Investigation of the Direct Heterogenous Electron Transfer of Hemoglobin and Cytochrome P450 Enzymes and Bioanalytical Application
Author: Nikitas Bistolas
Publisher:
Published: 2005
Total Pages: 120
ISBN-13:
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Cytochrome P450
Author: Paul R. Ortiz de Montellano
Publisher: Springer Science & Business Media
Published: 2007-02-05
Total Pages: 702
ISBN-13: 0387274472
DOWNLOAD EBOOKCytochrome P450: Structure, Mechanism, and Biochemistry, third edition is a revision of a review that summarizes the current state of research in the field of drug metabolism. The emphasis is on structure, mechanism, biochemistry, and regulation. Coverage is interdisciplinary, ranging from bioinorganic chemistry of cytochrome P450 to its relevance in human medicine. Each chapter provides an in-depth review of a given topic, but concentrates on advances of the last 10 years.
Oriented Attachment of Cytochrome P450 2C9 to a Self-Assembled Monolayer on a Gold Electrode as a Biosensor Design
Author: Elizabeth Schneider
Publisher:
Published: 2011
Total Pages: 214
ISBN-13:
DOWNLOAD EBOOKCytochrome P450s (CYPs) are a family of enzymes implicated in the metabolism of drugs in the body. Consequently, P450 reactions are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened as potential substrates of CYPs. The P450 reaction involves electron transfer to an iron heme via NADPH and the electron transfer partner enzyme P450 reductase (CPR). By immobilizing CYPs on an electrode however, NADPH and CPR are potentially no longer needed and the immobilized CYP can act as a biosensor by accepting electrons directly from the electrode. Such a biosensor could be used as an initial screening tool for CYP reactivity of pharmaceuticals in development. In this study, the drug-metabolizing enzyme CYP 2C9 was immobilized to a self-assembled monolayer (SAM) on a gold electrode in three different orientations to investigate the effect that orientation has on the direct electrochemistry of CYP and to evaluate oriented attachment of CYP to an electrode as a biosensor design. Three attachment methods were investigated: random attachment via amine coupling to a carboxy-terminated SAM, oriented attachment via C-terminal His-tag coupling to a Ni-NTA-functionalized SAM, and oriented attachment via maleimide/thiol coupling to a maleimide-functionalized SAM. Three 2C9 mutants (R125C, R132C, and K432C) were developed with a single cysteine mutation at the binding site for CPR on the side of the enzyme closest to the heme; attachment of these mutants to a gold electrode via maleimide/thiol coupling would orient the enzyme such that electron transfer occurs on the electrode in the same orientation that it does in vivo with CPR. Therefore, we expected oriented attachment via maleimide/thiol coupling to produce the most electroactive CYP biosensor. Electrochemical analysis and surface characterization of the SAMs on gold electrodes confirmed that electron transfer occurs through the SAMs, and activity assays of the 2C9 electrodes confirmed that wild-type 2C9 and the single Cys mutants R125C, R132C, and K432C were immobilized to the gold electrode via all three attachment methods. Cyclic voltammetry of the 2C9 electrodes revealed however, that direct electron transfer to 2C9 was not possible for all three attachment methods. Similar redox processes were observed for both the 2C9 electrodes and no-enzyme electrodes modified only with SAMs, suggesting that the redox process observed on the 2C9 electrodes is related to the underlying SAM. Thus, we were unable to make any conclusions regarding the effectiveness of oriented attachment in creating a 2C9 biosensor. However, to our knowledge, there are no examples in the literature of the oriented attachment of 2C9 to an electrode via coupling of an engineered cysteine to a maleimide-functionalized SAM on gold and therefore this study represents the first attempt towards such an electrode system.
Investigation of Surface Interactions Between Cytochrome B5 and Major Cytochrome P450 Isoforms
Author: Chunsheng Zhao
Publisher:
Published: 2012
Total Pages: 206
ISBN-13:
DOWNLOAD EBOOKCytochrome b5 (cyt b5 or holo b5) is known as one of the key components in the microsomal cytochrome P450 (CYP) monooxygenase system that metabolizes structurally diverse endogenous and exogenous compounds. It has been reported to modulate many CYPs activity and the effect is both CYP isoform and substrate dependent. However, to date no consensus has been made on the underlying mechanism. In the present study, the surface interactions between cyt b5 and major hepatic CYP isoforms 3A4, 2C9, 2A6 and 2D6 were investigated. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions on protein surfaces of cyt b5 and CYPs. Subsequently, the interaction models of cyt b5-CYPs were built using these identified cross-linking sites as constraints. For the first time, holo b5 and apo b5 (cyt b5 devoid of heme) surface interactions with CYPs were compared. The models suggest both of them bind to the same the groove on CYPs with very small difference in their orientations. The closest distances between the heme groups of cyt b5 and each CYP isoform are beyond direct electron transfer distance, indicating that cyt b5 likely modulates these CYP isoforms activity through allosteric effect in addition to the potential electron transfer role. In order to confirm that the residues involved in cross-linking are functionally important for cyt b5-CYP interaction, site-directed mutagenesis of CYP3A4 were carried out with the identified Lys residues on CYP3A4 being substituted with neutral residue Ala. In addition, the importance of Arg446 on CYP3A4 at the interface of the cyt b5-CYP3A4 complex model was also accessed by single-point mutation. Mutation of these residues reduced or abolished cyt b5 binding affinity, suggesting that electrostatic interactions on the interface of the two protein are functionally important and the chemical cross-linking coupled with mass spectrometric analysis serves as a useful tool to study protein-protein interaction.
Direct Electron Transfer of Human Hemoglobin Molecules on Glass/Tin-Doped Indium Oxide
Author: Flavio Dolores Martínez-Mancera
Publisher:
Published: 2017
Total Pages:
ISBN-13:
DOWNLOAD EBOOKInterfacial electron transfer kinetics of the haem (FeIII/FeII) group in human hemoglobin molecules were investigated on glass/tin-doped indium oxide electrodes. Factors such as surface roughness, crystallinity, hydrophilicity and partial polarization of the working electrode played an important role to provide a more compatible microenvironment for protein adsorption. Results suggested that direct electron transfer from electrode to haem (FeIII)-H2O intermediate is coupled to proton at near physiological pH (I = 0.035, pH = 7.2).
Colloidal Gold
Author:
Publisher: Elsevier
Published: 2012-12-02
Total Pages: 517
ISBN-13: 0323139027
DOWNLOAD EBOOKSince its introduction in 1971, the development and application of colloidal gold as a marker in electron microscopy has been phenomenal. Colloidal gold has become the method of choice in immunocytochemistry and many areas of cell biology. This universal method is applicable to most microscopical systems including optical microscopy; scanning, transmission, and high voltage electron microscopy; photoelectron, photon, fluorescent darkfield, and epipolarization microscopy. Colloidal gold allows high and low resolution studies, enzyme and nucleic acid labeling, study of dynamic cellular processes, and virus detection. This book is among the first available to cover the principles and methodology of colloidal gold in microscopy. Methods are described step by step, to enable researchers to learn these complex procedures solely by reference to these books Problems and limitations of techniques are discussed Guides users to avoid problems and choose the correct procedures for specific applications Contributors are eminent authorities in their fields
Bioelectrochemistry
Author: Richard C. Alkire
Publisher: John Wiley & Sons
Published: 2013-09-25
Total Pages: 411
ISBN-13: 3527644121
DOWNLOAD EBOOKBioelectrochemistry is a fast growing field at the interface between electrochemistry and other sciences such as biochemistry, analytical chemistry and medicinal chemistry. In the recent years, the methods and the understanding of the fundamentals have seen significant progress, which has led to rapid development in the field. Here, the expert editors have carefully selected contributions to best reflect the latest developments in this hot and rapidly growing interdisciplinary topic. The resulting excellent and timely overview of this multifaceted field covers recent methodological advances, as well as a range of new applications for analytical detection, drug screening, tumor therapy, and for energy conversion in biofuel cells. This book is a must-have for all Electrochemists, Biochemists, Analytical Chemists, and Medicinal Chemists.
Multi-copper Oxidases
Author: Albrecht Messerschmidt
Publisher: World Scientific
Published: 1997
Total Pages: 477
ISBN-13: 9810227116
DOWNLOAD EBOOKThe biological activation of dioxygen is a key reaction in biological systems. Enzymes involved in direct oxygen activation are oxidases and oxygenases. Multi-copper oxidases are an important class of oxidases reducing dioxygen in a four-electron reduction to water with concomitant one-electron oxidation of the reducing substrate. The progress in the characterization and understanding of the structure and function of these enzymes has advanced so tremendously over the last ten years that the publication of a book documenting these achievements has been overdue.Especially the recent discovery of a key role of the FET3 protein of Saccharomyces cerevisae, a multi-copper oxidase, in iron metabolism of this eukaryote has underpinned the function of the plasma multi-copper oxidase ceruloplasmin in vetebrate iron transport. The lately determined x-ray structure of human ceruloplasmin confirms its close structural relatedness to the plant multi-copper oxidases ascorbate oxidase and laccase and due to strong amino-acid sequence similarities has allowed to construct a useful model of the more distantly related blood-clotting factor VIII.This book contains review articles from experts in the field, dealing with modern spectroscopy, enzyme kinetics, bioinorganic chemistry, x-ray crystallography, electron transfer reactions, molecular biology, medical aspects and potential industrial applications of the three main members of multi-copper oxidases, i.e., laccase, ascorbate oxidase and ceruloplasmin.
Frontiers in Biosensorics I
Author: F.W. Scheller
Publisher: Springer Science & Business Media
Published: 1996-12-01
Total Pages: 302
ISBN-13: 9783764354756
DOWNLOAD EBOOKVolume I provides an in-depth discussion of the most recent developments of crucical biosensor components. It concentrates on the interface between the analyte phase and the detector, namely, the implementation of novel recognition elements, including nucleic acids, and of leading-edge technology in the construction of responsive thin layers. Thus, the reader can obtain a foretaste of achievable future progress in the field.
Point-of-care testing
Author: Peter Luppa
Publisher: Springer
Published: 2018-07-18
Total Pages: 451
ISBN-13: 3662544970
DOWNLOAD EBOOKThe underlying technology and the range of test parameters available are evolving rapidly. The primary advantage of POCT is the convenience of performing the test close to the patient and the speed at which test results can be obtained, compared to sending a sample to a laboratory and waiting for results to be returned. Thus, a series of clinical applications are possible that can shorten the time for clinical decision-making about additional testing or therapy, as delays are no longer caused by preparation of clinical samples, transport, and central laboratory analysis. Tests in a POC format can now be found for many medical disciplines including endocrinology/diabetes, cardiology, nephrology, critical care, fertility, hematology/coagulation, infectious disease and microbiology, and general health screening. Point-of-care testing (POCT) enables health care personnel to perform clinical laboratory testing near the patient. The idea of conventional and POCT laboratory services presiding within a hospital seems contradictory; yet, they are, in fact, complementary: together POCT and central laboratory are important for the optimal functioning of diagnostic processes. They complement each other, provided that a dedicated POCT coordination integrates the quality assurance of POCT into the overall quality management system of the central laboratory. The motivation of the third edition of the POCT book from Luppa/Junker, which is now also available in English, is to explore and describe clinically relevant analytical techniques, organizational concepts for application and future perspectives of POCT. From descriptions of the opportunities that POCT can provide to the limitations that clinician’s must be cautioned about, this book provides an overview of the many aspects that challenge those who choose to implement POCT. Technologies, clinical applications, networking issues and quality regulations are described as well as a survey of future technologies that are on the future horizon. The editors have spent considerable efforts to update the book in general and to highlight the latest developments, e.g., novel POCT applications of nucleic acid testing for the rapid identification of infectious agents. Of particular note is also that a cross-country comparison of POCT quality rules is being described by a team of international experts in this field.
