RESUMO
Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.
Assuntos
Sistema Enzimático do Citocromo P-450 , Microssomos Hepáticos/enzimologia , Análise Espectral Raman , Adsorção , Animais , Coloides , Citocromos , Eletrodos , Heme , Lipossomos/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Nonoxinol , Oxirredução , Fenobarbital/farmacologia , Polietilenoglicóis , Coelhos , Prata , SolubilidadeRESUMO
The use of glycosaminoglycans for coating of medical implants had it's beginning in heparin coating of coronary stents and catheters to improve their haemocompatibility. Until now glycosaminoglycans proved as very potent in a variety of medical applications. Glycosaminoglycans not only show anticoagulant properties, also their crystal growth inhibiting effects in urology, inhibition of bacterial adherence and influence on the proliferation behaviour of human cells could be demonstrated. Beside an overview over the development of glycosaminoglycan coatings so far, recent outcomes of a bacteriological and a cell proliferation study with coated surfaces are presented.
Assuntos
Materiais Revestidos Biocompatíveis , Glicosaminoglicanos , Próteses e Implantes , Animais , Aderência Bacteriana , Divisão Celular/fisiologia , Humanos , Ácido Hialurônico , Teste de Materiais , Microscopia de Fluorescência , Stents , Propriedades de SuperfícieRESUMO
Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Mitocôndrias Cardíacas/enzimologia , Paracoccus denitrificans/enzimologia , Conformação Proteica , Animais , Bovinos , Ácido Cólico , Ácidos Cólicos , Detergentes , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Heme/análise , Octoxinol , Análise Espectral Raman/métodosRESUMO
Capsular contracture around breast implants with smooth surfaces continues to be an unpredictable complication. Some surgeons believe that silicone implants covered with porous polyurethane foam have a lowered potential to contract. These textured implants are not as biocompatible as silicone. Recently, silicone implants with textured surfaces have been introduced with the hope that the incidence of unacceptable implant contracture will be reduced. Using a rat implant model, the tissue reaction to textured implant surfaces was assessed. The implant surfaces evaluated were Silastic II, Siltex, MISTI, Biocell, Silastic MSI, and Même. Disks of each implant material were implanted under the dorsal skin of rats for a period of 28 days. Each implant with its surrounding tissue was excised, processed for histological analysis, and assessed for the tissue's response to the implant with particular emphasis on the formation of a continuous collagen capsule. The results indicated that the magnitude of surface texturing influenced the development of a complete capsule. Implant surfaces with a texture of less than 150 microns in height or depth (Silastic II, Siltex, and MISTI) resulted in the formation of complete capsules. An implant (Biocell) with irregular texturing (200-350 microns) produced an organized capsule over most of its surface with localized interruptions of the capsule at the sites of its deepest cavities. Implant surfaces with texturing that exceeded 350 microns in height or depth (Silastic MSI and Même) resulted in inhibition of the formation of a continuous capsule during this 28-day study.