The cytochrome P450 enzymes represent an important class of heme containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological based chips for standardizing cytochrome P450 metabolism or for high throughput screening of pharmaceutical agents. Previous studies have generally immobilize P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed. The cytochrome P450 enzymes represent an important class of heme containing enzymes. They are responsible for a significant portion of xenobiotic metabolism and have been extensively studied for mechanistic and practical reasons. More recently there has been interest in immobilizing these enzymes on a surface to study the electron transfer in a single enzyme, 1 homodimer or heterodimer interactions, 2 or for construction of biological based chips for high throughput screening of pharmaceutical agents.Previous efforts to immobilize P450 enzymes have been made 3 including CYP2E1 on gold electrodes 1 bacterial P450 BM3 on graphite, 4 P450cam immobilized in sol-gel films, 5 pgannett@hsc.wvu.edu. CYP1A2 and CYP3A4 in polyion films, 6 and CYP119 in dimethyldidodecylammonium poly (p-styrene sulfonate). 5 In these studies the detection of substrate binding is often made electrochemically by observing shifts in the redox potential upon substrate binding. 7 Enzymelike metabolic reactions have also been observed by the application of an electrochemical current. 1 However, to be amenable to study and manipulation at the single molecule level it may be desirable to minimize the surrounding matrix. Also, it is highly desirable for the enzyme to metabolize substrates utilizing endogenous enzymes and co-factors to more closely model the corresponding biological system. To our knowledge, this has not been achieved with cytochrome P450 enzymes. NIH Public AccessIn this work we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is dependent upon the linkers used to attach CYP2C9 and to the presence of key substrates during the attachment. A novel method to detect substrate binding, namely superconducti...

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in mass spectrometry (MS) instrumentation, and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing, and characterization of glycoconjugates.

ABSTRACT:Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied.The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the MichaelisMenten constant (K m ) for the hydroxylation of warfarin (3 M), which is in good agreement with previous literature regarding K m values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.Cytochrome P450 (P450) enzymes are important for drug metabolism in humans, accounting for ϳ75% metabolism of drugs (Williams et al., 2004). Numerous factors affect the P450 metabolism rate and the resulting metabolite structure including electron transfer, protein-protein interactions, concentration and structure of the substrate, and the source and specific means whereby the P450 was prepared (Guengerich, 2007). In practice, it is difficult to isolate the individual contributions of these factors because they are often interdependent. Thus, it is of interest to devise model platforms that can be used to independently control these parameters to monitor their respective effects on metabolism.P450 catalysis requires a constant supply of NADPH as the electron source and cytochrome P450 reductase (CPR) to deliver the electrons.In attempts to obviate the requirement of these redox partners/cofactors for catalysis, P450 enzymes have been immobilized on electrodes so that the electrode supplies electrons to drive the P450 catalytic cycle (Estabrook et al., 1996;Reipa et al., 1997;Gilardi et al., 2002) with effective electrical communication between the electrode and the enzyme being critical (Yang et al., 2008). Direct adsorption of enzymes on bare electrodes, such as gold, platinum, and graphite, results in diminished biocatalytic activity (Habermüller et al., 2000;Joseph et al., 2003;Shumy...

Recent developments in bioanalytical instrumentation, mass spectrometry (MS) detection and computational data analysis approaches have provided researchers with capabilities for interrogating the complex cellular glycoproteome, to help gain a better insight into the cellular and physiological processes that are associated with a disease, and to facilitate the efforts centered on identifying disease-specific biomarkers. This review describes the progress achieved in the characterization of protein glycosylation by using advanced capillary and microfluidic-MS technologies. The major steps involved in large-scale glycoproteomic analysis approaches are discussed, with special emphasis given to workflows that have evolved around complex MS detection functions. In addition, quantitative analysis strategies are assessed, and the bioinformatics aspects of glycoproteomic data processing are summarized. The developments in commercial and custom fabricated microfluidic front-end platforms to ESI- and MALDI-MS instrumentation, for addressing major challenges in carbohydrate analysis such as sensitivity, throughput and ability to perform structural characterization, are further evaluated and illustrated with relevant examples.