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1.
Philip A. Wheeler Michael Brungs Graham R. Atkins 《Journal of Sol-Gel Science and Technology》2003,27(3):321-326
Organically modified silicates (ormosils) have been viewed as possible materials for optical signal processing devices for communications networks. One significant problem obstructing their use in this application is the absorption of infrared light by hydroxy species (present for example in silanol groups (SiOH) and water) around the communications wavelengths (1310 and 1550 nm). We have investigated several ormosil compositions, synthesised via an aqueous sol-gel method, to determine if residual hydroxy groups can be removed more effectively via reducing the connectivity of the material by increasing the number of organic groups (such as Si—CH3). Furthermore, we have investigated the effect of different inert atmospheres on the drying process. It was found that reducing the connectivity does promote the removal of hydroxy absorption, mainly via the condensation of residual silanol groups, but also by removal of residual water, depending on the other precursors in the system. It was also found that the drying atmosphere used (nitrogen or helium) had no effect on the drying process. 相似文献
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Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited
for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the
bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel
chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein
(493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids,
46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus
of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were
developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models
indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s.
The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis,
and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural
diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed
evolution. 相似文献
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Dabrowski MJ Schrag ML Wienkers LC Atkins WM 《Journal of the American Chemical Society》2002,124(40):11866-11867
Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates. 相似文献
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Alves GA Amato S Anjos JC Appel JA Astorga J Bracker SB Cremaldi LM Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Halling AM Jedicke R Karchin PE Kwan S Leuking LH Mantsch PM de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Passmore D Rafatian A dos Reis AC Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wallace A Wu Z 《Physical review D: Particles and fields》1994,49(9):R4317-R4320
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Alves GA Amato S Anjos JC Appel JA Astorga J Bracker SB Cremaldi LM Dagenhart WD Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Jedicke R Karchin PE Kennedy C Kwan S Lueking LH de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Passmore D Rafatian A dos Reis AC Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wallace A Wu Z 《Physical review letters》1996,77(12):2388-2391
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Alves GA Amato S Anjos JC Appel JA Bracker SB Cremaldi LM Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Jedicke R Kaplan D Karchin PE Kwan S Leedom I Lueking LH Luste GJ Mantsch PM de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Rafatian A dos Reis AC Reucroft S Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wu Z 《Physical review letters》1993,70(6):722-725