Adsorption of bovine hemoglobin onto spherical polyelectrolyte brushes monitored by small-angle X-ray scattering and Fourier transform infrared spectroscopy

The adsorption of bovine hemoglobin (BHb) onto colloidal spherical polyelectrolyte brushes (SPBs) is studied by a combination of small-angle X-ray scattering (SAXS) and Fourier transform infrared spectroscopy (FTIR). The SPBs consist of a polystyrene core onto which long chains of poly(styrene sulfonic acid) are grafted. Hemoglobin is a tetrameric protein that disassembles at low pH's and high ionic strengths. The protein is embedded into the brush layer composed of strong polyacids. Thus, the protein is subjected to a pH and ionic strength that largely differs from the bulk solution. At low ionic strengths up to 650 mg of BHb per gram of SPB could be immobilized. The analysis of the particles loaded with protein by SAXS demonstrates that the protein enters deeply into the brush. A large fraction of hemoglobin is bound at the surface of the polystyrene core. We attribute this strong affinity to hydrophobic interactions between the protein and the polystyrene core. The other protein molecules are closely correlated with the polyelectrolyte chains. The secondary structure of the protein within the brush was studied by FTIR spectroscopy. The analysis revealed a significant disturbance of the secondary structure of the tetrameric protein. The content of alpha-helix is significantly lowered compared to the native conformation. Moreover, there is an increase of beta-sheet structure as compared to the native conformation. The partial loss of the structural integrity of the hydrophobic protein is due to hydrophobic interactions with the hydrophobic polystyrene core. Hydrophobic interactions with the phenyl groups of the poly(styrene sulfonate) chains influence the secondary structure as well. These findings indicate that changes of the secondary structure play a role in the uptake of hemoglobin into the poly(styrene sulfonate) brushes.     

Adhesion of colloidal polyelectrolyte complexes to wet cellulose

The adhesion of wet regenerated cellulose to colloidal complexes formed between carboxymethyl cellulose (CMC) and polyvinylamine (PVAm) was evaluated by measuring the force to delaminate pairs of regenerated cellulose membranes bound together with polyelectrolyte complex. The most important parameter was the surface composition of the colloidal complex particles. High delamination forces corresponded to using complexes coated with excess PVAm whereas low adhesion was observed for both CMC coated complexes and complexes in which the PVAm was replaced with polymer bearing quaternary amine groups. Adhesion with complexes was highest at pH 4 and rather insensitive to pH from 6 to 9. Finally, mild TEMPO/NaBr/NaClO oxidation of the cellulose gave much stronger adhesion when PVAm was in excess but not with the CMC rich complexes.     

Bacteriolysis by immobilized enzymes.

Bacteriolytic enzymes produced by Achromobacter lunatus were immobilized in collagen membrane. Intact bacteria such as Pseudomonas solanacearum, Xanthomonas oryzae, Staphylococcus aureus, and Pseudomonas aeruginosa were lyzed with the bacteriolytic enzyme-collagen membrane. Relative activity of the bacteriolytic enzyme-collagen membrane against Pseu. solanacearum was about 2% of that of native bacteriolytic enzymes. No difference in the optimum pH was observed between immobilized enzymes and native enzymes. The bacteriolytic enzymes in the collagen membrane were stable against sodium chloride which was an inhibitor of the native bacteriolytic enzymes. Xanthomonas oryzae and Pseu. aeruginosa were continuously lyzed by a reactor containing the rolled bacteriolytic enzyme-collagen membrane.     

Carrier-free immobilized enzymes for biocatalysis

Methods for the preparation of carrier-free insoluble enzymes are reviewed. The technology of cross-linked enzyme aggregates has now been applied to a range of synthetically useful activities. Fusion proteins are also gaining momentum because they allow a relatively selective aggregation or even a specific self-assembly of the desired enzyme activity into insoluble particles in the absence of potentially denaturing chemicals required for precipitation and cross-linking. Recycling of insoluble protein particles for multiple rounds of batchwise reaction has been demonstrated in selected biotransformations. However, for application in a fully continuous biocatalytic process, low resistance to mechanical stress and high compressibility are issues for consideration on carrier-free enzyme particles.     

Glucose analysis utilizing immobilized enzymes

The four commercial instruments that measure glucose by incorporation of immobilized reagents are described and compared. The design of the immobilized enzyme or enzymes is shown to be related to the type of instrument. Three of the instruments are of the partitioned enzyme-sensor type requiring an immobilized enzyme capable of rapid, constant flow rate when inserted in a flowing stream. Moderately high enzyme loading is required if the instrument is designed to operate in the equilibrium mode while lower enzyme loading can be tolerated in kinetic mode. Only one instrument is an enzyme electrode in which the immobilized glucose oxidase is in the immediate vicinity of the electrochemical detector. In that case the immobilized enzyme must have very high enzyme activity per unit volume, but need not have high physical durability. The design of the instrument and immobilized enzyme(s) is also affected by whether the instrument is to be used in an industrial or a clinical laboratory.     

Synthesis of l-homophenylalanine with immobilized enzymes

A bi-enzyme process for the synthesis of l-homophenylalanine (l-HPA) from N-carbamoyl-d-homophenylalanine with immobilized N-acylamino acid racemase (racemase) and immobilized N-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) was demonstrated in this study. Upon covalent immobilization on Eupergit C, the operational pH range and temperature range were markedly broadened. The broadening of the range of operation pH bridges the gap between the optimal reaction pHs of the two free enzymes and thus makes possible the utilization of both enzymes in a single reactor. Under optimal conditions, the immobilized racemase and the immobilized l-N-carbamoylase exhibited a specific activity of 0.79 U/mg protein and 2.91 U/mg protein, respectively. The immobilized racemase had a lower activity retention but a significantly higher operation stability compared to the immobilized l-N-carbamoylase. The racemization activity of the immobilized racemase remained essentially unchanged after 40 cycles; the hydrolysis activity of the immobilized l-N-carbamoylase dropped by 40% after 14 cycles. In batch operation, quantitative conversion of N-carbamoyl-d-homophenylalanine to l-HPA with immobilized enzymes was achieved. However, the low stability of the immobilized l-N-carbamoylase complicated the development of repeated-batch or continuous processes. In continuous process, a stoichiometric excess of l-N-carbamoylase was used to extend the operation time of the system. The bi-enzyme process is a promising alternative for the synthesis of l-HPA from racemate of N-carbamoyl-d,l-homophenylalanine.     

Isolation and properties of prostaglandin H synthetase immobilized in insoluble polyelectrolyte complexes

Immobilization of prostaglandin-H-synthetase (EC 1.14.99.1) of the microsomal fraction of ram vesicular gland in the microparticles of insoluble polyelectrolyte complexes by non-covalent incorporation was studied. It was shown that immobilization occurs with a high efficiency and high activity yield (40-70%). Non-specific reversible inhibition of the enzyme by the polycationic component of the complex was demonstrated. The dependencies of activity of the native and immobilized enzymes on pH, temperature and substrate (adrenaline and arachidonic acid) were studied. The immobilized enzyme had an increased thermal stability as compared to the native one; the thermoinactivation rate constant was decreased 3-7 times. The biexponential type of the curve of the rependence of activity vs. time for the native enzyme and the transformation of the curve into a simple exponential form for the immobilized enzyme were observed.     

Adsorption of RNase A on cationic polyelectrolyte brushes: a study by isothermal titration calorimetry

We present a study of the adsorption of a positively charged protein to a positively charged spherical polyelectrolyte brush (SPB) by isothermal titration calorimetry (ITC). ITC is used to determine the adsorption isotherm as a function of temperature and of salt concentration (at physiological pH 7.2). At low ionic strength, RNase A is strongly adsorbed by the SPB particles despite the fact that both the SPB particles and the protein are positively charged. Virtually no adsorption takes place when the ionic strength is raised through added salt. This is strong evidence for counterion release as the primary driving force for protein adsorption. We calculated that ~2 counterions were released upon RNase A binding. The adsorption of RNase A into like-charged SPB particles is entropy-driven, and protein protonation was not significant. Temperature-dependent measurements showed a disagreement between the enthalpy derived via the van't Hoff equation and the calorimetric enthalpy. Further analysis shows that van't Hoff analysis leads to the correct enthalpy of adsorption. The additional contributions to the measured enthalpy are potentially sourced from unlinked equilibria such as conformational changes that do not contribute to the binding equilibrium.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.     

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

Summary Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutardialdehyde according to Morris, Campbell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O₂-uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent K_m-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same K_m-value.Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier.Dedicated to Professor Dr. Dr. U. Hagen on the occasion of his 60^th birthdayDAAD-Fellow from AustriaDAAD-Fellow from South-Korea