To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only ∼12% wild-type activity as measured by kcat(amine). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60−90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.
in parallel, enabling large data-sets to be generated for statistical analysis. One of the most convenient ways of generating large numbers of identical droplets is using microfluidic devices. [2] These can be used to create static arrays and continuously flowing droplets, and a wide range of techniques are available for onchip analysis. [3,4] Microfluidic systems are therefore attracting increasing attention for applications such as studying nucleation kinetics, [5,6] for screening protein crystallization conditions and generating large protein crystals for structural analysis, [7][8][9] and for exploring polymorphism in organic [10,11] and inorganic crystal systems. [12][13][14] A range of strategies have been used to achieve on-chip crystallization. Protein crystals are often highly soluble such that precipitation can be effectively achieved by combining protein and precipitant solutions at the point of droplet formation, [6,8] or controlling water removal from the plugs of protein solution. [15] More complex strategies have also been explored to promote the formation of high quality crystals including valve-based systems, [9,16] merging of alternate droplets containing protein and precipitant, and separation of nucleation and growth events, [17] where additional protein/ precipitant is added to existing plugs downstream of the point of initial droplet formation. Crystallization of soluble organics and inorganics has also been achieved via droplet-shrinkage [18] and on-chip temperature control. [11,13] Many crystal systems that are important to industry, the environment, and biology-such as calcium carbonate, sulfate, and phosphate-are highly insoluble, however, which results in short induction times and makes on-chip study of their precipitation more challenging due to problems with device fouling. [12] While precipitation within droplets rather than single-phase systems greatly reduces channel fouling, [19] the formation of supersaturated droplets by combining cation and anion solutions using a conventional "Y-shaped" channel or equivalent inevitably leads to precipitation at this junction. This can give rise to a range of problems including the introduction of crystal seeds into droplets. These issues can be minimized, or even eliminated, using droplet fusion-where pairs of droplets are fused using special channel architectures or applied fields [20] -or direct injection strategies, where use of a "picoinjector" to add solution to flowing droplets [21,22] offers Segmented flow microfluidic devices offer an attractive means of studying crystallization processes. However, while they are widely employed for protein crystallization, there are few examples of their use for sparingly soluble compounds due to problems with rapid device fouling and irreproducibility over longer run-times. This article presents a microfluidic device which overcomes these issues, as this is constructed around a novel design of "picoinjector" that facilitates direct injection into flowing droplets. Exploiting a Venturi junction to ...
This article presents a simple and highly reliable method for preparing PDMS microfluidic double emulsion devices that employs a single-step oxidative plasma treatment to both pattern the wettability of microchannels and to bond the chips. As a key component of our strategy we use epoxy glue to define the required hydrophobic zones and then remove this after plasma treatment, but prior to bonding. This novel approach achieves surface modification and device sealing in a single process, which reduces chip preparation times to minutes and eliminates the need for unreliable coating processes. The second key element of our procedure is the maintenance of the patterned surfaces, where we demonstrate that immediate filling of the prepared device with water or the use of solventextracted PDMS vastly extends the operational lifetimes of the chips. The reliability of this technique is confirmed by generating water-in-oil-in-water (W/O/W) double emulsion droplets with controlled core/shell structures and volumes, while its versatility is demonstrated by simply using a different placement of the epoxy glue on the same chip design to create oil-in-water-in-oil (O/W/O) double emulsion droplets. Both W/O/W and O/W/O double emulsion droplets can therefore be created from the same soft-lithography mould. This simple method overcomes one of the key problems limiting the wider use of double emulsions lack of reliability while its speed and simplicity will facilitate the high-throughput production of monodisperse double emulsions. Our method is demonstrated to produce double emulsion down to 55 µm in diameter and could be readily extended to produce microfluidic chips with more complex hydrophilic and hydrophobic patterns.
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