On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

The catalytic cycle of multicopper oxidases (MCOs) involves intramolecular electron transfer (IET) from the Cu-T1 copper ion, which is the primary site of the one-electron oxidations of the substrate, to the trinuclear copper cluster (TNC), which is the site of the four-electron reduction of dioxygen to water. In this study we report a detailed characterization of the kinetic and electrochemical properties of bilirubin oxidase (BOx) a member of the MCO family. The experimental results strongly indicate that under certain conditions, e.g. in alkaline solutions, the IET can be the rate-limiting step in the BOx catalytic cycle. The data also suggest that one of the catalytically relevant intermediates (most likely characterized by an intermediate oxidation state of the TNC) formed during the catalytic cycle of BOx has a redox potential close to 0.4 V, indicating an uphill IET process from the T1 copper site (0.7 V) to the Cu-T23. These suggestions are supported by calculations of the IET rate, based on the experimentally observed Gibbs free energy change and theoretical estimates of reorganization energy obtained by combined quantum and molecular mechanical (QM/MM) calculations

Imaging of Single Antigens, Antibodies, and Specific Immunocomplex Formation by Scanning Force Microscopy

The most sensitive analytical techniques available today for detecting immuno assay complexes are radio or enzyme immuno analytical techniques, by which quantities of 107-108 analyte molecules can be detected. With the introduction of scanning force microscopy, a new method for detecting biological processes became available. Here, we examine the feasibility of using scanning force microscopy as a biosensitive tool. We demonstrate that single or multiple rabbit anti-human serum albumin molecules form complexes with preadsorbed single human serum albumin molecules on mica. However, no interaction is observed between human immunoglobulin G molecules and preadsorbed single albumin molecules; only separate antigens and antibodies are observed at random positions on the mica. This shows the ability of scanning force microscopy to act as a biosensor for detection of immunocomplexes, and to act as a very powerful tool to study molecule-surface interactions in general

A New Method For The Determination Of Accommodation Coefficients.

PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/185323/2/6106414.pd

Electrospray ionization mass spectrometry in enzymology: uncovering the mechanisms of two- substrate reactions

The purpose of this review is to draw attention to the use of electrospray ionization mass spectrometry ( ESI-MS) for monitoring the course of enzyme substrate interactions, in the particular case of complex systems in which two substrates participate. The determination and characterization of intra-molecular reactions, especially those that occur in the enzyme active site, is not a trivial task in chemical kinetics, typically requiring long measurement times and relatively expensive techniques such as nuclear magnetic resonance (NMR), X-ray crystallography or electron microscopy ( EM). However, nowadays almost all laboratories are equipped with or else have access to the ESI-MS technique. The aim of this review is to focus on the possibilities of employing even quite simple MS equipment to tackle different applications in studies of complex enzymatic systems

Proteins in vacuo. A connection between mean overcrossing number and orientationally-averaged collision cross section

Recently, the structure of protein ions in vacuo has received a lot of interest. Experimentally, such structures can only be characterized by global, low-resolution descriptors like the orientationally-averaged collision cross section (σ) over bar. An alternative descriptor of chain entanglement, the mean overcrossing number (N) over bar, is a very useful tool because it can distinguish sensitively between different conformations based on aspects of their geometry and topology. An overcrossing number distribution arises from an ensemble of different projections of the protein structure onto a viewing plane, where for each projection, the number of crossings by the set of line segments joining sequence-adjacent Calpha atoms is computed. (N) over bar is the mean of this distribution. Despite their usefulness, overcrossing numbers depend on the atomic coordinates and thus cannot be measured for protein ions in vacuo as can collision cross sections. In this work, we explore theoretically whether collision cross sections are correlated with overcrossing numbers. Using a group of diverse proteins with the same chain length as lysozyme and known X-ray structures, we find a weak correlation between (σ) over bar and (N) over bar. Thus, a nontrivial range of orientationally-averaged cross section values can be consistent with the same mean overcrossing number. Since (N) over bar comprises a very sensitive tool for the study of conformational rearrangements, we checked also for correlations between (σ) over bar and (N) over bar during molecular dynamics simulated unfolding and relaxation pathways of lysozyme in vacuo. For a given pathway, the two quantities are found to be highly correlated. Although neither (σ) over bar nor (N) over bar maps one-to-one onto a particular tertiary fold, they are both sensitive descriptors to monitor the conformational state of a protein along a pathway of dynamical change

Energy and angular distributions of Rh atoms ejected due to ion bombardment from Rh{111}: A theoretical study

energy and angular distributions of Rh atoms ejected from a Rh[111] surface due to keV ion bombardment are predicted from classical dynamic calculations and are compared to those measured using a multiphoton resonance ionization scheme. The comparison is generally quite favorable. For example, the calculated distributions reproduce the changes in azimuthal anisotrophy which occur over an ejected-particle kinetic energy range of 5-50eV. The new detailed experimental data do, however, expose deficiencies in the pair potential, which we believe can be overcome with a many-body potential.Financial support from the National Science Foundation, the U.S. Office of Naval Research, the U.S. Air Force Office of Scientific Research, The Foundation Research Program of the Naval Postgraduate School, the IBM Corporation and the Camille and Henry Dreyfus FoundationApproved for public release; distribution is unlimited

Introduction to biological mass spectrometry: Determining identity and species of origin of two proteins

We describe a laboratory exercise that is designed to give advanced undergraduate students in analytical chemistry a meaningful introduction to biological mass spectrometry. We ask the students to solve a mystery: given two proteins, what are they, and from which animals do they originate? This exercise involves use of two mass spectrometers available in our institution: a fairly basic matrix-assisted laser desorption ionization time-of-flight (MALDI–TOF) unit and an electrospray ionization (ESI) ion-trap unit. Our exercise can be completed in two days (including analysis) and provides several interesting and educational surprises. The students, in small groups, are introduced to total mass measurement with ESI, proceed to peptide mass mapping with MALDI–TOF, and finally explore sequence information with ion-trap tandem (fragmentation) mass spectrometry. Even in the most advanced phase, we guide the students, but by this time they usually have their own ideas as to what avenues to pursue. We find that our students become excited after they solve the "mystery"of these proteins, and they receive enough education in the process to be able to join a research project involving biological mass spectrometry

Extraction and preconcentration of salbutamol and terbutaline from aqueous samples using hollow fiber supported liquid membrane containing anionic carrier

This paper presents a new three-phase liquid-phase microextraction (LPME) strategy for extraction and preconcentration of salbutamol (SB) and terbutaline (TB) from aqueous samples, including urine. The drugs were extracted from I I ml of aqueous sample (source phase; SP) into an organic phase with microliter volume located inside the pores of a polypropylene hollow fiber, and then back-extracted into 24 mu l of a second aqueous solution as the receiving phase (RP), located in the lumen of the hollow fiber. In preliminary experiments, we tried to transport the drugs using a pH gradient between the two sides of the hollow fiber. Due to the existence of both amine and phenolic groups on the drugs, very little transport occurred and enrichment factors (EF) less than one were obtained. Further experiments were done in the presence of bis(2-ethylhexyl) monohydrogenphosphoric acid (D2EHPA) or methyltrioctylammonium chloride (Aliquat 336) in the organic phase, to extract drugs from acidic and basic matrices, respectively. Results showed that transport of drugs from alkaline solution into I M of sodium bromide occurred when the membrane was impregnated with dihexyl ether containing 20% Aliquat 336. To optimize the EF, the effects of different parameters such as the nature of organic solvent used to impregnate the membrane, compositions and volumes of SP and RP, type and concentration of carrier, extraction time and stirring rate were investigated. Optimal results were obtained in the presence of 0.005 M of NaOH (pH 11.70) in the SP, I M of NaBr in the RP, 20% of Aliquat 336 in dihexyl ether as membrane impregnation solvent, stirring rate of 500 rpm and extraction time of 60 min. Under these conditions. enrichment factors of 52.9 and 213.1, dynamic linear ranges of 20-5000 and 10-5000, and limits of detection of 2.5 and 0.5 ng/ml were obtained for salbutamol and terbutaline, respectively. Also determination of drugs in environmental water and urine samples in the range of nanograms per millilitre with RSDs < 10% was possible using HPLC-photodiode array detection or HPLC-MS. (c) 2006 Elsevier B.V. All fights reserved

Analysis of triazines and associated metabolites with electrospray ionization field-asymmetric ion mobility spectrometry/mass spectrometry

Triazines comprise an important pollutant class owing to continued use in certain countries, and owing to strong environmental persistence that leads to problems even in countries like Sweden where the use of triazines has been prohibited for some years. We investigated mass-selective detection for analysis of triazines. More specifically, we studied the background reduction and sensitivity enhancement that result from the use of a new interface technique, field-asymmetric ion mobility spectrometry (FAIMS), in conjunction with electrospray ionization ion-trap mass spectrometry. This technique allows for ion sorting and discrimination against the considerable "chemical noise", nonspecific cluster and fragment ions, which are typically generated in electrospray ionization. This paper presents results of a pilot study of triazines and some metabolites in ideal solvents. Our long-range goal is automated analysis with mass-selective detection coupled to membrane-based sample cleanup and enrichment for additional enhancement in sensitivity