Studies of Peroxidase Refolding in the Presence of Specific Antibodies

A panel of eight monoclonal antibodies raised against horseradish root peroxidase was assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine chloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) oxidation. The yield of the active enzyme in the course of the refolding was increased by 1.5–1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.     

Antiperoxidase antibodies enhance refolding of horseradish peroxidase

The effect of monoclonal antibodies on protein folding was studied using horseradish peroxidase refolding from guanidine hydrochloride as a model process. Among the five antiperoxidase clones tested, one was found to increase the yield of catalytically active peroxidase after guanidine treatment. The same clone also increased the activity of the native peroxidase by a factor of 2-2.5. While peroxidase refolding under standard conditions resulted in the recovery of only 7-8% of the initial catalytic activity, antibody-assisted refolding increased the yield to 50-100% (or 20-40% from the activity of native enzyme with antibodies). Kinetics of autorefolding and antibody-assisted refolding differed significantly. In the course of autorefolding the catalytic activity was recovered within the first 2.5 min and did not change further within a 2.5- to 60-min interval, whereas in the course of antibody-assisted refolding maximal catalytic activity was attained only in 60 min. The yield of active peroxidase for the antibody-assisted refolding depended linearly on the antibody concentration. The observed effect was strongly specific. Other antiperoxidase clones tested as well as nonspecific antithyroglobulin antibody affected neither kinetics, no the yield of peroxidase refolding.     

Expression and Refolding of Tobacco Anionic Peroxidase from E. coli Inclusion Bodies

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.     

Role of protein-solvent interactions in refolding: effects of cosolvent additives on the renaturation of porcine pancreatic elastase at various pHs

The effects of cosolvent additives on the refolding of porcine pancreatic elastase were studied by comparing the enzymatic activity and the conformation of the enzyme renatured at various pHs with those of the native elastase under the same cosolvent and pH conditions. A lag period was observed before reaching the steady state of the hydrolysis of an amide substrate, and the lag period measured with the refolding enzyme was longer than that measured with the native elastase. Depending on the cosolvent studied (acetonitrile, dimethylsulfoxide, glycerol, methanol) there was or was not a dramatic increase in the duration of the lag period measured with the refolding enzyme, but not in the case of the native elastase. These results and additional kinetic data on inactivation of the enzyme demonstrated that dimethylsulfoxide, glycerol, and methanol enhance the stability of the intermediates able to refold into the native form, contrary to acetonitrile. In neither the case of the native enzyme nor that of the renatured enzyme, did the cosolvents modify the pK(app) of ionization of the amino acids that control enzymatic activity, indicating that they did not penetrate the core of the refolded elastase. Conversely, they shifted toward a more alkaline pH the structural transition of the native elastase, and the amplitude of the shift was comparable to that observed in bulk water with elastase whose Ser 195 has been acylated, suggesting that cosolvents stabilized the structure of the folded molecule by increasing its packing.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Characteristics of antigen-antibody interaction in the reversed micelles of surface-active agents in heptane

The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Monoclonal antibody epitope mapping describes tailspike beta-helix folding and aggregation intermediates

There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.     

The role of detergent in refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus Chinensis: the solubilization of aggregate and refolding in detergent solutions.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.     

Catalytic activity of steroid conjugates of various types of peroxidases in aqueous and micellar media

The horseradish peroxidase (HRP) conjugates with the sheep antirabbit antibodies and cortisol (COR) or progesterone (PROG) containing 9 to 40 steroid molecules per HRP molecule were synthesized. In aqueous media all the conjugates have lower catalytic activity in the o-phenylenediamine oxidation than the native enzyme. In reversed Aerosol OT micelles in heptane the HRP-COR and HRP-PROG conjugates containing 12 and 9 steroid molecules, respectively, have catalytic constants 2.6 and 2.7 times higher than the unmodified enzyme. The influence of the HRP hydrophobisation and its inactivation on the course of modification on catalytic properties of the enzyme is discussed.     

Spermine stabilization of folded ribonuclease T1

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form.     

Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase.

We have located the epitopes recognized by four different monoclonal antibodies which bind to partially unfolded creatine kinase (CK) (ATP: creatine N-phosphotransferase, EC 2.7.3.2) but not to the native enzyme. The epitopes appear to be buried within the CK structure in its native, proteinase-resistant, state. When the epitopes are made accessible to antibody by mild denaturation, CK becomes enzymically-inactive and can be cleaved by proteinase V8 into two large fragments which retain the epitopes and may represent domains. Epitopes on each V8 fragment are associated with highly conserved sequences and are brought physically close to the active site of the enzyme during the later stages of CK refolding and reactivation. The results suggest a catalytic site formed at the interface between two domains which carry the epitopes on their interacting surfaces. Separation of loosely associated domains before or during immunization may account for the origin of antibodies against buried epitopes.     

Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.     

Horseradish peroxidase renaturation is less efficient at lower protein concentrations

Renaturation of horseradish peroxidase from guainidine hydrochloride has been studied. Although refolding of the secondary structure was complete, only partial heme incorporation and recovery of enzymatic activity were observed. Heme capturing became less efficient at lower peroxidase concentrations: the refolding yield decreased from 60% at 1 microM to 10% at 0.1 microM concentration of the protein. Probing with conformation-sensitive antibodies indicated structural differences between peroxidase refolded at low concentration and the holo-enzyme.     

Chemical modification of epsilon-amino lysine groups in horseradish peroxidase. Its effect on catalytic properties and spatial structure of the enzyme]

Effect of chemical modification of horseradish peroxidase lysine epsilon-amino groups by propionic, butyric, valeric, succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) on catalytic properties of the enzyme is investigated. All the preparations of modified peroxidase have 100% peroxidase activity for o-dianizidine at pH 7.0, which indicates the absence of lysine epsilon-amino group in the enzyme active site. pH-dependencies of modified peroxidase relative activity are studied; modification by anhydrides of monobasic acids is not found to result in changes of the relative activity pH-profile, while modification by succinic anhydride widens it. Absorption and circular dichoism spectra of native and modified peroxidase within 260--270 nm are obtained, some changes in the enzyme tertiary structure after its epsilon-amino groups modification are observed. Modification of four epsilon-amino groups by buturic and succinic anhydrides and of three epsilon-amino groups by TNBS is found to increase the regidity of protein surrounding of heme, and modification of six epsilon-amino groups by TNBS results in more unwrapped enzyme structure as compared with its native molecule.     

Immunological similarity of the cationic and the anionic peanut peroxidase isozymes

Antibodies against both the native and the deglycosylated cationic peanut peroxidase (C.PRX) were used to probe the structural relationship of this isozyme with its anionic counterpart. Not only the native but also the deglycosylated forms of the cationic and the anionic peroxidases reacted with both antibodies. The activity of the cationic isozymes was inhibited by anti-native C.PRX. Similar but nevertheless distinct immunodetection patterns resulted from reaction of the partially digested cationic and anionic peroxidase peptides with antibodies directed to the deglycosylated as well as to the native C.PRX, suggesting a similarity in their polypeptide structures.     

Refolding of the full-length non-structural protein 3 of hepatitis C virus

An easy and reproducible procedure for purification and refolding of the full-length non-structural protein 3 (NS3) from hepatitis C virus has been developed. Refolding was achieved by simply diluting the protein into a suitable buffer. Low protein concentration, high pH, highly reducing conditions, the presence of detergent, and low viscosity were important parameters for high refolding efficiency. Refolding was insignificantly affected by the presence of Zn(2+) in the refolding buffer, while the addition of NS4A cofactor inhibited refolding. A comparison of the kinetic parameters showed that the refolded enzyme is not as catalytically competent as the native enzyme. Nevertheless, the activity of the refolded NS3 protease was dependent on the specific NS4A-peptide cofactor and was inhibited by the specific substrate-based NS3 protease inhibitor, which indicates that the refolded NS3 can be appropriate for inhibitor screening. The yield of pure protein from the insoluble fraction of cell lysate was 6 mg/L of bacterial culture, which is 18 times higher than obtained from the soluble fraction. Improvement of the refolding conditions has resulted in a 50-fold higher activity of the protease as compared to refolding in buffer with neutral pH and no additives.     

Formation of a misfolded conformation during refolding of HRPA1 in the presence of calcium

Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca2+-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, ICa2+, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence. The fraction of ICa2+ increases exponentially with increasing calcium concentration. The ICa2+ state is formed during refolding after calcium binding to the unfolded state, as reconstitution of HRPA1 from its apoprotein reveals that the affinity of the apoprotein to protoporphyrin IX is higher in the presence of calcium. If calcium is added after refolding only, the majority of HRPA1 molecules retain their native conformation, thus confirming the binding of calcium to the unfolded state.     

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.     

Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization of soluble porcine ALT by chemical modification with mono- and bis-imidates. The apparent transition temperatures (‘T_m’, the temperature where 50% of initial activity was lost in 10 min) for native and DMS-modified ALT were 46 and 56 °C respectively. The effects of water-miscible organic solvents (methanol, dimethylformamide, dimethylsulphoxide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in ALT catalytic activity was observed on reaching a certain threshold concentration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V_max and K_m for 2-oxoglutarate as substrate, determined in 10% (v/v) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 °C with no organic solvent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% (v/v) solvent at 35 °C, the DMS-modified ALT's half-life was up to 4.6 times greater than native enzyme's. DMS-modified ALT was also more stable in urea and guanidine HCl, and its refolding was more noticeable, than that of native enzyme.