Lemuel B. Wingard

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.

Modification of Riboflavin for coupling to glassy carbon

Abstract Riboflavin was acetylated and then modified at the position 8 methyl group to give the 8-bromo and then the 8-triphenylphosphonium bromide derivatives. The phosphonium salt, after the addition of a strong base, was incubated with pieces of glassy carbon that had been oxidized, acylated, and reduced to form surface aldehyde groups. The objective here was to couple the riboflavin derivative to the activated glassy carbon through a double bond bridge by a Wittig type reaction. Cyclic voltammetry measurements on the glassy carbon following the Wittig incubation, showed the appearance of new redox groups with E ′ o values of — 0.16 V and — 0.44 V, indicating that coupling may have been accomplished. NMR spectra of the riboflavin derivatives agreed with literature results where comparable data were available. Additional studies are planned for verification of the riboflavin to glassy carbon coupling and for subsequent testing of the coupled riboflavin for cofactor activity with an appropriate apoenzyme.

Cofactor modified electrodes

Abstract Studies are underway to see if flavin adenine dinucleotide or nicotinamide adenine dinucleotide cofactors can be attached to electrode surfaces to give rapid rates of electron transfer and in some cases reconstitution of enzyme activity with appropriate apoenzymes. Such electrodes may find widespread application in analytical chemistry.

Immobilization of flavins on electrode surfaces: Part II. Apparent conversion of glassy carbon-immobilized riboflavin to immobilized flavin adenine dinucleotide and partial reconstitution of apoglucos

Abstract The covalent attachment of flavin cofactors to electron-conducting supports serves both as a probe of the spatial requirements for flavinapoenzyme association as well as a novel route for the development of cofactor-apoenzyme electrodes. In this work, a procedure is described for the apparent conversion of riboflavin, immobilized at position 8 to glassy carbon, to flavin mononucleotide (FMN) and then to immobilized flavin adenine dinucleotide (FAD). The attached materials were characterized electrochemically and coenzymatically. FAD serves as the cofactor for the enzyme glucose oxidase. Partial restoration of enzyme activity was achieved by incubation of the FAD-electrode with apoglucose oxidase. This is the first reported covalent coupling of a coenzymatically active flavin to an electrode through the flavin position 8 group.

Effects of physostigmine, scopolamine, and mecamylamine on the sleeping time induced by ketamine in the rat.

Male Sprague-Dawley rats weighing 116–241 g were injected i.p. with ketamine hydrochloride, 80 mg per kilo of body weight. Immediately after loss of righting reflex, scopolamine, physostigmine, and mecamylamine were administered i.p. to different groups of rats. Control animals received sterile saline by the same route. The ketamine-induced sleeping time was significantly prolonged by physostigmine and scopolamine, but not by mecamylamine. After the delayed injection of physostigmine, the ketamine sleeping time was longer. These results, although too preliminary for a mechanistic interpretation, suggest that multiple neurotransmitter systems, probably including the cholinergic system, are involved in the mechanism of action of ketamine-induced narcosis.

Immobilization of flavins on electrode surfaces: Part I. attachment of riboflavin through 8-methyl to glassy carbon

Abstract The covalent immobilization of flavoenzyme cofactors on electronconducting supports can provide insight into the spatial aspects of flavinapoenzyme association in the holoenzyme as well as providing a novel route fo the development of cofactor-apoenzyme electrodes. In this work, the covalent attachment of riboflavin to the surface of glassy carbon electrodes through a carbon-carbon linkage at position 8 of the isoalloxazine ring system was carried out using an organolithium complex. Riboflavin was acetylated at room temperature with acetic anhydride in pyridine. The tetraacetate derivative was selectively brominated at position 8, as shown by Nuclear Overhauser Effect (NOE)/NMR. The bromo derivative was converted to an organolithium intermediate, which subsequently was attached to aldehyde-derivatized glassy carbon. Electrochemical methods were employed to characterize the attached flavin. The loading of attached flavin amounted to 3.1 × 10 −11 mol cm −2 .

Biosensor trends. Receptors, enzymes, and antibodies.

Most biosensor designs can be divided into two main elements-one to provide molecular-level recognition of the analyte being determined and the other to convert the recognition event into an output signal (transduction).’ Biological materials such as enzymes, immunocompounds (antibodies), portions of tissues or groups of cells, and (more recently) receptors serve as the molecular recognition element to impart selectivity for the analyte of interest, while rejecting other compounds of similar structures or related properties. Selective binding of the desired analyte with the biological recognition element is the mechanism whereby the selectivity normally takes place. The transduction element often does not involve any biological material, but utilizes physical or chemical changes to convert the selective binding of the analyte into a readable output signal. The two elements are summarized in TABLE 1. Although many enzyme-based electrochemical-type biosensors*~’ and some enzyme-, antibody-, or lectin-based optical biosen~ors**~ have been developed, extremely few so far have achieved practical acceptance and still fewer biosensors have attained large market penetration. Still, the possibilities for future innovative developments in the field of biosensors appear very bright; however, the timetables for practical developments are becoming more realistic. The purpose of this report is to point out several trends taking place in biosensor research and development and to describe efforts under way to explore receptor proteins as biological recognition elements (and possibly also transducers) for use in biosensors. The major output modes for biosensors are listed in TABLE 2. Present trends are evident in research involving a t least four of these output modes: amperometric, electronic (field effect transistor), piezoelectric, and combined fiber optic-fluorescence techniques. Each of these four trends is discussed separately. One of the present trends arises from the high selectivity and high sensitivity associated with amperometric enzyme electrodes. This is still a widely used transductionoutput mode. However, more efficient electron transfer techniques are needed with oxidoreductase enzymes to overcome the drawbacks with the use of soluble electron transfer mediators. Electron tunneling with ferrocene electron transfer relays absorbed within the enzyme matrix has been applied with some success to glucose oxidase.’ In related discussions, several laboratories in 1988 showed enhanced electron transfer

[8] Immobilized flavin coenzyme electrodes for analytical applications

Publisher Summary Flavins are compounds that typically contain the isoalloxazine ring system. The flavins serve as the coenzyme for well over 100 oxidoreductase enzymes and are active redox compounds by themselves. The application of flavins in analytical chemistry can be based either on their coenzyme properties or on their nonenzymatic chemical reactivity. A flavin-enzyme electrode must contain a coenzyme, an apoenzyme, and a substrate. When the substrate undergoes oxidation, the electrons (and often protons) are transferred to the coenzyme to give reduced coenzyme. With flavoenzymes, the coenzyme usually remains attached to the apoenzyme during the course of the enzymatic reaction. Several approaches can be used to measure either the amount or the rate of formation of the reduced coenzyme. A small molecular weight mediator molecule can be selected to react with the reduced coenzyme to form a reduced mediator. The mediator then must diffuse to the electrode surface and be sufficiently electroactive to undergo oxidation at the electrode surface and, thereby produce a current. However, mediator diffusional transport resistances and the presence of other materials that might interact with the mediator can cause problems. A second approach is to apply an appropriate potential to the electrode surface with the expectation that the flavin coenzyme that is now embedded within the apoenzyme matrix will undergo oxidation and transfer the released electrons to the electrode surface. An alternative approach that may overcome some of the above problems is to attach the flavin coenzyme to the electrode surface, either by adsorption or by covalent coupling.

Possible Roles of Enzymes in Development of a Fuel Cell Power Source for the Cardiac Pacemaker

Many types of “permanent” pacemaker devices and techniques have evolved since successful clinical application was first reported (Senning, 1959; Chardack et al.,1960; Zoll et al.,1961). These efforts have had an enormous impact upon the treatment of cardiac arrhythmias; however, they also have brought a host of new problems for the management of patients receiving pacers (Grendahl et al.,1969; Goldstein et al., 1970; Barold, 1973). Among the problems is that of providing a suitable power source for long-term trouble-free pacemaker function. This chapter will be devoted largely to discussion of one type of device potentially capable of solving this problem, namely, the biofuel or, more specifically, bioautofuel cell. Particular emphasis will be given to the role that enzyme catalysts might play. The term “bioautofuel cell” refers to a biofuel cell which can be implanted in the host and which then relies on the host for the delivery of fuel and the removal of wastes. Such an implantable fuel cell may incorporate enzymes to catalyze one or more of the reactions or to produce the fuel. A general introduction to cardiac pacing and a brief description of alternate power sources is included to place the enzyme-containing systems in perspective.

Cofactor Modified Electrodes for Energy Transfer

Considerable interest has evolved over the past 10 years in the development of enzyme catalyzed devices for the transfer of chemical energy to electrical energy, i.e. enzyme fuel cells. Some of these devices have been of the direct type, where the enzyme is an integral part of one of the electrodes, while others have been of the indirect type, where an enzyme or microorganisms are used to convert a complex substrate for use in an inorganic fuel cell (1–6). Our interest has been in the direct type enzyme fuel cell, with the glucose oxidase-flavin cofactor system as a model anoidc catalyst. The objective of this paper is to discuss the chemical modification of the electrode surface, as an approach to improved enzyme catalyzed fuel cells, with special emphasis on flavin cofactor systems.