RESUMO
Both infrared and Raman are increasingly applied in biomolecular structure characterization, thus gaining acceptance alongside traditional bioanalytical techniques. Physical principles and biological sampling requirements are reviewed. Advanced applications are used to illustrate vibrational spectroscopy capabilities and limitations in bioanalysis.
Assuntos
Fatores Biológicos/análise , Produtos Biológicos/análise , Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodosRESUMO
Substrate-free Delta9-18:0-acyl carrier protein desaturase (abbreviated to Des)[E.C. # 1.14.99.6] was 2-electron reduced with E'0=-0.03 +-0.01 V; the presence of spinach ferredoxin (SpFd) induces an additional 1-electron reduction wave at E'0=-0.21 +-0.02 V, which shifts by 0.106 V upon substrate binding.
Assuntos
Ácidos Graxos Dessaturases/química , Ferredoxinas/química , Oxigenases de Função Mista/química , Proteínas de Plantas/química , Eletroquímica , Elétrons , Oxirredução , SolubilidadeRESUMO
We successfully conjugated 1-2 nm diameter silicon nanoparticles to a 5'-amino-modified oligonucleotide (60mer) that contains a C6 linker between amide and phosphate groups. The conjugation was implemented via two photoinduced reactions followed by a DNA labeling step through formation of a carboxamide bond. Photoluminescence of the conjugates is dominated by two blue bands (400 and 450 nm maximal) under 340 nm excitation. The quantum yield of oligonucleotide-conjugated nanoparticles was determined to be 0.08 as measured against quinine sulfate in 0.1 M HClO(4) as a reference standard. We report a conjugation process that allows labeling of Si nanoparticles to an oligonucleotide in aqueous solutions. Ways to further optimize the procedure in order to achieve narrower and brighter photoluminescence are discussed.
Assuntos
DNA/metabolismo , Medições Luminescentes , Nanotubos/química , Silício/metabolismo , DNA/química , Silício/químicaRESUMO
Putidaredoxin (Pdx), a [2Fe-2S] redox protein of size M(r) 11,600, transfers two electrons in two separate steps from the flavin containing putidaredoxin reductase to the heme protein, cytochrome CYP101 in the P450cam catalytic cycle. It has recently come to light, through NMR measurements, that there can be appreciable differences in the Pdx conformational dynamics between its reduced and oxidized states. The redox reaction entropy, deltaS(0')rc = (S(0')Pdx(r)-S(0')Pdx(0)), as determined from measurements of the variation in formal potential with temperature, E0'(T), provides a measure of the strength of this influence on Pdx function. We designed a spectroelectrochemical cell using optically transparent tin oxide electrodes, without fixed or diffusible mediators, to measure E0'(T) over the temperature range 0-40 degrees C. The results indicate that the redox reaction entropy for Pdx is biphasic, decreasing from -213 +/- 27 J mol(-1) K(-1) over 0-27 degrees C, to -582 +/- 150 J mol(-1) K (-1) over 27-40 degrees C. These redox reaction entropy changes are significantly more negative than the changes reported for most cytochromes, although our measurement over the temperature interval 0-27 degrees C is in the range reported for other iron-sulfur proteins. This suggests that Pdx (and other ferredoxins) is a less rigid system than monohemes, and that redox-linked changes in conformation, and/or conformational dynamics, impart to these proteins the ability to interact with a number of redox partners.
Assuntos
Ferredoxinas/química , Eletroquímica/instrumentação , Entropia , Oxirredução , Soluções , Espectrofotometria , TemperaturaRESUMO
Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.
Assuntos
Cânfora 5-Mono-Oxigenase/metabolismo , Eletrodos , Compostos de Epóxi/metabolismo , Estireno/metabolismo , Cânfora 5-Mono-Oxigenase/genética , Catálise , Hidroxilação , Mutagênese Sítio-Dirigida , NAD/metabolismo , OxirreduçãoRESUMO
Electroreflectance (ER), ellipsometry, and surface-enhanced Raman spectroscopy (SERS) measurements were performed for immobilized tryptamine on polycrystalline gold electrodes. SERS study indicates that the indole ring does not interact directly with the gold surface. Ellipsometric measurements yielded a thickness of the tryptamine layer of (1.98 +/- 0.05) nm and an inferred surface concentration of 3.3 x 10(-6) mol/m2. ER measurements were performed in the wavelength region 200-300 nm using the NIST synchrotron ultraviolet radiation facility (SURF). The ER response was interpreted as a Stark shift in the tryptamine absorption line at 218 nm. Assuming that the tryptamine layer consists of two stacked tryptamine molecules associated at the indole rings, a unified analysis of ER and ellipsometric data gave the difference between the static dipole moments of the solvated ground and excited states of 0.65 x 10(-30) C m in low ionic strength buffer (0.01 M PBS) and 0.25 x 10(-30) C m in high ionic strength buffer (0.01 M PBS + 1.0 M NaClO4). The results point to the importance of the composition of the solvation shell. Semiempirical quantum chemistry calculations of tryptamine molecules gave a qualitative explanation of the ER response in the low ionic buffer. The large ER response suggests that the method could be applied to the study of the tryptophan environment in adsorbed proteins. Copyright 1998 Academic Press.
RESUMO
This paper reports measurements of the redox reaction rate for the {2Fe-2S} protein puditaredoxin (Pdx) on bare and bekanamycin-modified silver electrodes. The electron turnover rates were determined using electroreflectance (ER) and cyclic voltammetry (CV). The measurements were analyzed with two kinetic models: (1) strongly adsorbed protein layer and (2) freely diffusing protein. For the adsorbed layer model, the rates obtained with ER were almost two orders of magnitude greater than the rates obtained with CV. For the freely diffusing model, the rates determined by the two methods were similar, suggesting that this kinetic model is more appropriate. Modification of the Ag electrode with bekanamycin resulted in a negative 0.1-V shift of the Pdx formal potential, but showed only a small increase in the observed redox reaction rates. The measured rate constants were compared with the predictions of the Marcus theory of electron transfer.
RESUMO
The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor/nmol of CYP101 per min.
Assuntos
Reatores Biológicos , Cânfora 5-Mono-Oxigenase/metabolismo , Cânfora , Cânfora 5-Mono-Oxigenase/genética , Catálise , Eletrodos , Transporte de Elétrons , Desenho de Equipamento , Escherichia coli/genética , Oxirredução , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Compostos de EstanhoRESUMO
The Raman spectra of commercially available heparin disaccharide standards exhibit bands associated with the N-sulfate and the 6-O-sulfate groups of the glucosamine and the 2-O-sulfate of the iduronic acid. The N-sulfate has a strong band at 1039 cm-1. The 6-O-sulfate and the 2-O-sulfate exhibit bands at 1055 and 1065 cm-1, respectively. The pattern of these modes, which are assigned to the symmetric SO3 vibrations, was supported by semiempirical quantum mechanical calculations. The above bands were identified in the Raman spectrum of a commercial preparation of porcine mucosal heparin and were used to determine the relative proportion of the N-sulfate, 6-O-sulfate, and 2-O-sulfate groups in the heparin molecule. This information, which is complementary to that obtained by NMR spectroscopy, is of particular importance in relation to biological activity. This study also extends the usefulness of Raman spectroscopy to include structural details required for the quality assurance of pharmaceutical preparations of heparin.
Assuntos
Anticoagulantes/química , Heparina/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Dissacarídeos/química , Dados de Sequência Molecular , Análise Espectral Raman , SuínosRESUMO
Nuclear magnetic resonance spectroscopy (NMR), Raman spectroscopy, dynamic light scattering (DLS), and high performance exclusion chromatography (HPEC) were used to characterize two different commercial preparations of low molecular weight (LMW) heparin, produced either by peroxide cleavage or deaminative cleavage using nitrous acid. Proton NMR showed < 2% contamination by dermatan sulfate in the material produced by deaminative cleavage using nitrous acid and < 4% for the material produced by peroxide cleavage. The Raman spectra of the nitrous acid produced material showed an equivalent amount of O-sulfation to that in the material produced by peroxide, but about a 10% reduction in the content of N-sulfated glucosamine, as expected from the deamination reaction. DLS and HPEC indicated the presence of < 0.2% of very high molecular weight/aggregate material for the peroxide preparation compared to 1% for the nitrous acid-prepared material. The weight average molecular weight (Mw) determined from HPEC was 5900 Da for the nitrous acid-prepared material and 6850 Da for the peroxide-produced material. The number average molecular weight (Mn) calculated from this data was 5200 Da for the nitrous acid preparation and 5300 Da for the peroxide-produced material. In addition, the nitrous acid-prepared material exhibited a much narrower size distribution of oligomeric species, as evidenced by the polydispersity (Mw/Mn) of 1.1 for the nitrous acid-prepared material, as compared with a value of 1.3 for the peroxide-prepared material. These studies demonstrate that significant differences between preparations of LMW heparin can be resolved using these techniques. This is of critical importance in the design of quality assurance methods.