Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins.
Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.
Yamaguchi
Satoshi
S
Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Mannen
Teruhisa
T
Zako
Tamotsu
T
Kamiya
Noriho
N
Nagamune
Teruyuki
T
D000327:Adsorption; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D015335:Molecular Probes; D009113:Muramidase / Q000032:analysis / Q000737:chemistry*; D017830:Octoxynol / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012031:Refractometry / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods; D000520:alpha-Glucosidases / Q000032:analysis / Q000737:chemistry*
D004800:Enzymes, Immobilized; D015335:Molecular Probes; D017830:Octoxynol; D009113:Muramidase; D000520:alpha-Glucosidases