10 research outputs found
Immobilization of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e on Mg2+/Al3+ Layered Double Hydroxide Particles
A novel biomaterial formed by the immobilization of the Aminopeptidase from Aeromonas proteolytica (AAP) on synthetic Mg2+ and Al3+ ion-containing layered double hydroxide (LDH) particles was prepared. Immobilization of AAP on the LDH particles in a buffered, aqueous mixture is rapid such that the maximum loading capacity, 1 × 10−9 moles of AAP/mg LDH, is achieved in a few minutes. X-ray powder diffraction of LDH samples before and after treatment with AAP indicates that the enzyme does not intercalate between the layers of LDH, but instead binds to the surface. Treatment of AAP/LDH with various amounts of salt in a buffered mixture demonstrates that between 15 and 20% of AAP can be removed from the LDH by washing the composite material in 0.2 M NaCl. However, the residual AAP remains bound to the LDH even at 1 M salt concentrations. A suspension of the AAP/LDH biomaterial in 10 mM Tricine buffered, aqueous solution (pH 8.0 and 25° C) catalyzes the hydrolysis of l-leucine-p-nitroanilide demonstrating that immobilized AAP remains available to substrate and retains its catalytic activity. Recycling experiments reveal that the AAP/LDH particles can be recovered and reused multiple times without appreciable loss of activity. This work provides the foundation for the development of materials that will function in the degradation or detection of peptide hormones or neurotoxins
Exploring the pH Dependence of L-leucine-p-nitroanilide Cleavage by AminopeptidaseAeromonasProteolytica: A Combined Buffer-Enzyme Kinetics Experiment for the General Chemistry Laboratory
The concepts of weak acids and bases, and buffers are often difficult ones for general chemistry students to grasp and appreciate. In order to assist our students in learning these concepts and understanding their significance in nature and research, we have developed a two-lab period experiment that combines the preparation and characterization of buffer solutions followed by their use in conducting a simple study of the effect of pH on the activity of an enzyme. Through this experiment, students gain experience with pH titrations, analyzing acid/base titration curves, preparing buffer solutions, and conducting colorimetric assays of an enzyme using a spectrophotometer. Though the experiment described here is geared for general chemistry students, it is readily expanded for use in an upper-level biochemistry class by examining other factors that affect the activity of the enzyme including the presence of inhibitors
Immobilization of the Aminopeptidase from Aeromonas Proteolytica on Mg2+/Al3+ Layered Double Hydroxide Particles
A novel biomaterial formed by the immobilization of the Aminopeptidase from Aeromonas proteolytica (AAP) on synthetic Mg2+ and Al3+ ion-containing layered double hydroxide (LDH) particles was prepared. Immobilization of AAP on the LDH particles in a buffered, aqueous mixture is rapid such that the maximum loading capacity, 1 × 10−9 moles of AAP/mg LDH, is achieved in a few minutes. X-ray powder diffraction of LDH samples before and after treatment with AAP indicates that the enzyme does not intercalate between the layers of LDH, but instead binds to the surface. Treatment of AAP/LDH with various amounts of salt in a buffered mixture demonstrates that between 15 and 20% of AAP can be removed from the LDH by washing the composite material in 0.2 M NaCl. However, the residual AAP remains bound to the LDH even at 1 M salt concentrations. A suspension of the AAP/LDH biomaterial in 10 mM Tricine buffered, aqueous solution (pH 8.0 and 25° C) catalyzes the hydrolysis of l-leucine-p-nitroanilide demonstrating that immobilized AAP remains available to substrate and retains its catalytic activity. Recycling experiments reveal that the AAP/LDH particles can be recovered and reused multiple times without appreciable loss of activity. This work provides the foundation for the development of materials that will function in the degradation or detection of peptide hormones or neurotoxins
Charge Separation Related to Photocatalytic H\u3csub\u3e2\u3c/sub\u3e Production from a Ru–Apoflavodoxin–Ni Biohybrid
The direct creation of a fuel from sunlight and water via photochemical energy conversion provides a sustainable method for producing a clean source of energy. Here we report the preparation of a solar fuel biohybrid that embeds a nickel diphosphine hydrogen evolution catalyst into the cofactor binding pocket of the electron shuttle protein, flavodoxin (Fld). The system is made photocatalytic by linking a cysteine residue in Fld to a ruthenium photosensitizer. Importantly, the protein environment enables the otherwise insoluble Ni catalyst to perform photocatalysis in aqueous solution over a pH range of 3.5–12.0, with optimal turnover frequency 410 ± 30 h–1 and turnover number 620 ± 80 mol H2/mol hybrid observed at pH 6.2. For the first time, a reversible light-induced charge-separated state involving a Ni(I) intermediate was directly monitored by electron paramagnetic resonance spectroscopy. Transient optical measurements reflect two conformational states, with a Ni(I) state formed in ∼1.6 or ∼185 μs that persists for several milliseconds as a long-lived charge-separated state facilitated by the protein matrix
Charge Separation Related to Photocatalytic H<sub>2</sub> Production from a Ru–Apoflavodoxin–Ni Biohybrid
The direct creation
of a fuel from sunlight and water via photochemical
energy conversion provides a sustainable method for producing a clean
source of energy. Here we report the preparation of a solar fuel biohybrid
that embeds a nickel diphosphine hydrogen evolution catalyst into
the cofactor binding pocket of the electron shuttle protein, flavodoxin
(Fld). The system is made photocatalytic by linking a cysteine residue
in Fld to a ruthenium photosensitizer. Importantly, the protein environment
enables the otherwise insoluble Ni catalyst to perform photocatalysis
in aqueous solution over a pH range of 3.5–12.0, with optimal
turnover frequency 410 ± 30 h<sup>–1</sup> and turnover
number 620 ± 80 mol H<sub>2</sub>/mol hybrid observed at pH 6.2.
For the first time, a reversible light-induced charge-separated state
involving a NiÂ(I) intermediate was directly monitored by electron
paramagnetic resonance spectroscopy. Transient optical measurements
reflect two conformational states, with a NiÂ(I) state formed in ∼1.6
or ∼185 μs that persists for several milliseconds as
a long-lived charge-separated state facilitated by the protein matrix
Resolution of Electronic and Structural Factors Underlying Oxygen-Evolving Performance in Amorphous Cobalt Oxide Catalysts
Non-noble-metal,
thin-film oxides are widely investigated as promising
catalysts for oxygen evolution reactions (OER). Amorphous cobalt oxide
films electrochemically formed in the presence of borate (CoBi) and
phosphate (CoPi) share a common cobaltate domain building block, but
differ significantly in OER performance that derives from different
electron–proton charge transport properties. Here, we use a
combination of L edge synchrotron X-ray absorption (XAS), resonant
X-ray emission (RXES), resonant inelastic X-ray scattering (RIXS),
resonant Raman (RR) scattering, and high-energy X-ray pair distribution
function (PDF) analyses that identify electronic and structural factors
correlated to the charge transport differences for CoPi and CoBi.
The analyses show that CoBi is composed primarily of cobalt in octahedral
coordination, whereas CoPi contains approximately 17% tetrahedral
CoÂ(II), with the remainder in octahedral coordination. Oxygen-mediated
4<i>p</i>–3<i>d</i> hybridization through
Co–O–Co bonding was detected by RXES and the intersite <i>dd</i> excitation was observed by RIXS in CoBi, but not in CoPi.
RR shows that CoBi resembles a disordered layered LiCoO<sub>2</sub>-like structure, whereas CoPi is amorphous. Distinct domain models
in the nanometer range for CoBi and CoPi have been proposed on the
basis of the PDF analysis coupled to XAS data. The observed differences
provide information on electronic and structural factors that enhance
oxygen evolving catalysis performance
Immobilization of the Aminopeptidase from Aeromonas proteolytica
[Image: see text] A novel biomaterial formed by the immobilization of the Aminopeptidase from Aeromonas Proteolytica (AAP) on synthetic Mg(2+) and Al(3+) ion-containing layered double hydroxide (LDH) particles was prepared. Immobilization of AAP on the LDH particles in a buffered, aqueous mixture is rapid such that the maximum loading capacity, 1 × 10(-9) moles of AAP/mg LDH, is achieved in a few minutes. X-ray powder diffraction of LDH samples before and after treatment with AAP indicates that the enzyme does not intercalate between the layers of LDH, but instead binds to the surface. Treatment of AAP/LDH with various amounts of salt in a buffered mixture demonstrates that between 15 and 20% of AAP can be removed from the LDH by washing the composite material in 0.2 M NaCl. However, the residual AAP remains bound to the LDH even at 1 M salt concentrations. A suspension of the AAP/LDH biomaterial in 10 mM Tricine buffered, aqueous solution (pH 8.0 and 25 ° C) catalyzes the hydrolysis of l-leucine-p-nitroanilide demonstrating that immobilized AAP remains available to substrate and retains its catalytic activity. Recycling experiments reveal that the AAP/LDH particles can be recovered and reused multiple times without appreciable loss of activity. This work provides the foundation for the development of materials that will function in the degradation or detection of peptide hormones or neurotoxins
Initial invasive or conservative strategy for stable coronary disease
BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used
Health-status outcomes with invasive or conservative care in coronary disease
BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline