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.

A series of experiments with male rats clearly demonstrated the hypocholesterolemic activity of dietary chitosan. On feeding a high cholesterol diet for 20 days, addition of 2 to 5% chitosan resulted in a significant reduction, by 25 to 30%, of plasma cholesterol without influencing food intake and growth. The concentration of liver cholesterol and triglyceride also decreased significantly. Plasma, but not liver cholesterol-lowering effect, was roughly comparable with that of cholestyramine. Chitosan at the 10% level further reduced plasma cholesterol, but depressed growth. Also, finer chitosan particles tended to restrain growth even at the 2% level. In rats fed a cholesterol-free diet containing 0.5% chitosan for 81 days, the concentration of serum cholesterol was the same with that of the corresponding control, but relatively more cholesterol existed as high-density lipiproteins and less as very low-density lipoproteins. Dietary chitosan increased fecal excretion of cholesterol, both exogenous and endogenous, while that of bile acids remained unchanged. There was no constipation or diarrhea. A proper supplementation of chitosan to the diet seemed to be effective in lowering plasma cholesterol.

Kinetic measurements of the interaction between an oligosaccharide and various lectins were performed using a biosensor based on surface plasmon resonance (SPR). A glycopeptide, prepared from asialofetuin and having a nearly homogeneous N-linked sugar chain, was immobilized on the surface of a sensor chip via the amino groups of its peptide moiety. The interactions of this bound glycopeptide with six lectins [Sambucus sieboldiana lectin, Maackia amurensis lectin, Aleuria uuruntia lectin, Ricinus communis agglutinin-1 20 (RCA,,,), Datura stramonium lectin (DSA) and Phaseolus vulgaris leukoagglutinating lectin] were monitored in real-time with the change in the SPR response. Of these lectins, only RCA,,, and DSA showed an increase in the SPR response, indicating that these two lectins bound specifically to the immobilized glycopeptide. The other lectins did not show any significant changes in the SPR response. These results are in good agreement with the binding specificity previously demonstrated with affinity chromatography. The association-rate constant (k,,,) and the dissociation-rate constant (k,,,,) for the glycopeptide-RCA,,, interaction were 3.4X lo5 M-' s-' and 2.1 X lop3 s-', respectively. The k,,, and kdlSs determined for DSA were S.7X105 M-' s-' and 1.3X 10-3 sp', respectively. Furthermore, the relative binding molar ratio to the glycopeptide was three times higher for RCA,,, than for DSA, suggesting that this sugar chain possesses three binding sites for RCA,,, and one for DSA. These parameters are expected to provide useful information for defining the interaction between oligosaccharides and lectins.Many aspects of the interactions between lectins and glycoproteins have been studied, especially regarding the recognition of molecules. These interactions have been used for the detection of the oligosaccharides of glycoproteins. In conventional methods, the recognition and adsorption properties of lectins with the specific sugar residues of glycoproteins are used for chromatographic purification and electrophoretic analytical techniques [l-31. However, it is not easy to elucidate the reaction mechanisms. Furthermore, for kinetic studies of the interactions between lectins and glycoproteins, it is usually necessary to label the molecules, either lectins or carbohydrates [4, 51. This has been a time-consuming step in these studies. The method introduced here requires no labeling, and the interaction phenomena of biological molecules can be monitored directly by the surface plasmon resonance (SPR).The phenomena of SPR was studied by Otto [6] and Kretschman and Raether [7], and it was used as a chemical Correspondence to Y. Shinohara,

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

No abstract

The mechanism underlying molecular recognition of lectins was elucidated by a novel solid phase binding assay system based on surface plasmon resonance. When the apparent affinities of interactions between chitooligosaccharides and wheat germ agglutinin were compared between lectin-immobilized and oligosaccharide-immobilized assay systems, the affinity constants (Ka) calculated for the former system were in good agreement with the previously reported values measured in solution. On the other hand, in the latter system, the calculated Ka could be more than 10,000 times higher than the values in solution at lower lectin concentrations. To elucidate the reason for this, we systematically investigated the effects of the oligosaccharide immobilized density and the lectin valence on the apparent affinity in the oligosaccharide-immobilized assay system. Both the apparent association (k[ass]) and dissociation rate constants (k[diss]) showed a tendency to decrease as the oligosaccharide density increased. This effect was most remarkable for the interaction possessing an extremely fast intrinsic k(ass). Oligomerization of lectin enhanced the avidity due to a significant reduction in k(diss). These phenomena could be explained by considering the nonhomogeneous conditions under which binding occurred. The reaction in a nonhomogeneous state is limited by the mass transport effect, and the effect of rebinding becomes so large that it cannot be disregarded. These findings are the first to demonstrate the importance of the mass transport effect in modulating the affinity of lectin for oligosaccharides on a solid phase surface.

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.