Studying the interaction of alpha-gal carbohydrate antigen and proteins by quartz-crystal microbalance

The interaction of alpha-galacosyl antigen and its binding protein (anti-Gal antibody) has been studied by quartz crystal microbalance through a SAM immobilization technique which overcomes the nonspecific interactions and molecule trapping for current piezoelectric biosensors.     

Dynamic analyses on induced-fit gaseous guest binding to organic crystals with a quartz-crystal microbalance

The inclusion behavior of gaseous guest molecules in a solid apohost, an orthogonal anthracene-bis(resorcinol)tetraol (1), was investigated with a quartz-crystal microbalance (QCM). Compound 1 forms crystals composed of molecular sheets bound together by an extensive hydrogen-bonded network. An apohost of 1 was cast onto a QCM and the binding of gaseous guest molecules was followed as a function of time by observing the decrease in the oscillation frequency, which is directly related to the increase in mass. Ethyl acetate and methyl ethyl ketone were significantly included into the apohost, whereas benzene and cyclohexane were simply adsorbed onto the surface of the solid; all these guests have similar vapor pressures at 25 degrees C. On the other hand, a host analogue 2, a tetramethoxy derivative of 1, barely included these guest molecules. The inclusion amount and the rate of inclusion of ethyl acetate or methyl ethyl ketone showed a drastic increase above a threshold concentration of guests in the gas phase. Thus, the structure of the apohost changed cooperatively in order to bind guest molecules above the threshold guest concentration. This cooperativity of the binding behavior was kinetically analyzed.     

Transient kinetic studies of protein hydrolyses by endo- and exo-proteases on a 27 MHz quartz-crystal microbalance

We have compared endo- and exo-type protease reactions and characterized the enzymatic reaction mechanisms by determining all kinetic parameters (k(on), k(off), k(cat), K(d) = k(off)/k(on), and K(m) = (k(off) + k(cat))/k(on)) by following the mass change of the formation and the decay of the enzyme-substrate (ES) complex (k(on) and k(off)), and the formation of the product (k(cat)) on a 27 MHz quartz-crystal microbalance in aqueous solutions. The K(m) value was nearly equal to the K(d) value for the endo-type protease (subtilisin and alpha-chymotrypsin); however, in the case of exo-type protease (carboxypeptidase P), the K(m) value was quite different from the K(d) value, due to k(cat) > k(off).     

Enzymatic preparation of biotinylated naturally-occurring sialylglycan and its molecular recognition on a quartz-crystal microbalance

A biotinylated sialylglycan was prepared enzymatically by endo-M, and binding behavior of an SSA lectin was studied on a different coverage of a sialylglycan-immobilized 27 MHz quartz-crystal microbalance (QCM).     

Simultaneous anomalous reflection and quartz-crystal microbalance measurements of protein bindings on a gold surface

Protein bindings onto a gold surface were detected simultaneously by QCM (delta F(water)) and anomalous reflection (deltaR) of gold on the same surface in aqueous solutions; the obtained delta F(water)/deltaR values correlated with surface areas and viscosity of proteins.     

Direct monitoring of DNA cleavages catalyzed by an ATP-dependent deoxyribonuclease on a 27 MHz quartz-crystal microbalance

Kinetic studies of enzymatic DNA cleavage reactions (the enzyme binding, hydrolysis along DNA strands, and then release of the enzyme from the completely hydrolyzed ssDNA) were carried out on a 27 MHz quartz-crystal microbalance.     

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.     

Adsorption of liposomes and emulsions studied with a quartz crystal microbalance

The adsorption from phospholipid liposome solutions (1.2%) and phospholipid stabilized oil-in-water emulsions (20% purified soybean oil) with the same phospholipid liposome concentration, has been followed by means of a quartz crystal microbalance allowing the simultaneous determination of changes in resonance frequency and energy dissipation. Both the fundamental resonance frequency and the third overtone were used for following the interfacial processes. The adsorption from the liposome solution resulted in formation of a phospholipid bilayer with an additional and incomplete outer layer of liposomes. The outer layer was removed by dilution leaving a bilayer of phospholipids on the surface. The adsorption process observed from the concentrated emulsion solution was considerably more complex. A slow spreading process that also resulted in some expulsion of material from the interface followed the rapid initial adsorption of emulsion droplets. After rinsing with water a phospholipid bilayer was retained on the surface.     

Enantioselective absorption of chirally doped liquid crystalline networks studied by the use of an electronic microbalance

A polydomain cholesteric elastomer was obtained by cross-linking a nematic side-chain polysiloxane in the presence of a chiral dopant. After extraction of the chiral dopant, sorption experiments were performed, by the use of an electronic microbalance, in the presence of each enantiomer of a chiral amine molecule. The sorption kinetics corresponds to a Fickian diffusion behavior. They allowed us to determine the diffusion coefficients and to show that the doped polymer has a more pronounced affinity toward one of the enantiomers.     

In vitro selection and evaluation of RNA aptamers that recognize arginine-rich-motif model peptide on a quartz-crystal microbalance

To study RNA-peptide interactions, we performed an in vitro selection of RNA on a simple alpha-helical peptide-immobilized quartz-crystal microbalance (QCM) and evaluated the association constants (10(7) M-1) of the selected RNA to the model peptide on the same QCM plate.     

A combined electrochemical quartz-crystal microbalance probe beam deflection (EQCM-PBD) study of solvent and ion transfers at a poly

The oxidative polymerisation of the complex2,3-dimethyl-N,N'-bis-(salicylidene)butane-2,3-diaminatonick-el(II), [Ni(saltMe)], was monitored by the electrochemical quartz microbalance (EQCM) and crystal impedance techniques. Polymerisation efficiency was maintained throughout deposition of a film, which behaved rigidly, on the electrode. A combined EQCM-PBD (probe beam deflection) study of the redox process of the film exposed to a monomer-free solution of 0.1 M tetraethylammonium perchlorate (TEAP) in acetonitrile showed an electroneutrality mechanism dominated by anion movement accompanied by co-transfer of solvent above 0.8 V. The individual contributions of all the mobile species involved in the redox switching of the poly[Ni(saltMe)] film were determined quantitatively by temporal convolution analysis; the estimated solution-phase diffusion coefficient of the exchanged species was 1.24 x 10(-5) cm2s-1.     

Multilayers of charged polypeptides as studied by in situ ellipsometry and quartz crystal microbalance with dissipation

The buildup of poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces, with and without an initial layer of polyethyleneimine (PEI), was investigated and characterized by means of in situ ellipsometry and quartz crystal microbalance with dissipation. A two-regime buildup was found in all systems, where the length of the first slow-growing regime is dependent on the structure of the initial layers. In the second fast-growing regime, the film thickness grows linearly while the mass increases more than linearly (close to exponentially) with the number of deposited layers. The film refractive indices as well as the water contents indicate that the film density changes as the multilayer film builds up. The change in film density was proposed to be due to polypeptides diffusing into the multilayer film as they attach. Furthermore, the use of PEI as the initial layer was found to induce a difference in the thickness increments for PGA and PLL.     

Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy

Interfacial peptides and proteins are critical in many biological processes and thus are of interest to various research fields. To study these processes, surface sensitive techniques are required to completely describe different interfacial interactions intrinsic to many complicated processes. Sum frequency generation (SFG) spectroscopy has been developed into a powerful tool to investigate these interactions and mechanisms of a variety of interfacial peptides and proteins. It has been shown that SFG has intrinsic surface sensitivity and the ability to acquire conformation, orientation, and ordering information about these systems. This paper reviews recent studies on peptide/protein-substrate interactions, peptide/protein-membrane interactions, and protein complexes at interfaces and demonstrates the ability of SFG on unveiling the molecular pictures of complicated interfacial biological processes.     

Carbamino group formation with peptides and proteins studied by mass spectrometry

At high pH and in the presence of dissolved CO₂, the N-terminus and ε-amino groups of amino acids, peptides, and proteins can form carbamino adducts with CO₂, R-NH₂ + CO₂ ↔ R-NHCOO⁻ + H⁺. We report the first study of carbamino group formation by electrospray ionization (ESI) mass spectrometry (MS). Angiotensin II, bradykinin, substance P, and insulin have been studied. A careful optimization of the instrumental parameters was necessary to allow the transfer of the fragile adducts into vacuum for mass analysis. Particularly, dissociation of the adducts in the ion sampling process and pH changes in ESI must be minimized. With these precautions, levels of carbamino group formation of angiotensin II and bradykinin determined from mass spectra agree with those expected to be in solution, calculated from literature equilibrium constants. Thus, ESI MS can quantitatively measure ratios of carbamino adduct to total peptide concentration in solution. Values of equilibrium constants for carbamino group formation with substance P (pK_c = 4.77 ± 0.18) and insulin (pK_c = 4.99 ± 0.05) are reported for the first time.     

Buildup of polyelectrolyte multilayers of polyethyleneimine and microfibrillated cellulose studied by in situ dual-polarization interferometry and quartz crystal microbalance with dissipation

Polyethyleneimine (PEI) and Microfibrillated cellulose (MFC) have been used to buildup polyelectrolyte multilayers (PEM) on silicone oxide and silicone oxynitride surfaces at different pH values and with different electrolyte and polyelectrolyte/colloid concentrations of the components. Consecutive adsorption on these surfaces was studied by in situ dual-polarization interferometry (DPI) and quartz crystal microbalance measurements. The adsorption data obtained from both the techniques showed a steady buildup of multilayers. High pH and electrolyte concentration of the PEI solution was found to be beneficial for achieving a high adsorbed amount of PEI, and hence of MFC, during the buildup of the multilayer. On the other hand, an increase in the electrolyte concentration of the MFC dispersion was found to inhibit the adsorption of MFC onto PEI. The adsorbed amount of MFC was independent of the bulk MFC concentration in the investigated concentration range (15-250 mg/L). Atomic force microscopy measurements were used to image a MFC-treated silicone oxynitride chip from DPI measurements. The surface was found to be almost fully covered by randomly oriented microfibrils after the adsorption of only one bilayer of PEI/MFC. The surface roughness expressed as the rms-roughness over 1 microm2 was calculated to be 4.6 nm (1 bilayer). The adsorbed amount of PEI and MFC and the amount of water entrapped by the individual layers in the multilayer structures were estimated by combining results from the two analytical techniques using the de Feijter formula. These results indicate a total water content of ca. 41% in the PEM.     

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.     

Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins

RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins.     

Study of hydrogen bonding interactions relevant to biomolecular structure and function

Ab initio molecular orbital calculations were used to study hydrogen bonding interactions and interatomic distances of a number of hydrogen bonded complexes that are germane to biomolecular structure and function. The calculations were carried out at the STO-3G, 3-21G, 6-31G*, and MP2/6-31G* levels (geometries were fully optimized at each level). For anionic species, 6-31 + G* and MP2/6-31 + G* were also used. In some cases, more sophisticated calculations were also carried out. Whenever possible, the corresponding enthalpy, entropy, and free energy of complexation were calculated. The agreement with the limited quantity of experimental data is good. For comparison, we also carried out semiempirical molecular orbital calculations. In general, AM1 and PM3 give lower interaction enthalpies than the best ab initio results. With regard to structural results, AM1 tends to favor bifurcated structures for O? H-O and N? HO types of hydrogen bonds, but not for hydrogen bonds involving O-H? S and S-H? O, where the usual hydrogen bond patterns are observed. Overall, AM1 geometries are in general in poor agreement with ab initio structural results. On the other hand, PM3 gives geometries similar to the ab initio ones. Hence, from the structural point of view PM3 does show some improvement over AM1. Finally, insights into the formation of cyclic or open formate–water hydrogen bonded complexes are presented. © 1992 by John Wiley & Sons, Inc.     

Electrochemically induced transformations of heteropolyanions: new electroactive metal oxide films studied by the electrochemical quartz crystal microbalance

New oxide films have been electrodeposited from [P₂Mo₁₈O₆₂]⁶⁻ by potential cycling in mildly acidic aqueous media. To obtain an adherent and persistent film, it is necessary that more than six electrons/molecule be fixed on the framework of the heteropolyanion. The film is then studied in pure supporting electrolyte. In this medium, a remarkable current increase is observed during the potential cycling. Whether the film is deposited on a glassy carbon electrode or on the gold electrode of an electrochemical quartz crystal microbalance (EQCM), exactly the same steady current increase up to a maximum is obtained in cyclic voltammetric measurements. The EQCM reveals a steady mass increase during the continuous cycling of the film in the supporting electrolyte. This behaviour is interpreted as featuring an irreversible water and electrolyte intake into the film, up to a maximum, after which the phenomena observed during reduction and oxidation processes are taken as featuring intercalation/deintercalation, respectively. This behaviour is much the same as described in the literature for WO₃ and MoO₃ bronzes, except that the present films seem very stable and have shown no tendency to dissolve or deactivate. Received: 2 December 1998 / Accepted: 26 January 1999