title: Self-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases.
abstract: A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry and reflection absorption infrared spectroscopy, respectively. When the SAM-modified gold electrode was incubated with a solution of aspartyl protease, pepsin, a decrease in both anodic and cathodic peak currents and an increase in potential difference were observed in the cyclic voltamogram of hydroquinone as a probe, whereas a coexistence of free pepstatin fragment inhibited these phenomena, indicating the specific binding of pepsin to the fragment at the exterior of the SAM. The binding rate of the enzyme to the SAM was largely dependent on the surface density of the fragment moiety in the SAM. Furthermore, when the SAM of DTUA-Pepsta(h) on a gold colloid array deposited on an amino group-modified glass plate was immersed in a pepsin solution, absorption of the glass plate at 550 nm corresponding to a localized surface plasmon resonance of the gold colloid abruptly increased and slightly red-shifted, and a further addition of pepstatin A gradually decreased the absorbance. From the increasing and decreasing profiles of absorbance, the association constant (K(assoc)) for pepsin with the fragment on the SAM was determined. Similar phenomena were observed upon immersion of the fragment-modified SAM in a solution of HIV-1 protease, suggesting a usability of the pepstatin fragment SAM for the detection and removal of the enzyme from biological fluids.
authors.affiliation: Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan. kitano@eng.toyama-u.ac.jp
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kitano
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Makino
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sumi
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D010434:Pepsin A / Q000494:pharmacology; D010436:Pepstatins / Q000737:chemistry / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
chemical_list: D010436:Pepstatins; D010446:Peptide Fragments; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C031375:pepstatin