacity and separation efficiency; however, both arm lengths were effective. Hydrophobic interactions between the arm and the enzyme contribute to the separation.

Ultraviolet spectra of polyfluoropolyphenyls

Abstract The ultraviolet absorption spectra of a series of polyfluoropolyphenyls (4H-nonafluorobiphenyl, 4H-tridecafluoroterphenyl and 4H-septadecafluoroquatraphenyl) are determined and compared to their nonfluorinated analogs. The spectra are interpreted in terms of a resonance interaction between skewed benzene rings.

[42] Electrochemical applications of oxireductases

Publisher Summary This chapter discusses the occurrence and bioanalytical significance of oxireductases. Because of the formation or disappearance of an electroactive species by enzymic action of oxidases, this class of enzymes readily lends itself to electroanalytical methods. It reviews that the interest in the analytical uses of oxidases has steadily increased. A major difficulty in the analytical schemes involving enzymes in general is the inherent instability of biological materials. Many processes, such as oxidation, thermal unfolding, and dissociation of cofactor, result in gradual loss of enzymic activity. Significant stability can be confirmed on an enzyme by immobilizing it on or in a stable matrix. This also eliminates the necessity of adding the enzyme to the solution to be measured. The chapter discusses methods that are directly applicable to enzyme electrodes. It describes some selected significant developments and provides experimental methods that may enable the reader to further exploit the use of immobilized oxidases for analytical clinical chemistry. There are two basic approaches to the construction of a system for electrochemical measurements with immobilized oxidoreductases. The enzyme may be brought into intimate contact with the sensor—an enzyme electrode—or the enzymatic reaction may be conducted at a separate site and the reaction products are then transported to the sensor, an arrangement refer to as a microflow immobilized enzyme reactor (IMER).

Derivatives of Controlled Pore Glass for Affinity Chromatography

Many of the characteristics of controlled pore glass (CPG) that are advantageous for the preparation of immobilized enzymes (1) also are appropriate to carriers for affinity chromatography. We have prepared a series of dibasic acid derivatives of alkylamine CPG and examined several members of the series as carriers for the affinity chromatographic purification of β-galactosidase. The affinity ligand was p-aminophenyl-β-D-thiogalactopyranoside (PAPTG) which is a weak competitive inhibitor for the enzyme and was used by Cuatrecasas (2) in conjunction with several Sepharose derivatives.