Binding of a denatured heme protein and ATP to erythroid spectrin

Spectrin is a large, worm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed significant decrease in the reactivation yield, after 30 min of refolding, in presence of increasing concentrations of spectrin from that in the absence. This indicated that spectrin could bind denatured HRP and inhibit their refolding. In presence of 1 mM ATP and 10 mM MgCl(2) the spectrin binding of denatured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refolding in the presence of spectrin, pretreated with Mg/ATP, showed about 40% increase in the reactivation yield compared to the same in absence of spectrin. Fluorescence spectroscopic studies indicated binding of ATP to native spectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin was estimated to be 1.1 mM. A high affinity binding of spectrin with denatured HRP has been characterized (K(d) = 16 nM). Since these properties are similar to those of established molecular chaperone proteins, these data indicate that spectrin might have a chaperone-like function in erythrocytes.     

Effects of gramicidin-A on the adsorption of phospholipids to the air-water interface

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air-water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (H_II) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and ³¹P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the H_II phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.     

Effect of polar head groups on the activity of aspartyl protease adsorbed to lipid membranes

The proteolytic activity of an aspartyl protease of Mucor miehei was correlated with the adsorption of the protease to lipid vesicles. It was observed that the presence of phosphatidylethanolamines (PE's) in the membrane increased the enzyme activity in a 20% in the gel phase and 10% in the fluid phase. The effects of protease on the surface pressure of monolayers composed by dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPE) were measured at constant temperature as a function of the surface pressure. At low surface pressures, the major changes were induced by protease on DOPC and DMPC monolayers. However, the effect were much lower when the monolayer was composed by DMPE. The low hydration and strong head-head interaction between the phosphates and the amine groups of adjacent PE's would result in an area per molecule much lower in PE than in phosphatidylcholine (PC) in concordance with the lower penetration in PE. Protease adsorption on PE membranes increases the proteolytic activity in which condition is less susceptible to inhibition by pepstatin. However, PC's do not alter the enzyme activity being the action of inhibitor unaffected.     

(Na~++K~+)—ATP酶与磷脂膜的相互作用

本文研究了不同磷脂对兔肾外髓质(Na~++K~+)-ATP酶活性的影响、结果表明,DOPC、PG重组活性最高,用DMPC重组导致酶失活,酸性磷脂有利于维持该酶活性.DSC及自旋标记ESR实验结果示出(Na~++K~+)-ATP酶有选择地与酸性磷脂相互作用.     

Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl₂ in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Bilayer phase transitions of N-methylated dioleoylphosphatidylethanolamines under high pressure

The bilayer phase transitions of four kinds of unsaturated phospholipids with different-sized polar head groups, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-N-methylethanolamine (DOMePE), dioleoylphosphatidyl-N,N-dimethylethanolamine (DOMe2PE) and dioleoylphosphatidylcholine (DOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. DSC thermogram and light-transmittance curve for each phospholipid vesicle solution exhibited only one phase transition under ambient pressure, respectively. The light-transmittance of DOPC solution at pressure higher than 234 MPa abruptly increased stepwise at two temperatures, which corresponds to the appearance of stable subgel and lamellar gel phases under high pressure in addition to the liquid crystal phase. The constructed temperature (T)-pressure (p) phase diagrams were compared among these phospholipids. The phase-transition temperatures of the phospholipids decreased stepwise by N-methylation of the head group. The slops of the T-p phase boundary (dT/dp) of DOPE, DOMePE and DOMe2PE bilayers (0.127-0.145 K MPa-1) were found to be close to that of the transition from the lamellar crystal (or subgel; Lc) phase to the liquid crystal (Lalpha) phase for DOPC bilayer (0.131 K MPa-1). On the other hand, the dT/dp value of the main transition from the lamellar gel (Lbeta) phase to the Lalpha phase for DOPC bilayer (0.233 K MPa-1) was significantly different from that of the Lc/Lalpha transition, hence both curves intersected with each other at 234 MPa. The thermodynamic quantities associated with the phase transition of DOPE, DOMePE and DOMe2PE bilayers had also similar values to those for the Lc/Lalpha transition of DOPC bilayer. Taking into account of the values of transition temperature, dT/dp and thermodynamic quantities compared with the corresponding results of saturated phospholipids, we identified the phase transitions observed in the DOPE, DOMePE and DOMe2PE bilayers as the transition from the Lc phase to the Lalpha phase although they have been regarded as the main transition in the previous studies. The Lbeta phase is probably unstable for DOPE, DOMePE and DOMe2PE bilayers at all pressures, it exists as a metastable phase at pressures below 234 MPa while as a stable phase at pressures above 234 MPa in DOPC bilayer. The difference in phase stability among the phospholipid bilayers is originated from that in hydration structure of the polar head groups.     

Effects of GM1 on brain spectrin-aminophospholipid interactions

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure – area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin.     

Artificial chaperone-assisted refolding of chemically denatured alpha-amylase

It is now well established that alpha-cyclodextrin (alpha-CD) is a valuable folding agent in refolding processes of several denatured enzyme solutions. The refolding of Gu-HCl denatured alpha-amylase in the dilution-additive mode revealed that alpha-CD enhanced the refolding yield by 20-30% depending upon alpha-CD concentration. However, the refolding efficiency of the Gu-HCl denatured alpha-amylase through the artificial chaperone-assisted method indicated that alpha-CD enhanced the activity recovery of denatured alpha-amylase by almost 50% and also increased the reactivation rate constant relative to the unassisted control sample. The higher refolding efficiency should be due to different mechanism played by alpha-CD in this technique. In addition, our data indicated that higher refolding yields are obtained when the residual Gu-HCl concentration is low in the refolding environment and when the capture agent is removed not in a stepwise manner from the protein-detergent complexes in the stripping step of the whole process. Collectively, the results of this investigation expand the range of procedural variations used to refold different denatured proteins through artificial chaperone-assisted method.     

Technical refolding of proteins: Do we have freedom to operate?

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate.     

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV–Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.     

Peroxidase immobilized on phospholipid bilayers supported on au (111) by DTT self‐assembled monolayers: Application to dopamine determination

In this work, horseradish peroxidase (HRP) was immobilized on dimyristoylphosphatidylcholine (DMPC) bilayers supported on Au (111) by dithiotreitol (DTT) self‐assembled monolayers and used as a nanostructured electrochemical biosensor to dopamine determination. The morphology of the phospholipid bilayers and the immobilization of HRP to these layers were characterized by atomic force microscopy (AFM). Square‐wave voltammetry (SWV) experiments were done to investigate the performance of the HRP‐modified electrode. The AFM images indicate that the enzyme is adsorbed at the external layer of the lipid bilayer and, although the electrical charges on the surface were not measured, the enzyme and phospholipids surface interaction occurs probably by electrostatic forces due to the pH used in the experiments. Interestingly, the present system can be used as one‐shot sensor for the rapid detection of dopamine. The analytical performance of this system was linear for dopamine concentrations from 3.3 × 10^?5 to 1.3 × 10^?3 mol L^?1 (r = 0.9997) with a detection limit of 2.0 × 10^?6 mol L^?1. Our results indicate that the use of HRP‐DMPC bilayer system may be useful not only in developing new nanostructured materials for technological purposes, but could be very useful in fundamental studies to investigate the interactions between different micro‐and macromolecules, even with soluble proteins, and lipid membranes. Biotechnol. Bioeng. 2013; 110: 374–382. © 2012 Wiley Periodicals, Inc.     

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca²⁺ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.     

Hydroimidazolone modification of human αA-crystallin: Effect on the chaperone function and protein refolding ability

AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), αA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of αA-crystallin by MGO (with 20–500 µM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of β_L- and γ-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured β_L- and γ-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of αA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound αA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of αA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the αA-crystallin responsible for the chaperone function and refolding are the same in the native αA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance αA-crystallin's ability to refold denatured proteins.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Effect of liposomal model membrane composition on immunogenicity

We have examined the effect of composition on the immunogenicity in mice of liposomal model membranes sensitized with dinitrophenyl-epsilon-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) derivatives. Neither cholesterol content nor incorporation of exogenous charged amphiphile (dicetylphosphate, stearylamine) exerted a significant influence on the in vivo anti-DNP response as measured by the appearance of direct plaque-forming cells in the spleen. Similarly, the nature of the fatty acids (saturated vs unsaturated) present in DNP-Cap-PE had no effect. In contrast, the nonpolar region of the basic phospholipids comprising the liposomal bilayers played an important role as revealed by a comparative study of model membranes prepared with beef sphingomyelin (SM), egg phosphatidylcholine (PC), and synthetic distearoyl-, dimyristoyl-, dilauroyl-, and dioleoyl-phosphatidylcholines (DSPC, DMPC, DLPC, DOPC). Thus, liposomes with a large content of phospholipids possessing a high transition temperature (e.g., beef SM, DSPC) were more immunogenic than those containing phospholipids of low transition temperature (e.g., egg PC, DOPC). This correlation held for both unsonicated and sonicated liposomes. These findings may have a bearing on the phenomenon of membrane-localized antigen expression.