Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.
We have developed a convenient and effective method for biotinylation of oligosaccharides at their reducing ends. A novel biotin hydrazide having a phenyl group produced the biotin adduct of N-acetyllactosamine (LacNAc) by simple incubation at 90 degrees C for 1 h. Although the biotin adduct was obtained as a mixture of several stereoisomers, one of the isomers, cyclic beta-glycoside, became predominant upon letting the reaction mixture stand in a weakly acidic state (pH 3.5). This conversion may be very advantageous for functional analysis of oligosaccharides because natural N-linked oligosaccharides exist in the cyclic beta form. The limit of detection of labeled LacNAc in reversed-phase chromatography was 330 fmol and showed good linearity in the range from 330 fmol to 261 pmol. When this procedure was applied to complex type and high mannose type N-linked oligosaccharides, the labeled oligosaccharides were easily detected and separated by reversed-phase, gel filtration, and anion exchange chromatographies. Furthermore, these labeled oligosaccharides were able to be immobilized onto the solid phase using avidin-biotin technology and were stable enough to allow the binding assay to be performed repeatedly and under the conditions for in situ exoglycosidase digestion. These results suggest that this derivatization technique might be useful for both separation and functional analysis of oligosaccharides.
Despite high theoretical sensitivity, low-cost manufacture, and compactness potentially amenable to lab-on-a-chip use, practical hurdles have stymied the application of the quartz crystal microbalance (QCM) for aqueous applications such as detection of biomolecular interactions. The chief difficulty lies in achieving a sufficiently stable resonance signal in the presence of even minute fluctuations in hydrostatic pressure. In this work, we present a novel versatile planar sensor chip design (QCM chip) for a microliter-scale on-line biosensor. By sealing the quartz resonator along its edges to a flat, solid support, we provide uniform support for the crystal face not exposed to solvent, greatly decreasing deformation of the crystal resonator under hydrostatic pressure. Furthermore, this cassette design obviates the need for direct handling when exchanging the delicate quartz crystal in the flow cell. A prototype 27-MHz sensor signal exhibited very low noise over a range of flow rates up to 100 microL/min. In contrast, signals obtained from a conventional QCM sensor employing an O-ring-based holder were less stable and deteriorated even further with increasing flow rate. Additional control designs with intermediate amounts of unsupported undersurface yielded intermediate levels of stability, consistent with the interpretation that deformation of the crystal resonator under fluctuating hydraulic pressure is the chief source of noise. As a practical demonstration of the design's high effective sensitivity, we readily detected interaction between myoglobin and surface-bound antibody.
We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.
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