Absence Of Mediators Research Articles

Direct electrochemistry of membrane-entrapped horseradish peroxidase.: Part I. A voltammetric and spectroscopic study

This paper reports the electrochemical behaviour, relative to the Fe(III)–Fe(II) conversion, of horseradish peroxidase (HRP) entrapped within a solid matrix, at a pyrolytic graphite electrode. The results indicate that (i) immobilization enhances the electron exchange between the protein and the electrode surface; (ii) reversible electron transfer (eT) is achieved within a wide pH range (pH 3.0–12.0) even in the absence of mediators, (ii) the embedded protein shows native-like structural properties and increased stability. The results obtained may be of potential value, since they represent a first step for engineering a novel `solid-state' electrode system, of importance for basic and applied biochemistry.

On the determination of redox midpoint potential of the primary quinone electron acceptor, Q A, in Photosystem II

Redox titrations of Q A, the first quinone electron acceptor, have been performed on Photosystem II (PS II) membranes which were either active or inactive in terms of oxygen evolution. The redox state of Q A was monitored by measuring the chlorophyll fluorescence yield. When titrations were done at room temperature in the absence of mediators, an E m value of approx. −80 mV was obtained for active centres and approx. +65 mV for inactive centres. These values confirm earlier reports (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) in which measurements were made under comparable conditions. In addition, we found that these E m values were independent of pH from pH 5.5 to pH 7.5, the range of pH over which the O 2-evolving enzyme is stable. To understand better the scattered values for the E m of Q A which exist in the literature and to assess the validity of the present values, experiments were performed under a range of different titration conditions. Two main experimental factors were found to have a strong influence on the measured E m of Q A. First, the presence of redox mediators at low ambient potentials led to an irreversible shift from the low-potential (active) form to the high-potential (inactive) form. This is attributed to the reduction of the Mn cluster which is thought to remain out of equilibrium when titrations are done without mediators. Secondly, upon freezing of samples poised at low potential a change in the redox state of Q A occurred, as measured by EPR and fluorescence at low temperature. Freezing and thawing of active PS II at potentials where Q A is chemically reduced results in an irreversible change in the E m of Q A from the low-potential to the high-potential form. This is accompanied by inhibition of oxygen evolution. It is suggested that this effect might also be related to the reduction of the Mn cluster which is, in this case, induced by freeze-thawing in the presence of chemically reduced Q A −. Based on these observations, it is suggested that most titrations of Q A in active PS II that have been reported previously suffer from one or both of these unexpected technical difficulties. Thus, the E m values obtained at room temperature and without mediators are probably those which should be taken into account in understanding the energetics of PS II.

AMPEROMETRIC BIOSENSORS BASED ON BIOCATALYST ELECTRODES. MEDIATED AND MEDIATORLESS ENZYME ELECTRODES

An enzyme-modified electrode is constructed by immobilizing an oxidoreductase behind a dialysis membrane on a carbon paste electrode containing a redox compound. The redox compound functions as an electron transfer mediator between the electrode and the immobilized enzyme, thus the electrode can oxidize or reduce the substrate electro-enzymatically. Many oxidoreductases, especially dehydrogenases which use artificial electron acceptors are the enzymes suitable for constructing the enzyme-modified electrodes. They can be used as mediated amperometric biosensors which are insensitive to oxygen tension in test solutions. Examples of the mediated enzyme electrodes are given and characteristics of the current response of the electrodes to the substrates are described. Use of the mediated enzyme electrodes with high sensitivity are also described. Enzyme-modified electrodes based on oxidoreductases from bacterial membranes can oxidize the substrates electro-enzymatically in the absence of mediators. Such electrodes can be used as mediatorless enzyme electrodes.

Adrenergic and cholinergic regulation of phospholipid release in sublingual salivary gland in vitro

1. 1. The role of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion was investigated using rat sublingual acinar cells maintained in the presence of [ 3H]choline. 2. 2. The release of [ 3H]choline containing phospholipids was enhanced by β-adrenergic agonist, isoproterenol to a greater extent than the cholinergic agonist, pilocarpine. 3. 3. The effect of isoproterenol on phospholipid release was observed even after 5 min and by 30 min a 1.7-fold increase in secretion occurred, whereas pilocarpine evoked 1.2-fold increase by 30 min with no discernible effect in 5 min. The isoproterenol effect was blocked by alprenolol, and an inhibition of pilocarpine effect was observed with atropine. 4. 4. In the absence of mediators, 82% of secreted labeled phospholipids were represented by phosphatidylcholine (PC), 4.3% by lysophosphatidylcholine (LPC) and 12.1% by sphingomyeline (Sph), those secreted in the presence of isoproterenol showed significantly lower (37%) content of LPC, while those secreted in response to pilocarpine were substantially richer in LPC (2.5 times) and Sph (27%), and contained less (17%) PC. 5. 5. The results provide first evidence for the involvement of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion.

Direct electrochemistry of the blue copper proteins pseudoazurin, plantacyanin, and stellacyanin

Direct electrochemistry of three blue copper proteins. Achromobacter cycloclastes IAM 1013 pseudoazurin, Cucumis sativus plantacyanin, and Rhus vernicifera stellacyanin, is achieved at a glassy-carbon electrode over the pH range 5-11 in the absence of mediators and promoters

Direct electron transfer of redox proteins at the bare glassy carbon electrode

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 micrograms, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric-acid-pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from $293 mV in 1 mM potassium phosphate to $266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH-dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi-reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4 C; 3 X Em = -0.32 mV, Em = -0.26 V), and cytochrome c553 (pI = 9.3; Em = +60 mV), and from Megasphaera elsdenii rubredoxin (pI congruent to 3; Em = -353 mV). The latter protein absorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin-linked enzymes.

A spectroelectrochemical study of carboxymethylated cytochrome- c

Direct electrochemical reduction of dicarboxymethylated cytochrome c [(Cm)-cyt c] has been carried out to investigate the effect of the displacement of methionine 80 (Met 80) from the sixth coordination position on the redox potential of heme iron. Differential pulse and cyclic voltammetry were employed in the present study. At gold microelectrodes or gold-coated RVC electrodes, the reduction process occurs in the absence of mediators. The rate of heterogeneous charge transfer for (Cm)-cyt c appears to be greater than that operative in native cytochrome c. A value of −0.218 versus s.h.e. at neutral pH was determined for the formal standard potential ( U′ 0,7) from spectroelectrochemical measurements. The large change in U′ 0 of the modified cytochrome c with respect to the native protein (Δ U ⋍0.5 V) is in agreement with rearrangement of the tertiary structure induced by rupture of the Met 80-heme iron bond, which leads to a high degree of heme exposure to the solvent. In addition, the results support the important role of Met 80 in assuring stability, through its bond with the heme iron, to the close crevice structure.

THE PHYSIOLOGY OF HOST–PARASITE RELATIONS: VI. OXIDATION–REDUCTION CHANGES IN WHEAT LEAF SAP CAUSED BY RUST INFECTION

Extracts of healthy and rusted leaves of Little Club and Khapli wheats were made under anaerobic conditions. Oxidation–reduction potentials in the extracts were measured at two different levels both in the presence and in the absence of both methylene blue and riboflavin as redox mediators. At the lower level (i.e. in the absence of mediators) a reversible rise in potential was noted during the course of rust infection on Little Club; with Khapli the rise was similar but was not reversed. The response to rust infection of resistant and susceptible plants was also reflected in characteristic changes in the average potentials at higher levels (i.e. in the presence of mediators). After infection the oxidation–reduction balance of susceptible tissue was reorganized by the introduction of new systems which were found in the reduced state only. After infection the oxidation–reduction potential of resistant tissue was only poorly poised as indicated by a rise in the average potentials and as demonstrated by oxidation– reduction titrations.The results are discussed and the possibility is considered that a rust-induced catalyst may “short circuit” the chain of hydrogen transport, thus initiating all the observed redox changes.