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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...

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The effects of hydrogen (H2) and deuterium (D2) absorption were studied in two Co/Pd multilayers with perpendicular magnetic anisotropy (PMA) using polarized neutron reflectivity (PNR). PNR was measured in an external magnetic field H applied in the plane of the sample with the magnetization M confined in the plane for 6.0 T and partially out of plane at 0.65 T. Nominal thicknesses of the Co and Pd layers were 2.5 Å and 21 Å, respectively. Because of these small values, the actual layer chemical composition, thickness, and interface roughness parameters were determined from the nuclear scattering length density profile and its derivative obtained from both x-ray reflectivity and PNR, and uncertainties were determined using Monte Carlo analysis. The PNR showed that although D2 absorption occurred throughout the samples, absorption in the multilayer stack was modest (0.02 D per Pd atom) and thus did not expand. Direct magnetometry showed that H2 absorption decreased the total M at saturation and increased the component of M in the plane of the sample when not at saturation. The PNR magnetic scattering length density revealed that the Pd layers in the multilayer stack were magnetized and that their magnetization was preferentially modified upon D2 absorption. In one sample, a modulation of M with twice the multilayer period was observed at 0.65 T, which increased upon D2 absorption. These results indicate that H2 or D2 absorption decreases both the PMA and total magnetization of the samples. The lack of measurable expansion during absorption 2 indicates that these changes are primarily governed by modification of the electronic structure of the material.