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1.
T Tsujita  J M Smaby  H L Brockman 《Biochemistry》1987,26(25):8430-8434
The physical specificity of adsorption of porcine pancreatic carboxylester lipase to mixed-lipid surfaces was examined by using films at the argon-buffer interface. They were comprised of 1-palmitoyl-2-oleoylphosphatidylcholine and triolein, 1,3-diolein, methyl oleate, oleonitrile, oleyl alcohol, or 13,16-docosadienoic acid. Under conditions where the surfaces are thermodynamically well-defined, each of these binary systems exhibits the formation of a lipid-lipid complex that is completely miscible with uncomplexed non-phospholipid [Smaby, J. M., & Brockman, H. L. (1985) Biophys. J. 48, 701-707]. Initial rates of adsorption of enzyme to the complexes were less than or equal to 5% of those measured in the absence of phospholipid and comparable to its rate of adsorption to phospholipid alone. This occurred despite there being up to 46% of the surface area occupied by non-phospholipid in the complexes. Equilibrium binding measurements were made at a composition where phospholipid-fatty acid complex was the predominant species. These showed that the low rates were due to an absence of adsorption sites relative to surfaces of fatty acid alone. With diolein or fatty acid and phospholipid, equilibrium binding was also measured at compositions intermediate between that of the complex and pure non-phospholipid. In both systems surface concentrations of enzyme varied nonideally with respect to either the mole fraction or area fraction of complex and uncomplexed diolein or fatty acid in the film. At area fractions of uncomplexed lipid of 0.35 and 0.67, dissociation constants for enzyme adsorption were increased 10-20-fold relative to pure fatty acid or diolein.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
1. A direct method for determining the binding of triated water to lipids is described. The experimental conditions were practically identical to those previously employed (1974) in the determination of the cofactor activities of a series of oleyl-lipids in reactivation of the C55-isoprenoid alcohol phosphokinase apoprotein. 2. Active cofactor lipids (dioleyl lecithin, sodium oleate, 1-monoolein, 1-monomyristin)bound between 2.3 and 5.3 nmol 3H2O per nmol lipid, whereas less than 0.14 nmol 3H2O were bound per nmol of the inactive lipids (1,2- and 1,3-diolein, triolein, oleyl alcohol, methyl oleate, cholesteryl oleate). 3. When exposed to 3H2O vapour, the active lipids adsorbed between 1 and 2 nmol 3H2O per nmol lipid, whereas the inactive lipids adsorbed less than 0.1 nmol 3H2O per nmol lipid. 4. The active lipid cofactor, egg lecithin, bound more than twice as much 3H2O as egg phosphatidylethanolamine which was devoid of cofactor activity in the absence of detergent. 5. Appropriately hydrated lipid polar groups are concluded to be required for an alignment with polar amino acid side chains of the enzyme apoprotein in the formation of a mixed micellar lipoprotein complex. The enzyme reaction might occur at the resulting lipoprotein/water interface.  相似文献   

3.
The lipase-catalyzed exchange of the carboxyl oxygens of 13,16-cis,cis-docosadienoic acid (DA) was studied in the presence of a nonsubstrate matrix lipid, 1-palmitoyl-2-oleoylphosphatidylcholine. For mixed lipid films at the argon-water interface exposed to pancreatic carboxylester lipase (EC 1.1.1.13), the extent of oxygen exchange showed an abrupt increase as the abundance of DA in the interface was increased from 0.5 to 0.6 mole fraction. This compositional range was independent of the level of enzyme used and of the surface pressure, i.e., lipid packing density, of the film. Concomitant with the transition was a change in the apparent mechanism of exchange from coupled to random sequential. Like the extent of oxygen exchanged, the shift in mechanism was independent of all variables except the lipid composition of the interface. The absence of any chemical or physical change accompanying the exchange reaction precludes mechanistic explanations based on the generation of reaction products by the enzyme. Instead, the results suggest that the lateral distribution of DA in phosphatidylcholine-DA interfaces regulates the expression of carboxylester lipase activity and its apparent mechanism. Preliminary measurements give an average cluster size of 1825 molecules of DA when its mole fraction is 0.35. As the DA content of the interface reaches 0.5-0.6, there appears to be a lipid head-group based percolative transition in which DA becomes the continuum. Because this transition involves the lateral organization of the lipids themselves, other interfacially active enzymes may be regulated similarly.  相似文献   

4.
The miscibility of 1-palmitoyl-2-oleoyl phosphatidylcholine with triolein, 1,2-diolein, 1,3-diolein, 1(3)-monoolein, oleyl alcohol, methyl oleate, oleic acid, and oleyl cyanide (18:1 lipids) was studied at the argon-water interface. The isothermal phase diagrams for the mixtures at 24 degrees were characterized by two compositional regions. At the limit of miscibility with lower mol fractions of 18:1 lipid, the surface pressure was composition-independent, but above a mixture-specific stoichiometry, surface pressure at the limit of miscibility was composition-dependent. From the two-dimensional phase rule, it was determined that at low mol fractions of 18:1 lipids, the surface consisted of phospholipid and a preferred packing array or complex of phospholipid and 18:1 lipid, whereas, above the stoichiometry of the complex, the surface phase consisted of complex and excess 18:1 lipids. In both regions of the phase diagram, mixing along the phase boundary was apparently ideal allowing application of an equation of state described earlier (J. M. Smaby and H. L. Brockman, 1984, Biochemistry, 23:3312-3316). From such analysis, apparent partial molecular areas and hydrations for phospholipid, complex, and 18:1 lipid were obtained. Comparison of these calculated parameters for the complexed and uncomplexed states shows that the aliphatic moieties behave independently of polar head group. The transition of each 18:1 chain to the complexed state involves the loss of about one interfacial water molecule and its corresponding area. For 18:1 lipids with more than one chain another two water molecules per additional chain are present in both states but contribute little to molecular area.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Adsorption kinetics of (125)I-radiolabeled alpha-chymotrypsin at pH 8.6 was studied in a laminar regime between two walls of poly(ethyleneterephthalate) tracked films and membranes. Adsorption kinetics in the presence of solution (10 microg/mL), desorption by rinsing with buffer, and the following exchange of proteins by flowing unlabeled solution were measured. At pH 8.6, alpha-chymotrypsin is almost neutral and can be mostly removed from the film surface, contrary to positive lysozyme adsorbed at pH 7.4. Results suggest that alpha-chymotrypsin is irreversibly adsorbed in pores, while desorption and exchange occur on membrane flat faces. A method is proposed to determine adsorption kinetics in the pores. Kinetics of desorption and exchange of alpha-chymotrypsin from the film surface can be described by stretched exponential functions in the examined time domain with the same exponent, beta approximately 0.62, which does not depend also on the former adsorption duration. However, the mean residence time at the interface is about 2.5 times greater in the presence of only the buffer than that in the presence of solution. This effect could be explained by a fast exchange at the arrival of unlabeled solution for a part of the adsorbed population.  相似文献   

6.
The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.  相似文献   

7.
Adsorption of viruses to charge-modified silica.   总被引:4,自引:2,他引:2       下载免费PDF全文
The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.  相似文献   

8.
Escherichia coli RNA polymerase holoenzyme forms two-dimensional crystals when adsorbed to positively charged lipid layers at the air/water interface. Adsorption of the protein is driven by electrostatic interactions between the positively charged lipid surface and the polymerase molecule, which has a net negative charge. Crystallization is dependent on the adsorption and concentration of RNA polymerase on fluid lipid surfaces. Image analysis of electron micrographs of crystals in negative stain, which diffract to 30 A resolution, shows irregularly shaped protein densities about 100 x 160 A, consistent with the dimensions of single polymerase molecules.  相似文献   

9.
Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.  相似文献   

10.
Adsorption isotherms of BSA at the solid-water interfaces have been studied as a function of protein concentration, ionic strength of the medium, pH and temperature using silica, barium sulphate, carbon, alumina, chromium, ion-exchange resins and sephadex as solid interfaces. In most cases, isotherms for adsorption of BSA attained the state of adsorption saturation. In the presence of barium sulphate, carbon and alumina, two types in the isotherms are observed. Adsorption of BSA is affected by change in pH, ionic strength and temperature of the medium. In the presence of metallic chromium, adsorbed BSA molecules are either denatured or negatively adsorbed at the metallic interface. Due to the presence of pores in ion-exchange resins, adsorption of BSA is followed by preferential hydration on resin surfaces in some cases. Sometimes two steps of isotherms are also observed during adsorption of BSA on the solid resins in chloride form. Adsorption of BSA, beta-lactoglobulin, gelatin, myosin and lysozyme is negative on Sephadex surface due to the excess adsorption of water by Sephadex. The negative adsorption is significantly affected in the presence of CaCl2, KSCN, LiCl, Na2SO4, NaI, KCl and urea. The values of absolute amounts of water and protein, simultaneously adsorbed on the surface of different solids, have been evaluated in some cases on critical thermodynamic analysis. The standard free energies (delta G0) of excess positive and negative adsorption of the protein per square meter at the state of monolayer saturation have been calculated using proposed universal scale of thermodynamics. The free energy of adsorption with reference to this state is shown to be strictly comparable to each other. The magnitude of standard free energy of transfer (delta G0B) of one mole of protein or a protein mixture at any type of physiochemical condition and at any type of surface is observed to be 38.5 kJ/mole.  相似文献   

11.
Malate dehydrogenase was adsorbed onto monomolecular lipid films, using a multicompartment trough. The quantity of adsorbed protein and its enzymatic activity were studied with monolayers of various electrical charge densities and subphases of various electrolyte compositions. A closely packed layer of enzyme molecules was adsorbed onto negatively charged films, whereas considerably less protein was adsorbed onto neutral and positively charged monolayers. Electrolytes reduce the quantity of adsorbed protein. The adsorption was found to be irreversible even at high ionic strength. When adsorbed to uncharged lipid films the enzyme is nearly inactive, whereas negatively charged lipid headgroups enhance the specific activity of the enzyme.  相似文献   

12.
Functional similarities between colipase and apolipoprotein C-II (apoC-II) in activating lipases suggest that apoC-II may, like colipase, preferentially interact with interfaces containing the substrates and products of lipolysis. To test this hypothesis, the binding of a peptide comprising residues of the cofactor implicated in lipid binding, apolipoprotein C-II(13-56), and, to a lesser extent, apoC-II, to monomolecular lipid films was characterized. The lipids used were a diacylphosphatidylcholine, a diacylglycerol, and a fatty acid. The peptide had an affinity for the argon-buffer interface and for all lipids consistent with a dissociation constant of <10 nM. Changes in surface pressure accompanying peptide binding were comparable to those reported for native apoC-II and indicate peptide miscibility with each of the lipids tested. The capacity of the surfaces to accommodate the peptide decreased with increasing lipid concentration in the interface, indicating competition between lipid and peptide for interfacial occupancy. At a lipid acyl chain density of 470 pmol/cm2, or 35 A2 per acyl chain, a lower limit of peptide adsorption was reached with all lipids. The limiting level of adsorption to phosphatidylcholine was only 1 pmol/cm2 compared with 6;-7 pmol/cm2 for fatty acid and diacylglycerol. Similar results were obtained with apoC-II.The difference in the extent of protein adsorption to lipid classes suggests that the distribution of apoC-II among lipoproteins will depend on their lipid composition and surface pressure.  相似文献   

13.
Trichlorophenols are weak acids of high hydrophobicity and are able to transport protons across the mitochondrial membrane. Thus the proton motive force is dissipated and the ATP production decreased. In situ Fourier Transform Infrared-Attenuated Total Reflection (FTIR-ATR) experiments with 2,4,5-trichlorophenol (TCP) adsorbed to model membranes resulted in good evidence for the formation of the TCP-heterodimer. Two surfaces were examined: a dipalmitoyl phosphatidic acid (DPPA) monolayer and a planar DPPA/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. TCP was adsorbed from 1 to 3 mM solutions at pH 6.0 to the lipid layers leading to surface layers at the water/lipid interface. Difference spectra showed an effect on DPPA acyl chains even when it was covered with POPC. Time-resolved measurements revealed two distinct adsorption processes, which were assigned to TCP and its deprotonated anion (phenoxide), respectively. For DPPA/POPC bilayers, the adsorption of TCP was faster than that of its phenoxide, whereas adsorption of both species to DPPA monolayers proceeded with similar velocity. In both cases, phenoxide formation at the membrane was found to be delayed with respect to phenol adsorption. Phenoxide and phenol were retained after replacing the TCP solution with buffer. For the retained species, we estimated a phenol/phenoxide molar ratio of 1 at pH 6.0 (pKa=6.94 for TCP), demonstrating strong evidence for heterodimer formation.  相似文献   

14.
ADSORPTION OF FULVIC ACID ON ALGAL SURFACES AND ITS EFFECT ON CARBON UPTAKE   总被引:1,自引:0,他引:1  
Adsorption of Suwannee River fulvic acid (SRFA) to algal surfaces of three green algae was studied at environmentally relevant pH values (4 –7) and SRFA concentrations (5–100 mg·L 1). The influence of adsorbed SRFA on carbon uptake of Scenedesmus subspicatus Chodat was also examined. Although no adsorption was observed at neutral pH values (pH 6 and 7), at pH 4 up to 31 mg SRFA·m 2 and at pH 5 up to 4 mg SRFA·m 2 was adsorbed to the algal surfaces. Electrophoretic mobility measurements of S. subspicatus demonstrated an increase in the negative surface charge of the alga in the presence of SRFA at pH 4. The adsorbed SRFA also influenced 14C uptake in S. subspicatus; in this case, enhanced carbon uptake could be related to the amount of adsorbed SRFA. The binding of humic substances by algal surfaces was interpreted as the result of hydrogen bonding and hydrophobic interactions.  相似文献   

15.
In the study of hydrolysis of tributyrin by the lipase of Candida cylindracea, it is shown that initial rates of hydrolysis are directly proportional to the amount of enzyme adsorbed at the substrate-water interface. As a consequence of understanding the role of the physical state of the substrate in aqueous reaction media, it was hypothesized that the inclusion of synthetic (nonsubstrate) surfaces into the reaction media may enhance the hydrolysis rate of simple liquid lipids which are partly soluble in water, like triacetin. Nonpolar n-hydrocarbons having 5-11 carbon atoms were used to create interfaces in the hydrolysis of triacetin in the soluble range. All of the C(5)-C(11) hydrocarbons showed an activating effect. For quantitative evaluation of the effects of n-hydrocarbons, n-heptane was chosen as the model n-hydrocarbon. Interrelations between the reaction kinetics and adsorption of the enzyme at the n-heptane-water interface were experimentally determined by the use of the same in-line filtration device used for the tributyrin-water system. At 35 degrees C and pH 6 the relative values of the rate constants for the decomposition of enzyme-interface-substrate complexes were calculated as 12 and 1 for the tributyrin and n-heptane-triacetin systems, respectively. The nature of activation at the solvent surfaces were accounted for by a kinetic model which assumes simultaneous adsorption of enzyme and triacetin molecules at the n-heptane-water interface. Making use of the proposed model, the value of a the apparent Michaelis constant for the soluble triacetin-n-heptane system at constant n-heptane concentration, 2 vol %, was calculated as 0.044 mol/L.  相似文献   

16.
Conformational characteristics and the adsorption behavior of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133 degrees C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.  相似文献   

17.
Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

18.
Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of beta-sheet is found in these two systems. Formation of interfacial beta-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein beta-lactoglobulin and lysozyme.  相似文献   

19.
The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.  相似文献   

20.
The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.  相似文献   

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