Biomedical subjects

Hiromi Kitano

Publications and source records attributed to Hiromi Kitano.

18 recordsLinked to original sources

PubMed2006

Functionalization of single-walled carbon nanotube by the covalent modification with polymer chains.

A single-walled carbon nanotube (SWNT), which had been oxidized by incubation with a mixture of nitric acid and sulfuric acid to afford carboxyl groups at its ends, was incubated with an azo-type radical initiator carrying poly(2-methacryloyloxyethyl D-glucopyranoside) blocks at both ends (PMEGlc-initiator). Due to its high radical trapping activity, the SWNT could be coated with glycopolymers corresponding to the cloven macro-initiator (PMEGlc-SWNT). The PMEGlc-SWNT indicated a lectin (concanavalin A, Con A)-induced aggregation, and a buckey sheet composed of PMEGlc-SWNT could be used for the recovery of Con A from its aqueous solution. Furthermore, the carboxylated SWNT was also incubated with a terminal-aminated poly(N-isopropyl acrylamide) (PIPA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl salt (PIPA-SWNT). The PIPA-SWNT indicated a definite temperature-responsiveness in the turbidity of its dispersion. These methods would be promising to modify SWNT with various functional polymers.

Journal Article↗

PubMed2006

Resistance of surface-confined telomers with pendent glucosylurea groups against non-specific adsorption of proteins.

A disulfide-carrying random telomer with pendent D-glucosylurea groups (Cys-poly(glucosylureaethyl methacrylate-r-ureaethyl methacrylate), Cys-Poly(GUMA-r-UMA)) was obtained by UV-irradiation of GUMA and UMA in the presence of benzyl N,N-diethyldithiocarbamoyl (BDC) derivative which shows the abilities of initiation, transfer, and termination (iniferter). The disulfide-carrying telomer was accumulated on a gold electrode and colloidal gold-immobilized glass substrate, and the binding of various proteins to the surface of the polymer brush was examined by both cyclic voltammetry (CV) with hydroquinone as a probe, and localized surface plasmon resonance (LSPR) absorption method. The Cys-Poly(GUMA-r-UMA) brush did not show a significant non-specific adsorption of proteins such as bovine serum albumin and egg white lysozyme. Furthermore, sugar binding proteins, concanavalin A (with an affinity for mannose and glucose) and wheat germ agglutinin (with an affinity for N-acetylglucosamine), were only slightly adsorbed to the GUMA-carrying brush, which is in contrast with the prompt and distinct binding of these proteins to the brushes composed of 2-methacryloyloxyethyl D-glucopyranoside and 1-(6'-methacrylamido)hexyl-2-N-acetoamido-2-deoxy-D-glucopyranoside, respectively. The glucosylurea group-carrying telomer brush prepared here might be useful to provide a "bio-inert" surface in bio-medical fields.

Adsorption↗

PubMed2006

State of irremovable water in solid polymer films examined by fourier transform infrared spectroscopy I: poly(ethylene glycol) dimethyl ether.

The state of the sorbed water, including the water that cannot be removed by the reduced pressure and water-sorption processes, into poly(ethylene glycol) dimethyl ether (PEG-DME) film was examined by Fourier transform infrared (FT-IR) spectroscopy. The spectrum of the irremovable water could be obtained without a thermal treatment frequently used as the dehydration procedure. It was found that the irremovable water mainly existed in the crystalline region of PEG-DME film, and that its hydrogen-bonding (HB) structure differed from that of the water sorbed from the air. Moreover, the amount of water having the same HB structure as the irremovable water increased with the water contents. These findings could not be revealed by the spectrum of the sorbed water obtained by the conventional dehydration procedure. The experimental procedure examined here allowed us to investigate the true aspects of the irremovable water and the water-sorption processes.

Ethers↗

PubMed2006

Raman spectroscopic study of the structure of water in aqueous solutions of amphoteric polymers.

Methacrylic acid (MA) and [3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC) were polymerized to give amphoteric copolymers with various compositions. The structure and H-bonding of water in an aqueous solution of the copolymer were analyzed using the contours of the O-H stretching in the polarized Raman spectra. For comparison, the H-bonded network structure of aqueous solutions of homopolymers (polyMA and polyMAPTAC) was also examined. From the relative intensity of the collective band (C value) corresponding to a long range coupling of the O-H stretching in the aqueous polymer solutions, the number of H-bonds disrupted due to the presence of one monomer residue of the polymers (Ncorr) was determined. The Ncorr value for polyMA was largely positive, and with an increase in the content of the MAPTAC residue, the Ncorr value became smaller, and after passing a minimum (which was still slightly positive) at a roughly equivalent molar ratio, the Ncorr value increased again. This is in significant contrast with the larger positive Ncorr values for the homopolymers (both polyMA and polyMAPTAC), and other ordinary polyelectrolytes such as sodium polyethylenesulfonate, poly-L-lysine hydrobromide and sodium polyacrylate. Furthermore, the Ncorr value for the copolymer (MA ratio MAPTAC = 56:44) became much smaller by the neutralization of MA residues in the copolymer with sodium hydroxide, and comparable to those for neutral polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone) and zwitterionic polymers such as poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[3-sulfo-N,N-dimethyl-N-(3'-methacryloylaminopropyl)propanaminium inner salt]. The present results clearly indicate that the amphoteric polymers with comparative contents of cationic and anionic groups do not significantly disturb the H-bonded network structure of water, probably due to the counteraction of the electrostatic hydration effect by the proximity between the anionic and cationic side groups.

Journal Article↗

PubMed2006

Sensing capabilities of colloidal gold monolayer modified with a phenylboronic acid-carrying polymer brush.

A dithiolated random copolymer with pendent phenylboronic acid residues [Cys-poly(3-acrylamidophenylboronic acid-co-N,N-dimethylaminopropyl methacrylamide), Cys-poly(APBA-co-DMAPMA)] that shows the abilities of initiation, transfer, and termination (iniferter) was obtained by using a benzyl N,N-diethyldithiocarbamoyl (BDC) derivative. The obtained disulfide-carrying copolymer was accumulated on a colloidal gold-immobilized glass substrate, and the usefulness of the polymer brush as a sensing element for glycoproteins such as ovalbumin (OVA) was examined by UV-visible spectrophotometry with the help of localized surface plasmon resonance (LSPR). The sensor showed a concentration-dependent binding of OVA with a detection limit of 100 nM, and it had a very high stability at high ionic strength. The sensor chip could be used for a detection of another glycoprotein, avidin, as well. Furthermore, the binding of biotin-modified human serum albumin (biotinylated HSA) to the avidin-phenylboronic acid- (PBA-) carrying polymer brush complex and further specific binding of anti-HSA immunoglobulin G to the biotinylated HSA-avidin-PBA-carrying polymer brush ternary complex could clearly be observed. The polymer-brush-coated device examined here not only was useful as a simple sensor chip, but also is expected to open a new perspective on interfacial phenomena performed by various functional polymer brushes fixed to colloidal gold on glass substrates.

Boronic Acids↗

PubMed2005

Correlation between the structure of water in the vicinity of carboxybetaine polymers and their blood-compatibility.

The structure and hydrogen bonding of water in the vicinity of carboxybetaine homopolymer (poly[1-carboxy-N,N-dimethyl-N-(2'-methacryloyloxyethyl)methanaminium inner salt] (PolyCMB), and a random copolymer of CMB and n-butyl methacrylate, Poly(CMB-r-BMA), with various molecular weights were analyzed in their aqueous solutions and thin film with contours of O-H stretching of Raman and attenuated total reflection infrared (ATR-IR) spectra, respectively. The relative intensity of the collective band (C value) corresponding to a long-range coupling of O-H stretchings of the Raman spectra for aqueous solution of Poly(CMB-r-BMA) was very close to that for pure water, which is in contrast with the smaller C value in aqueous solution of ordinary polyelectrolytes. The number of hydrogen bonds collapsed by the presence of one monomer residue (N(corr) value) of PolyCMB and Poly(CMB-r-BMA) (CMB, 45 mol %) (M(w), 1.14 x 10(4) and 1.78 x 10(4), respectively) could be calculated from the C value. The N(corr) values were much smaller than those for ordinary polyelectrolytes and close to those for nonionic water-soluble polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone). Furthermore, a water-insoluble Poly(CMB-r-BMA) with a large BMA content (M(w) = 347 kD, CMB 27 mol %) could be cast as a thin film (thickness, ca. 10 microm) on a ZnSe crystal for the ATR-IR analyses. At an early stage of sorption of water into the Poly(CMB-r-BMA) film, the O-H stretching band of IR spectra for the water incorporated in the film was similar to that for free water, which is in contrast with the drastic change in the O-H stretching band of water incorporated in polymer films such as poly(methyl methacrylate) (PMMA) and poly(n-butyl methacrylate) (PBMA). The theoretical vibrational frequency for water molecules hydrating a betaine molecule calculated by using a density functional method supported the experimental results. The adhesion of human platelets to Poly(CMB-r-BMA) films was much less than that to PMMA and PBMA. With an increase in the content of CMB residue, the number of platelets adhered to the Poly(CMB-r-BMA) film drastically decreased and then gradually increased, probably due to the increase in the roughness of the film surface. These results suggest that the carboxybetaine monomer residues with a zwitterionic structure do not significantly disturb the hydrogen bonding between water molecules in both aqueous solution and thin film systems, resulting in the excellent blood-compatibility of the carboxybetaine polymers.

Betaine↗

PubMed2005

Inclusion of bisphenols by a self-assembled monolayer of thiolated calix[6]arene on a gold surface.

The molecular recognition of various kinds of bisphenols (BPs) by a self-assembled monolayer (SAM) of thiolated calix[6]arene on a gold electrode was examined using cyclic voltammetry (CV). Based on the inhibitory effect of BPs on the inclusion of hydroquinone (HQ) as a probe bythe surface-confined calix[6]arene, the association constants (Kassoc) of BPs with the immobilized calix[6]arene were estimated. The Kassoc values for BPs with the SAM of thiolated hexasodium calix[6]arenehexasulfonic acid (thioSCX6) were much smaller than those in the free calix[6]arene derivative systems reported previously. The order of the Kassoc values for BPs with thioSCX6 was bisphenol A (BPA) > bisphenol S (BPS) > bisphenol B (BPB) > bisphenol F (BPF). The Kassoc values for BPA and BPS with thioSCX6 were larger than that for BPB, despite the larger hydrophobicity of BPB than that of BPA and BPS. This is probably because the inclusion phenomena in this system are not simply driven by the hydrophobic interaction, but are significantly affected by several steric and structural factors with immobilization of the host. Those are (1) a decrease in flexibility of the SCX6 cavity by the formation of SAM, (2) a decrease in inclusion ability of the SCX6 by the presence of Au surface just beneath it, and (3) a repulsion of hydrophobic guests bythe presence of sulfonate groups at the top of the SAM. Moreover, the association phenomena (adsorption and desorption processes) of a bisphenol with the SCX6 SAM were examined directly by using localized surface plasmon resonance spectroscopy.

Calixarenes↗

PubMed2005

Structure of water incorporated in sulfobetaine polymer films as studied by ATR-FTIR.

The structure and hydrogen bonding of water in the vicinity of a thin film of a sulfobetaine copolymer (poly[(N,N-dimethyl-N-(3-sulfopropyl)-3'-methacrylamidopropanaminium inner salt)-ran-(butyl methacrylate)], poly(SPB-r-BMA)), were analyzed with band shapes of O-H stretching of attenuated total reflection infrared (ATR-IR) spectra. The copolymer could be cast as a thin film, of approximate thickness 10 microm, on a ZnSe crystal for the ATR-IR spectroscopy. At an early stage of sorption of water into the polymer film, the O-H stretching band of the IR spectra for the water incorporated in the film was similar to that for free water. This is consistent with the tendency for another zwitterionic polymeric material, poly[(2-methacryloyloxyethylphosphorylcholine)-ran-(butyl methacrylate)] (poly(MPC-r-BMA). It is, however, contradictory to the drastic change in the O-H stretching band for water incorporated into films of polymers such as poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate) and poly(butyl methacrylate). These results suggest that polymers with a zwitterionic structure do not significantly disturb the hydrogen bonding between water molecules incorporated in the thin films. The investigation into the blood-compatibility of both the poly(SPB-r-BMA) and the poly(MPC-r-BMA) films indicate a definite correlation between the blood-compatibility of the polymers and the lack of effect of the polymeric materials on the structure of the incorporated water.

Betaine↗

PubMed2005

Raman spectroscopic study on the structure of water in aqueous solution of alpha,omega-amino acids.

The structure and hydrogen bonding of water in an aqueous solution of various alpha,omega-amino acids were analyzed using the contours of the OH stretching in the polarized Raman spectra. From the relative intensity of the collective band (C value) corresponding to a long-range coupling of the OH stretching in the aqueous amino acid solutions, the number of hydrogen bonds disrupted due to the presence of one amino acid molecule (N(corr) value) was evaluated. The N(corr) value for glycine was slightly positive, whereas with an increase in the number of methylene groups between ammonium and carboxylate groups, the N(corr) value gradually increased. These results suggest that the species with proximal anionic and cationic groups do not disturb the hydrogen-bonded network structure of water significantly, probably due to the counteraction of the electrostatic hydration effect attributable to the anionic and cationic groups.

Amino Acids↗

PubMed2005

Self-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases.

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.

Aspartic Acid Endopeptidases↗

PubMed2005

Resistance of zwitterionic telomers accumulated on metal surfaces against nonspecific adsorption of proteins.

Telomers of N,N-dimethyl-N-(3-sulfopropyl)-3'-methacryloylaminopropanaminium inner salt (SPB), 2-methacryloyloxyethyl phosphorylcholine (MPC), and N,N-dimethyl-N-(1-carboxymethyl)-2'-methacryloyloxylethanaminium inner salt (CMB) were prepared by UV irradiation in the presence of N,N,N',N'-tetraethylthiuram disulfide and a disulfide-group-carrying iniferter (a compound which pursues initiation, chain transfer, and termination), Cys-BDC, which had been prepared by coupling N,N-diethyldithiocarbamoylmethylbenzoic acid succinimidyl ester with cystamine dihydrochloride. The telomers formed a self-assembled monolayer (SAM) on a gold electrode and a monolayer of colloidal gold deposited on a glass plate, as confirmed by the increase in potential difference (DeltaE(p)) of the voltammogram for hydroquinone as a probe using cyclic voltammetry (CV) and the increase in absorbance using localized surface plasmon resonance (LSPR) absorption spectroscopy, respectively. Nonspecific adsorption of various proteins onto the surfaces of various telomer SAMs was examined from the decrease in peak current (DeltaI) using CV and the absorption increase at 550 nm using LSPR absorption spectroscopy. The zwitterionic telomer SAM in general did not adsorb proteins significantly, suggesting the usability of zwitterionic polymer SAMs and brushes to coat various materials used in biomedical fields. The correlation between the structure of water in the vicinity of zwitterionic telomers and the resistance of the zwitterionic telomer SAMs against the nonspecific adsorption of proteins was discussed.

Absorption↗

PubMed2005

Orientational effect of surface-confined cyclodextrin on the inclusion of bisphenols.

The molecular recognition of various kinds of bisphenols (BPs) and a bisphenol A-polymer conjugate (BPA-polymer) by a self-assembled monolayer (SAM) of thiolated beta-cyclodextrin (CD) on a gold electrode was examined using cyclic voltammetry (CV). Based on the inhibitory effect of BPs on the inclusion of hydroquinone (HQ) as a probe by the surface-confined CD, the association constants (K(assoc)) of BPs with the immobilized beta-CD were estimated. The K(assoc) values for BPs with the SAM of 3-dithiobis(undecanoylamido)-3-deoxy-beta-cyclodextrin (DTUA-beta-CD) were smaller than those in the free beta-CD system reported previously. A similar tendency was obtained when 6-(lipoylamido)-6-deoxy-beta-cyclodextrin (LP-beta-CD) was used in place of DTUA-beta-CD. The K(assoc) values for all the BPs except for bisphenol B with the SAM of LP-beta-CD were always larger than those with the SAM of DTUA-beta-CD, due to a difference in the orientation of the beta-CD moiety in the SAMs. Furthermore, adsorption and desorption processes of the BPA-polymer from the surface-confined beta-CD was followed using local surface plasmon resonance spectroscopy.

Amides↗

PubMed2004

Fishing of beta-amylase with a SAM of alpha-cyclodextrin-poly- (ethylene glycol) conjugate.

Alpha-cyclodextrin (alpha-CD) with an amino group was conjugated to an alpha, omega-dicarboxylated poly(ethylene glycol) (PEG). The inhibition constant (Ki) of the alpha-CD-PEG conjugate for the catalysis by beta-amylase was larger than that of alpha-CD, due to a steric obstruction of the PEG moiety to the binding of alpha-CD moiety to beta-amylase. alpha-CD-PEG was further modified with cystamine (CD-PEG-Cys) or cysteamine methyl disulfide (CD-PEG-MDS), and the disulfide-carrying alpha-CD-PEG was accumulated on a gold surface as a self-assembled monolayer (SAM). The binding of beta-amylase to the alpha-CD-PEG SAM was followed by a decrease in cathodic peak current in the voltammogram of hydroquinone as a probe using a cyclic voltammetry (CV). The beta-amylase bound to the alpha-CD-PEG SAM was desorbed by the addition of free alpha-CD, and the ratio of desorbed beta-amylase from the SAM of alpha-CD-PEG-Cys to the total amount of the enzyme bound to the SAM was 40% whereas that from the alpha-CD-PEG-MDS SAM was 83-85%. The percentage of desorption was increased to 100% by the treatment of the alpha-CD-PEG-MDS SAM-carrying electrode with 2-hydroxyethyldisulfide prior to the immersion in the enzyme solution. Adsorption and desorption processes of beta-amylase to the surface of alpha-CD-PEG-MDS SAM were clearly observed using localized surface plasmon resonance absorption spectroscopy. The binding constant of the enzyme to the surface-confined alpha-CD-PEG was much larger than that to free alpha-CD, probably due to a large local concentration of the alpha-CD moiety on the gold surface.

Catalysis↗

PubMed2004

Sensing capabilities of colloidal gold modified with a self-assembled monolayer of a glucose-carrying polymer chain on a glass substrate.

A disulfide-carrying polymer with pendent glucose residues (poly(2-methacryloyloxyethyl D-glucopyranoside)) was obtained by using a benzyl N,N-diethyldithiocarbamoyl derivative which shows the abilities of initiation, chain transfer, and termination (iniferter). The disulfide-carrying polymer was accumulated on a colloidal Au-immobilized glass substrate, and the usefulness of the polymer as a sensing element of concanavalin A (Con A) was examined by using a UV-visible spectrophotometer with the help of surface plasmon resonance. The sensor showed a concentration-dependent specific binding of Con A with a detection limit of 1.9 nM, and furthermore, it had a very high stability at high ionic strength. The polymer-coated device examined here was not only useful as a simple biosensor chip but is also expected to expand our knowledge of interfacial phenomena by introducing various functional polymers on colloidal Au.

Adsorption↗

PubMed2004

Accumulation of phenyl boronic acid-carrying telomers on a gold surface.

By coupling two equivalents of N,N-diethyldithiocarbamoylmethylbenzoic acid succinimidyl ester with cystamine dihydrochloride, a disulfide-carrying compound (Cys-BDC) was prepared and used as iniferter (a compound which pursues initiation, chain transfer, and termination) in the telomerization of N,N-dimethylacrylamide and 3-acrylamidophenylboronic acid. The telomerization was carried out in the presence of N,N,N',N'-tetraethylthiuram disulfide under photoirradiation at 365 nm. An aqueous solution of the telomer obtained showed responsiveness to both temperature and sugars as confirmed by the turbidity measurements. The telomer formed a self-assembled monolayer (SAM) on a gold surface as confirmed by both cyclic voltammetry (CV) and ellipsometry. The recognition of sugar residues by the telomer SAM constructed on the gold electrode was detected by the CV measurements. The usefulness of the iniferter to prepare various telomer-carrying SAMs with bio-related functions was strongly suggested.

Journal Article↗

PubMed2004

Raman spectroscopic study on the structure of water in aqueous solution of zwitterionic surfactants.

The structure and hydrogen bonding of water in aqueous solutions of various surfactants were analyzed using the contours of the O-H stretching in the polarized Raman spectra. From the relative intensity of the collective band (C value) corresponding to a long-range coupling of the O-H stretching in the aqueous surfactant solutions, the number of hydrogen bonds disrupted due to the presence of one surfactant molecule (N(corr) value) was evaluated. The N(corr) value for decylsulfobetaine was slightly negative, whereas those for ordinary ionic surfactants such as sodium dodecylsulfate and dodecyltrimethylammonium chloride were large positive values. Furthermore, the N(corr) for carboxybetaine surfactant was a small positive value. These results suggest that zwitterionic surfactants do not disturb the hydrogen-bonded network structure of water significantly, probably due to the counteraction of the electrostriction effect by the proximity between the anionic and cationic groups.

Journal Article↗

PubMed2002

Catalytic effects of galactose oxidase on micelle-forming galactolipids.

Catalytic effects of galactose oxidase on the oxidation of beta-D-galactose-carrying lipids with an oligo-ethylene glycol spacer (number of ethylene glycol units (n)=1, 2, 3, 6, 9, 13, and 20) were examined. The affinity of galactose oxidase for the galactose residue in the amphiphile (estimated by the inverse of the Michaelis constant, K(m)) was much higher than those for free D-galactose and small beta-D-galactopyranosides, and dependent on the length of the ethylene glycol spacer. That is, both below and above the critical micellar concentration, the 1/K(m) values decreased with an increase in the n value. The effectiveness of the enzyme, which can be estimated by the k(cat)/K(m) value, showed the same tendency as the 1/K(m) value. These results could be attributed to the role of the nonpolar environment around the galactose residue in the binding by the enzyme. A significant enhancement of the enzymatic oxidation of galactose residue on the liposome surface was also observed.

Galactolipids↗

PubMed2002

Substrate monolayers as electrochemical sensing elements for alpha-chymotrypsin.

A disulfide, which carried two L-phenylalanyl p-nitroanilide (Phe-pNA) moieties at both ends, was prepared by the coupling of 11,11'-dithiodiundecanoic acid (DTUA) with Phe-pNA. The compound obtained (DTUA-Phe-pNA) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry, and reflection absorption spectroscopy, respectively. Incubation of alpha-chymotrypsin with the SAM-modified electrode induced both a decrease in anodic and cathodic peak currents (-DeltaIa and -DeltaIc) and an increase in potential difference (DeltaEp) in the cyclic voltammogram of potassium ferricyanide as a probe, which suggested the attack of the enzyme at the amide group between Phe and pNA groups of the SAM, resulting in the formation of an intermediate. The linear relationship between the initial rate of increase in the amount of enzyme bound to the SAM and in both DeltaIa (absolute value of the decrease in anodic peak current) and DeltaEp values was confirmed by the quartz crystal microbalance method. The binding rate of the enzyme to the Phe-pNA SAM was dependent on the surface density of the Phe-pNA group in the SAM. The alpha-chymotrypsin-induced increases in the DeltaIa and DeltaEp values were inhibited by the addition of N-acetyl-D-phenylalanine methyl ester (N-Ac-D-Phe-OMe). In contrast with alpha-chymotrypsin, trypsin did not show a significant increase in the DeltaIa and DeltaEp values upon incubation with Phe-pNA-carrying SAM. These results could be attributed to the specific attack of alpha-chymotrypsin to the amide group in the SAM. The inhibition constant for N-Ac-D-Phe-OMe in the SAM system was quite similar to that in the free substrate system, showing that the enzymatic reaction above the SAM proceeds in a similar way to that in the homogeneous solution system.

Anilides↗