Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the synthesis of polymeric cross-linking agents, poly(glutamic acid) poly(phosphorothioates), and their use in the cross-linking and stabilization of proteins upon treatment with alkaline phosphatase. We have shown that poly(phosphorothioates) are excellent substrates of alkaline phosphatase, yielding thiolated polymers which react covalently with electrophilic groups introduced into the proteins. Three proteins of different structure and function were cross-linked using this method: calf intestinal alkaline phosphatase, glucose oxidase (Aspergillus niger), and (R)-phycoerythrin. The cross-linking of alkaline phosphatase is self-catalyzed since this enzyme catalyzes the hydrolysis of phosphates, unmasking thiolates which react with the maleimide prederivatized alkaline phosphatase. Incubation of buffered solutions of native alkaline phosphatase at 45 degreesC for 7-14 days resulted in a 35% higher loss of enzymatic activity compared to that of cross-linked enzyme. The effect of cross-linking glucose oxidase is even more notable, ranging from 800% stabilization at 37 degrees C and pH 9.0 to 3000% at 37 degrees C and pH 7.4. (R)-Phycoerythrin cross-linked with 1-3 equiv of poly(phosphorothioates) and incubated at 45 degrees C for 45 days was 20% more fluorescent than the native (R)-phycoerythrin subjected to the same conditions. The stabilizing effect of cross-linking was confirmed by comparing the rate of loss of quaternary structure of the cross-linked (R)-phycoerythrin with that of the native protein.
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PMID:Alkaline phosphatase activatable polymeric cross-linkers and their use in the stabilization of proteins. 957 14

This paper describes the employment of a novel phenoxy-substituted acridinium ester (di-ortho-bromophenyl-AE) as a chemiluminescent endpoint indicator for ligand binding assays. The reactivity of this compound is such that it is capable of generating a high-intensity chemiluminescent signal at neutral pH. Under these conditions, when present in excess, it has been used as an indicator of hydrogen peroxide generation by the action of glucose oxidase (GOx, EC 1.1.3.4) on glucose substrate. The resulting chemiluminescent signal is a long-lived glow. The magnitude of the chemiluminescent signal is directly proportional to the quantity of GOx present and has been used to measure GOx with a sensitivity of 1.8 x 10(-16) mol. In addition, this ability to monitor GOx activity has been utilized in an alkaline phosphatase (ALP, EC 3.1.3.1) amplification cascade assay. Here ALP catalyzes the formation of FAD from a prosthetogenic substrate FADP. FAD, a cofactor for a number of oxidase enzymes, then converts inactive apo-GOx to holo-GOx, the activity of which is monitored by the chemiluminescent endpoint and facilitates detection of ALP over the range 10(-15) to 4.1 x 10(-19) mol. The clinical utility of this system has been demonstrated by application to the assay of human thyrotrophin (TSH, sensitivity 0.005 mU/liter).
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PMID:Employment of a phenoxy-substituted acridinium ester as a long-lived chemiluminescent indicator of glucose oxidase activity and its application in an alkaline phosphatase amplification cascade immunoassay. 960 55

A new procedure is described to deposit paramagnetic beads on surfaces to form microscopic agglomerates. By using surface-modified beads, microscopic structures with defined biochemical activity are formed. The shape and size of agglomerates were characterized by scanning electron microscopy (SEM), and the biochemical activity was mapped with scanning electrochemical microscopy (SECM). This approach is demonstrated using beads modified with anti-mouse antibodies (Ab). After allowing them to react with a conjugate of mouse IgG and alkaline phosphatase (ALP), the beads were deposited as agglomerates of well-defined size and shape. The biochemical activity was recorded in the generation-collection SECM mode by oxidizing 4-aminophenol formed in the ALP-catalyzed hydrolysis of 4-aminophenyl phosphate at the surface of the beads. The signal height correlated with both the amount of beads present in one agglomerate and the proportion of Ab binding sites saturated with the ALP mouse IgG conjugate. The feedback mode of the SECM was used to image streptavidin-coated beads after reaction with biotinylated glucose oxidase.
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PMID:Spatially addressed deposition and imaging of biochemically active bead microstructures by scanning electrochemical microscopy. 1065 27

Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.
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PMID:Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity. 1086 46

Fluorescence microscopy was used to visualize the accumulated fluorescent product of the enzyme alkaline phosphatase to indicate where active covalently bound enzyme remained on the surface after application of a Nd: YAG laser interference pattern to a surface that was first globally derivatized with the covalently bound enzyme. The electrochemical kinetics of the same carbon fiber surface were examined through the electrogenerated chemiluminescence of Ru(bpy)(3)2+ to determine that electron-transfer sites were indeed segregated from the enzyme-binding sites. The enzyme-derivatized areas are determined to be separate and distinct from the areas of enhanced electron transfer. Two other enzymes, glucose oxidase and malic dehydrogenase, were then covalently bound to carbon fiber microelectrode surfaces in order to verify the change in detection limit of their respective cofactors, NADH or H2O2, under a variety of surface conditions. The S/N of an enzyme-modified electrode after laser interference pattern photoablation and electrocatalytic treatment is improved by more than 1 order of magnitude over that observed at an electrode that is globally enzyme modified.
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PMID:Laser interference pattern ablation of a carbon fiber microelectrode: biosensor signal enhancement after enzyme attachment. 1105 9

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.
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PMID:Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots. 1150 9

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.
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PMID:Design and characterization of immobilized enzymes in microfluidic systems. 1181 12

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.
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PMID:Patterning enzymes inside microfluidic channels via photoattachment chemistry. 1505 41

We report a new approach to the measurement of alkaline phosphatase concentration based on the use of a disposable poly(aniline) microelectrochemical transistor. The measurement is carried out in a two cell configuration in which the poly(aniline) microelectrochemical transistor operates in acid solution and is connected to the alkaline buffer solution containing the analyte by a salt bridge. Disposable microelectrochemical transistors were reproducibly fabricated by electrochemical deposition of poly(aniline) onto photolithographically fabricated gold microband arrays. Using these devices alkaline phosphatase was detected by employing p-aminophenyl phosphate as the substrate for the enzyme and using glucose and glucose oxidase to recycle the p-aminophenol generated upon enzyme catalysed hydrolysis of the phosphate. Recycling the p-aminophenol with glucose and glucose oxidase amplified the detection of alkaline phosphatase approximately tenfold. Using this approach we obtain linear calibration curves for alkaline phosphatase up to 5 nM within 70 s on single use devices.
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PMID:The measurement of alkaline phosphatase at nanomolar concentration within 70 s using a disposable microelectrochemical transistor. 1521 47

Protein adsorption on surfaces is a complex phenomenon that is described by the balance of convective/diffusive transport of the protein species to the surface and its adsorption/desorption at the surface. The extent of binding depends on a variety of factors such as protein/surface interactions, availability of binding sites, localized concentrations of protein near biomaterial surfaces and flow characteristics of the protein in that region. Factors such as time-varying flows, complex device geometries, presence of multiple competitive species, or possible denaturing of proteins when they attach to the surface make it extremely difficult to quantitatively analyze protein interactions with surfaces. Adsorption/desorption rate constants are often inferred using simplistic models which neglect mass transport and have limited use across different microfluidic systems and flow protocols. In this work, we have developed and demonstrated a fluidics-resolved model that evaluates protein adsorption, accounting for both the fluidic transport and the biochemical kinetics in complex biomicrofluidic devices. The model is valid for both flow and static conditions. An automated procedure was also developed to extract the "intrinsic" mass-transport-independent adsorption kinetic rate constants from experimental data using a least squares optimization method. The automated data extraction methodology is applied to two proteins (alkaline phosphatase and glucose oxidase) that have been brought into contact with poly(etheretherketone) and Teflon capillaries. The applicability of the procedure in analyzing flow and adsorption in complex microfluidic structures is also demonstrated.
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PMID:Fluidics-resolved estimation of protein adsorption kinetics in a biomicrofluidic system. 1526 24


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