Enzyme-mediator systems

Oxygen-utilizing organisms have generally evolved specific enzyme-mediated systems that serve to protect the cell from such reactive species. These enzymes include SOD and catalase or glutathione peroxidase (GSH-px), which catalyse the following reactions ... 

Scheme 3.5 Electron transfer between the substrate and enzyme/mediator system in biosensors.

Biomechanical straw pulps treated with enzyme mediator systems have been studied using mid-lR spectroscopy [108]. IR spectra indicated that a manganese-based enzyme system in combination with hydrogen peroxide generated the glucose oxidase couple, which removed aromatic ring structures. 

T Vares, A Hatakka, J Dorado, G Ahnendros, P Bocchini, GC Galletti, and AT Martinez. Microhandsheet Evaluation and Chemical Analysis of Wheat-Straw Pulp Treated with Enzyme-Mediator Systems. 7th International Conference on Biotechnology in the Pulp and Paper Industry, Montreal, 1998, A, pp. 149-152. 

Scheme 2.6 Different enzyme-mediator systems used for the regeneration of NAD(P)+ cofactors (a) xylose reductase (Ct-XR) H-9,10-phenanthrenequinone (b) laccase-I-diaphorase (M/-LPD) H-2,5-dimethoxy-l,4-benzoquinone and (c) laccaseH-Meldola s blue.

FIGURE 6-5 Second-generation enzyme electrode sequence of events that occur in a mediated system. (Reproduced with permission from reference 12.)... 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

The water-soluble vitamins generally function as cofactors for metabolism enzymes such as those involved in the production of energy from carbohydrates and fats. Their members consist of vitamin C and vitamin B complex which include thiamine, riboflavin (vitamin B2), nicotinic acid, pyridoxine, pantothenic acid, folic acid, cobalamin (vitamin B12), inositol, and biotin. A number of recent publications have demonstrated that vitamin carriers can transport various types of water-soluble vitamins, but the carrier-mediated systems seem negligible for the membrane transport of fat-soluble vitamins such as vitamin A, D, E, and K. 

Although the absence of paracellular transport across the BBB impedes the entry of small hydrophilic compounds into the brain, low-molecular-weight lipophilic substances may pass through the endothelial cell membranes and cytosol by passive diffusion [7]. While this physical barrier cannot protect the brain against chemicals, the metabolic barrier formed by the enzymes from the endothelial cell cytosol may transform these chemicals. Compounds transported through the BBB by carrier-mediated systems may also be metabolized. Thus, l-DOPA is transported through the BBB and then decarboxylated to dopamine by the aromatic amino acid decarboxylase [7]. 

In cases where the depuration of HOCs from BMOs involves enzyme-mediated biotransformations (Eq. 7.4) or active transport mechanisms, and environmental concentrations are high (e.g. near a point source), depuration rates have been shown to follow Michaelis-Menten kinetics (Spade and Hamelink, 1985). Michaelis-Menten kinetics is elicited when an enzyme or active transport system is saturated with a chemical. This type of kinetics is characterized by lower values of keS at sites with high HOC concentrations. If k s are unchanged at high concentration sites, Michaelis-Menten kinetics will result in elevated BAFs. However, if chemical concentrations become toxic, finfish likely avoid the area and sessile organisms such as mussels may close their valves for extended periods (Huckins et al., 2004). 

Equations 2.26 and 2.27 carmot be solved analytically except for a series of limiting cases considered by Bartlett and Pratt [147,192]. Since fine control of film thickness and organization can be achieved with LbL self-assembled enzyme polyelectrolyte multilayers, these different cases of the kinetic case-diagram for amperometric enzyme electrodes could be tested [147]. For the enzyme multilayer with entrapped mediator in the mediator-limited kinetics (enzyme-mediator reaction rate-determining step), two kinetic cases deserve consideration in this system in both cases I and II, there is no substrate dependence since the kinetics are mediator limited and the current is potential dependent, since the mediator concentration is potential dependent. Since diffusion is fast as compared to enzyme kinetics, mediator and substrate are both approximately at their bulk concentrations throughout the film in case I. The current is first order in both mediator and enzyme concentration and k, the enzyme reoxidation rate. It increases linearly with film thickness since there is no... 

Figure 5. Oxidation of methanol to carbon dioxide by a three-enzyme system consisting of alcohol (ADH), aldehyde (AldDH), and formate (FDH) dehydrogenases. Each enzyme is NAD+-dependent, and the NAD+ is regenerated by the anode via a redox mediator system. Redrawn with permission from ref 82. Copyright 1998 Elsevier Science S.A.

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

There are inherent problems associated with enzyme-mediated methods, regardless of the method used. The right conditions must be met, of course, for the enzyme action to take place. Unlike fluorochromes or gold particles (two other marker compounds), enzymes need to act chemically for the assay to work. Also, the enzyme action must only represent the marker molecule. Endogenous enzyme or enzyme-like activity can create problems only realized in systems that use enzymes. Also, the use of enzymes demands more attention to detail because of the increase in sensitivity that is often obtained. The problem of unwanted reactivity is enhanced in enzyme-mediated reactions more so than in others, in part because of the additional level of sensitivity brought about by the continuous action on a substrate. 

The pyridinium salt NAD 19a and its reduced form NADH 20a are important co-factors for many enzymes, fhe reduced form is involved in enzyme mediated reductions where it is converted to NAD. In natural systems, NAD is converted back to NADH by another enzyme-controlled process. Attempts to effect the direct electrochemical conversion of NAD to NADH are not very successful. Reduction on a mercury cathode at -1.1 V see on the first one-electron reduction wave leads to the radical-zwitterion, which reacts further to give dimers. Three stereoisomers of the 4,4 -dimer account for 90 % of the mixture and three 4,6 -dimers form the remainder [78]. Reduction at -1.8 V on the second reduction wave produces only 50 % of enzymatically active 1,4-NADH. The NAD analogue 19b shows related behaviour and one-electron reduction affords two diastereoisomers... 

I will extend this argument to encompass the diversification of such systems in lineages of plants in an effort to correlate enzyme-mediated glycoside toxicity with the evolution of host plant specificity and the coevolution of plants and insects. 

There are a number of findings suggesting that agents that facilitate elimination of protein carbonyls (by either proteolytic elimination or by enzymically mediated chemical reduction) may suppress neurodegenerative conditions in model systems (Botella et al., 2004). Consequently, as carnosine may also react with protein carbonyls, it is theoretically possible that it could suppress formation and /the reactivity of protein carbonyls in the brain. Whether carnosine participates in carbonyl reductase activity has not been investigated but it is also a reasonable speculation. 

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