Binding of β-secretase to a peptide inhibitor-carrying SAM

A novel disulfide, which carried a specific inhibitor for β-secretase (KMI360) at both ends, was prepared by the coupling of 11,11′-dithiobisundecanoic acid (DTUA) with the inhibitor. The compound obtained (DTUA–KMI360) formed a self-assembled monolayer (SAM) on a gold electrode as proven by cyclic voltammetry (CV) using hydroquinone as a probe. Furthermore, DTUA–KMI360 could be accumulated as a SAM on a gold colloid deposited on a glass plate (Au colloid-glass chip) as proven by both the red-shift and the increase in absorbance of the gold colloid corresponding to localized surface plasmon resonance (LSPR). When the SAM-modified Au colloid-glass chip was immersed in a solution of aspartyl proteases, pepsin and β-secretase, the absorbance of the chip at 550 nm corresponding to LSPR of the gold colloid further increased and was slightly red-shifted, whereas coexistence of a free inhibitor obstructed these phenomena. Adsorption of the enzymes was promoted by the incorporation of a zwitterionic group into the SAM, while non-specific adsorption to the mixed SAM was significantly reduced. The optimal ratio of ω-zwitterionic alkanethiol, 3-[(6-mercaptohexyl)-N,N-dimethylamino]propane-1-sulfonic acid (C6-SPB), and DTUA–KMI360 in the SAM for the binding of enzymes was found to be DTUA–KMI360:C6-SPB = 1:11 using polarization modulation infrared reflection absorption spectroscopy (PM-IR-RAS). From increasing profiles of absorbance of the Au colloid-glass chip, the association constant (Kassoc) for pepsin with the inhibitor on the SAM was determined, whereas that for β-secretase could not be due to the strong binding of the enzyme to the inhibitor, resulting in the absence of the dissociation process. The results suggested that the SAM of the enzyme inhibitor can be used for both investigation of enzymes and removal of target enzymes from biological fluids.