A mix-and-click method to measure amyloid-? concentration with sub-micromolar sensitivity.
ABSTRACT: Aggregation of amyloid-? (A?) protein plays a central role in Alzheimer's disease. Because protein aggregation is a concentration-dependent process, rigorous investigations require accurate concentration measurements. Owing to the high aggregation propensity of A? protein, working solutions of A? are typically in the low micromolar range. Therefore, an ideal A? quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280?nm is frequently used to measure A? concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the A? sequence. Here we present a fluorescence method for A? quantification using fluorescamine, which gives high fluorescence upon reaction with primary amines. We show that, using hen egg white lysozyme as a standard, fluorescence correlates linearly with primary amine concentration across a wide range of fluorescamine concentrations, from 62.5 to 1000?µM. The maximal sensitivity of detection is achieved at a fluorescamine concentration of 250?µM or higher. The fluorescamine method is compatible with the presence of dimethyl sulfoxide, which is commonly used in the preparation of A? oligomers, and limits the use of absorbance at 280?nm due to its high background reading. Using aggregation kinetics, we show that the fluorescamine method gives accurate concentration measurements at low micromolar range and leads to highly consistent aggregation data. We recommend the fluorescamine assay to be used for routine and on-the-fly concentration determination in A? oligomerization and fibrillization experiments.
Project description:Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of A?40 and A?42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500?µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20-50?µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20?µM or lower. ThT at concentrations of 50?µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.
Project description:Highly fluorescent carbon dots (CDs) exhibiting molecular fluorescence were synthesized and successfully used for sensing ferricyanide based on fluorescence quenching. We conducted dialysis to purify the CDs and found that the dialysate is also fluorescent. From the mass spectra and quantum yield analyses of the dialysate, it is demonstrated that molecular fluorophores were also synthesized during the synthesis of CDs. By the comparison of fluorescence spectra between CDs and dialysate, it is established that the fluorescence emission of CDs partly originates from fluorophores that are attached to CDs' surface. The fluorescence quenching caused by ferricyanide is proved to be the overlap of absorption spectra between ferricyanide and CDs. The changes of the absorbance and fluorescence spectra are combined to enhance the detection sensitivity, and the limit of detection is calculated to be 1.7 μM. A good linear response of fluorescence-absorbance combined sensing toward ferricyanide is achieved in the range of 5-100 µM. This method is highly selective to ferricyanide among other common cations and anions, and it is also successfully applied in detecting ferricyanide in real water samples.
Project description:Light transmission aggregometry (LTA) can be performed with microtiter plates (96-well LTA). When conducting LTA, an agonist is added to platelet-rich plasma and the sample is shaken for minutes after which absorbance readings are done. Platelet aggregation is detected as decrease in absorbance. However, the classical method is cumbersome and therefore microtiter plates can be used for concomitant testing of multiple samples. Furthermore, it would be convenient to prepare the plate in advance of platelet aggregation testing. Aim: The aim of the present study was to establish a simplified 96-well LTA protocol, where plates were pre-coated with agonists and stored at -80 C until use.We developed and validated a protocol for 96-well LTA using a Victor X5 plate reader and pre-coated microtiter plates. The minimum requirement of platelet-rich plasma was 45 ?L per sample and the sample platelet count should not be below 100 x109/L. Optimal absorbance reading was 595 nm wavelengths. Platelet aggregation results were higher at 37°C than at room temperature. Platelet adherence to wells after stimulation was observed; it was not avoided by pre-coating of the wells with gelatin. A range of up to 7 concentrations for each agonist (collagen, arachidonic acid, adenosine diphosphate, thrombin receptor-activating peptide and protease-activated receptor-4) was tested concomitantly. A transient rise in platelet aggregation was observed after 2 minutes of shaking in some samples with low agonist concentration, and platelet aggregation was optimal after 10 minutes of shaking for samples with high agonist concentration. Plates could be stored at -80°C for 15 days without significant change in the platelet aggregation results.The 96-well LTA is suitable for platelet aggregation testing and a range of agonist concentrations can be concomitantly tested.
Project description:The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5'-[ beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.
Project description:Data presented in this article show the extent of the inner filter effect (IFE) in fluorescence measurements of wastewater and wastewater-impacted surface water samples. Particularly, data show the effectiveness of a commonly used method for IFE correction based on UV absorbance measurement to reinstate the linearity of the relationship between fluorescence intensities and absorbance values. Data report also the effect of nitrates in fluorescence measurements of wastewater samples. Finally, data presented in this work show the effect of total suspended solids (TSS) in the UV absorbance and fluorescence measurements of different waters. Particularly, data describe the TSS effect in fluorescence intensities acquired at different pairs of excitation-emission wavelengths, and in waters with different TSS concentration. Data of this article are related to the publication "M. Sgroi, E. Gagliano, F.G.A. Vagliasindi, P. Roccaro, Inner filter effect, suspended solids and nitrite/nitrate interferences in fluorescence measurements of wastewater organic matter, Sci. Total Environ., In press" . Raw data are available in a public repository (https://doi.org/10.17632/4zss49jycj.1).
Project description:Synthetic bacteriochlorins absorb in the near-infrared (NIR) region and are versatile analogues of natural bacteriochlorophylls. The utilization of these chromophores in energy sciences and photomedicine requires the ability to tailor their physicochemical properties, including the incorporation of units to impart water solubility. Herein, we report the synthesis, from two common bacteriochlorin building blocks, of five wavelength-tunable, bioconjugatable and water-soluble bacteriochlorins along with two non-bioconjugatable benchmarks. Each bacteriochlorin bears short polyethylene glycol (PEG) units as the water-solubilizing motif. The PEG groups are located at the 3,5-positions of aryl groups at the pyrrolic ?-positions to suppress aggregation in aqueous media. A handle containing a single carboxylic acid is incorporated to allow bioconjugation. The seven water-soluble bacteriochlorins in water display Qy absorption into the NIR range (679-819 nm), sharp emission (21-36 nm full-width-at-half-maximum) and modest fluorescence quantum yield (0.017-0.13). Each bacteriochlorin is neutral (non-ionic) yet soluble in organic (e.g., CH2Cl2, DMF) and aqueous solutions. Water solubility was assessed using absorption spectroscopy by changing the concentration ?1000-fold (190-690 µM to 0.19-0.69 µM) with a reciprocal change in pathlength (0.1-10 cm). All bacteriochlorins showed excellent solubility in water, except for a bacteriochlorin-imide that gave slight aggregation at higher concentrations. One bacteriochlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The antibody conjugate of B2-NHS displayed a sharp signal upon ultraviolet laser excitation (355 nm) with NIR emission measured with a 730/45 nm bandpass filter. Overall, the study gives access to a set of water-soluble bacteriochlorins with desirable photophysical properties for use in multiple fields.
Project description:Tetramethylrhodamine methyl ester (TMRM) is a fluorescent dye used to study mitochondrial function in living cells. Previously, we reported that TMRM effectively labeled mitochondria of neurons deep within mouse brain slices. Use of micromolar concentration of dye, which was required to get sufficient staining for two-photon imaging, resulted in typical fluctuations of TMRM. With prolonged exposure, we recorded additional responses in some neurons that included slow oscillations and propagating waves of fluorescence. (Note: We use the terms "fluctuation" to refer to a change in the fluorescent state of an individual mitochondrion, "oscillation" to refer to a localized change in fluorescence in the cytosol, and "wave" to refer to a change in cytosolic fluorescence that propagated within a cell. Use of these terms does not imply any underlying periodicity.) In this report we describe similar results using cultured rat hippocampal neurons. Prolonged exposure of cultures to 2.5 µM TMRM produced a spontaneous increase in fluorescence in some neurons, but not glial cells, after 45-60 minutes that was followed by slow oscillations, waves, and eventually apoptosis. Spontaneous increases in fluorescence were insensitive to high concentrations of FCCP (100 µM) and thapsigargin (10 µM) indicating that they originated, at least in part, from regions outside of mitochondria. The oscillations did not correlate with changes in intracellular Ca(2+), but did correlate with differences in fluorescence lifetime of the dye. Fluorescence lifetime and one-photon ratiometric imaging of TMRM suggested that the spontaneous increase and subsequent oscillations were due to movement of dye between quenched (hydrophobic) and unquenched (hydrophilic) compartments. We propose that these movements may be correlates of intracellular events involved in early stages of apoptosis.
Project description:Protein aggregation into insoluble fibrillar aggregates is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Commonly used methods to study aggregation inhibition or fibril destabilization by potential drugs include spectroscopic measurements of amyloidophilic dye molecule fluorescence or absorbance changes. In this work we show the cross-interactions of five different dye molecules on the surface of insulin amyloid fibrils, resulting in cooperative binding and fluorescence quenching.
Project description:Critical aggregation concentration (CAC) of surfactants is lowered when polyelectrolytes act as counterions. At a concentration in between the CACs of the surfactant and the polymer-surfactant complex, protein-induced disassemblies can be achieved. This is because, when proteins competitively bind to the polyelectrolytes, the surfactants are not capable of sustaining a micelle-type assembly at this concentration. Since these amphiphilic aggregates are capable of noncovalently sequestering hydrophobic guest molecules, the protein binding induced disassembly process also results in a guest release from these assemblies. We show here that the change in fluorescence with different proteins is dependent not only on the nature of the polymer-surfactant complex, but also on the fluorescent transducer. Two processes can be responsible for the observed fluorescence change: fluorophore guest release from the hydrophobic interior of the assembly and excited state quenching due to complementary components in the analyte. The latter mechanism is especially possible with metalloproteins. We show here that an excited state quenching is possible at nanomolar concentrations of the proteins, while the disassembly based fluorescence reduction is the dominant pathway at micromolar concentrations.
Project description:The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his40 and his57 ) quickly and completely, with an IC50 of 35?±?6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation.