Search

A small-volume (∼60 μl) spectroelectrochemical cell was constructed and characterized.The working electrode is An minigrid, and the spectral window is from 170 nm to 2 μm. The solution is held in a thin layer around the working electrode, providing the advantages of thin-layer electrochemistry. The cell can be constructed from common materials in about 6-h working time. The cell also has flow-through and vacuum capabilities, with only minor modifications. Characterization of the cell was done by cyclic voltammetry and coulometry on ferri-ferrocyanide and spectroelectrochemistry on cytochrome c and o-tolidine. The optical and electrochemical windows of this cell, as well as its convenient and versatile operation, make it useful for the study of a wide variety of biological redox components.

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.

Zone centrifugation experiments are occasionally ruined by convective processes which occur at one gravity subsequent to layering a solution of macromolecules on a supporting density gradient. The factors which contribute to this convection have been little understood. In most of the experiments described in this communication, Blue Dextran 2000 is used as the macromolecular component. The convective disturbance may be clearly seen and accurately measured. The effects of macromolecular concentration, composition and density of the supporting gradient, diffusion coefficient of the solute, and several other variables are studied, and an empirical equation constructed to describe the phenomenon. This convective disturbance appears to be minimized or prevented by layering and fractionating in the presence of a centrifugal field.

The E0′ values for the conversion of horse heart cytochrome c from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H2O and D2O. In H2O, the decrease in the E0′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D2O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H2O above 42°C but not in the more stable D2O (Kreishman, Foss, Inoue and Leifer (1976) Biochemistry, 15, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E0′.

A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 X 10(6) M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

The redox activation of galactose oxidase by various oxidants is characterized by using a unique thin-layer electrochemical cell. Activation is shown to be strictly a redox process and can be rapidly accomplished by using ferricyanide, cobalt terpyridine or tetracyanomonophenanthroline ferrate, and a control electrode to control solution potential. This oxidation is a one-electron process and does not result in modified galactose oxidase which exhibits enhanced activity. On the contrary, this oxidation is required for activity. The solution potential dependence of activity is independent of which of these mediator-titrants is used, the concentration used, and which of various substrates is used in the determination. The substrates used were acetol, dihydroxyacetone, glycerin, 2-propyn-1-ol, allyl alcohol, 2-butyne-1,4-diol, furfuryl alcohol, benzyl alcohol, 4-pyridylcarbinol, galactose, and stachyose. The E1/2 and n values obtained are 0.40 +/- 0.005 V vs. SHE and 0.9 +/- 0.1 electron at pH 7.3. E1/2 is defined as the potential at which half the maximal enzymatic activity is observed and probably reflects the E0' of the enzymic group involved in activation. A model is proposed in which activation occurs during turnover due to the redox buffering (by oxidants) of an enzymic Cu(II)/Cu(I) state which has a higher E0' than in resting galactose oxidase. The pH dependence of E1/2 is 60 mV/pH unit in the pH range 6.0-8.0. The data suggest that the deprotonation of an amino acid residue facilitates the one-electron oxidation (activation) of galactose oxidase.

Immunoassay is a very selective and sensitive approach to trace metabolite analysis (Thorell, 1978). As a consequence of binding geometries the interaction between antigen (Ag)† and antibody (Ab) molecules may be extremely specific under favorable conditions (Eisen, 1974). The utility of immunoassay in diagnostic medicine is well established with many routine clinical methods already in use for the determination of a wide variety of biologically important substances (Chalt and Ebersole, 1981).

The reaction between plasmin and alpha 2PI (alpha-2-plasmin inhibitor), which constitutes a major regulatory step in fibrinolysis, involves both interactions between the kringle structures of plasmin and a complementary site on the inhibitor, and also between the inhibitor reactive center and the enzyme active site. An attempt was made to distinguish calorimetrically the two functional domains of alpha 2PI. Only one transition was seen, both in the DSC and UV experiments, contrary to the expected two. This transition was unaffected by K4 of plasminogen but was abolished in the presence of anhydrotrypsin. This suggests the major domain structure in alpha 2PI is associated with the reactive center.

Enzyme immunoassays (EIAs) are methods which use the chemical amplification feature of an enzyme and the high specificity of an antibody to provide a highly sensitive and selective means for quantitating an analyte. The applications of EIAs are numerous and ever-expanding (Jarvis, 1979). The sensitivity of EIAs is as high as that of radioimmunoassays (Maggio, 1980) which have detection limits ranging from 1-500 fmol/mL assay fluid (Oellerich, 1980). EIAs can be classified as either heterogeneous or homogeneous.

Immunoassay is a powerful method exhibiting high selectivity and sensitivity for biologically important molecules (Chait and Ebersole, 1981). The utility of immunoassay in diagnostic medicine is well documented, and a number of routine clinical methods have been established for the determination of a wide variety of common antigens (Landon and Moffar, 1976). However, immunoassay has only recently gained prominence within the analytical community (Monroe, 1984).

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10 −8 to 1.0 × 10 −5 M, a detection limit of 2.4 × 10 −8 M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml −1 by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.