International Journal of Biological Macromolecules 38 (2006) 289295

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies
Mohd. Rehan, Hina Younus
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 28 December 2005; received in revised form 22 March 2006; accepted 22 March 2006 Available online 29 March 2006

Abstract Effect of six organic solventsmethanol, ethanol, propanol, dimethyl sulphoxide (DMSO), N,N-dimethyl formamide (DMF), and glycerol on the conformation and interaction of catalase and anticatalase antibodies were studied with the aim of identifying the solvents in which antigenantibody interactions are strong. The antigen binding activity of the antibodies in the various organic solvents increased in the following order: ethanol < methanol < no organic solvent < propanol < DMSO < DMF < glycerol. The structure of both the antibody and the antigen molecule was affected signicantly in 40% concentration of the organic solvents used in this study. Catalase activity was inhibited in DMSO. However, the enzyme was activated in DMF upto about 50% of its concentration. 2006 Elsevier B.V. All rights reserved.
Keywords: Organic solvents; Antigenantibody interaction; Conformation; Catalase

1. Introduction The ability of various organic solvents to interfere with the physico-chemical properties of proteins is well known [14]. The effects of such solvents are generally attributed to alteration of various non-covalent interactions in the protein, solvation of ionic groups and dipoles, hydrogen bonding and hydrophobic interactions. The conformation that a protein attains in a solvent depends on the ratio of hydrophobic and hydrophilic regions on its surface. For the stabilization of the native protein structure, this ratio should have a certain value. Changes in this ratio cause a rearrangement of hydrogen bonds that results in conformational changes of the whole molecule [5,6]. Some organic solvents have been used earlier to change the conformation of protein molecules for studying protein folding/unfolding. For example, triuoroethanol was used to induce formation of -helices in various proteins such as -lactoglobulin [7], porin [8] and tumor necrosis factor [9]. The alcohol-induced denaturation of some

Abbreviations: DMSO, dimethyl sulphoxide; DMF, N,N-dimethyl formamide; HRP, horse rabbit peroxidase; TMB, tetramethyl benzidine; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffer saline; CD, circular dichroism Corresponding author. Tel.: +91 571 2720 388; fax: +91 571 272 1776. E-mail address: hinayounus@rediffmail.com (H. Younus). 0141-8130/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.03.023

proteins e.g. cytochrome c results in the appearance of a partially structured state resembling the molten globule [10]. The addition of DMSO into the medium for the renaturation of lysozyme increases the rate of production of native like structures and formation of the nal native state [11]. It is widely accepted, however, that the catalytic efciency of enzymatic reactions in organic media is comparable to, and in some cases higher, than that displayed in aqueous media, as long as the enzymes are surrounded by a thin lm of water, which is necessary for the retention of their catalytic activity [3,4,12]. Thus organic phase enzyme electrodes have been developed for use in organic phases [13]. The operation of such devices can offer several advantages, such as extended concentration range, prevention of undesirable side reactions, improved operational stability and simplied immobilization techniques. In contrast to the above mentioned studies, there are only a few investigations on the behaviour of antibodies in organic solvents [1417]. It was demonstrated that the binding of a hapten, 4-aminobiphenyl, to its monoclonal antibody was strong and specic in such solvents [14]. The inhibition of binding between testosterone and its antibody in a variable range of water miscible solvents was found not to be correlated with some solvent properties, such as dielectric constant, polarity index and dipole moment, but was found to be inversely correlated to the molecular mass of the solvent [15]. A study on the effect of nine organic

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solvents on lysozyme-antilysozyme precipitin reaction showed that these solvents destabilize the antigenantibody complex [18]. Data on functional activation of protein molecules after their structural perturbation are very scarce [17,19]. Several proteins are extracted and utilized in organic solvents [2023]. However, studies on the conformation of proteins in organic solvents are rare. In the present study, the effect of six organic solvents (methanol, ethanol, propanol, DMSO, DMF and glycerol) on the conformation and interaction of antigen and the antibody molecule were studied. The aim was to identify the solvents in which antigenantibody interactions are strong. Such solvents may be utilized in immunoassays for the identication of trace amounts of antigen/antibody. Catalase was chosen as the test antigen because its activity may be easily monitored and its activity is reasonably stable in both aqueous and organic solvents [4,24]. The concentration of the organic solvents used was that which is generally employed for isolation and during chemical modication of proteins [25,26]. 2. Materials and methods 2.1. Materials Bovine liver catalase [9001-05-2] (EC 1.11.1.6) was purchased from Sigma (St. Louis, MO). It was homogenous on the basis of size and was used in the experiments without further purication. H2 O2 , methanol, ethanol, DMSO, DMF were obtained from Qualigens ne chemicals, India. Glycerol and propanol were supplied by Sisco Research Lab., India. Microtitre plates were purchased from Granier, USA. Horse rabbit peroxidase (HRP) conjugated goat anti-rabbit IgG and tetramethyl benzidine (TMB)/H2 O2 were the product of Genei laboratories, India. All the other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Assay for catalase activity A standard curve for determining the concentration of H2 O2 was constructed by taking increasing concentration of H2 O2 in a series of tubes in 1 ml of 20 mM sodium phosphate buffer, pH 7.0. Then 2 ml of dichromate/acetic acid reagent (5% potassium dichromate and acetic acid in the ratio of 1:3) was added to each tube and they were incubated at 100 C for 10 min. A green solution of chromic acetate appeared, whose absorbance was measured at 570 nm. A standard curve of the absorbance at 570 nm versus concentration of H2 O2 was plotted. The activity of catalase was measured using the above reaction mixture. Both H2 O2 and the enzyme were taken in 1 ml volume of the sodium phosphate buffer, pH 7.0. The reaction was started by the addition of the enzyme, followed by incubation at 30 C for 5 min. The degradation of H2 O2 was measured using the above standard curve. 2.2.2. Catalase activity measurement in organic solvents Catalase activity was determined in increasing concentration of DMSO or DMF (060%). The standard reaction mixture con-

sisted of H2 O2 , the enzyme, and the appropriate concentration of DMSO or DMF in a total volume of 1 ml of sodium phosphate buffer, pH 7.0. The degradation of H2 O2 was measured as described above. 2.2.3. Immunization of rabbits Healthy rabbits were injected subcutaneously with 500 g of catalase using freunds adjuvant. The animals were boosted on day 21 and subsequently bled after a week for monitoring the production of catalase specic antibodies. 2.2.4. Enzyme-linked immunosorbent assay (ELISA) The generation of catalase specic antibodies was measured in the sera of catalase immunized rabbits by the ELISA. Ninetysix well microtitre plates were coated overnight with 100 l of catalase (10 g/ml) in 0.05 M bicarbonate buffer, pH 9.6 at 4 C. After extensive washing with phosphate buffer saline (PBS)Tween 20, 100 l of blocking buffer (5% skimmed milk in PBS-Tween 20) was applied to the wells and the plates incubated at 37 C for 2 h. After removal of the blocking buffer, serially diluted test and control sera were added and binding allowed to proceed at 37 C for 2 h. The microtitre plates were washed and incubated with 100 l of HRP conjugated goat anti-rabbit IgG at 37 C for 1 h. After the usual washing steps, the peroxidase reaction was initiated by the addition of the substrate TMB/H2 O2 , arrested by the addition of 4.0 N H2 SO4 and absorbance measured at 492 nm in an ELISA reader. Catalase was immunogenic and readily elicited the formation of antibodies in rabbits. 2.2.5. Purication of antibodies The rabbit serum samples that exhibited a good titre of anticatalase antibodies were saturated with ammonium sulphate to 40% concentration. The precipitate thus obtained was separated out by centrifugation at 5000 rpm for 15 min and dissolved in minimal volume of sodium phosphate buffer (20 mM, pH 7.0) and then was dialyzed against the same buffer. The antibodies were further puried by ion exchange chromatography on DEAE-cellulose matrix equilibrated with the same buffer. The impurities bound to the matrix while pure IgG was left in the supernatant. The purity of the IgG was ascertained by SDSPAGE. Two bands were visible in the SDS-PAGE of IgG and their molecular mass corresponded to those of heavy (50 kda) and light chain (25 kda) of IgG. The cross-reactivity of the puried IgG with catalase was also proved by ELISA. 2.2.6. Measurement of antigenantibody interactions in organic solvents by ELISA Ninety-six well microtitre plates were coated overnight with 100 l of catalase (10 g/ml) in 0.05 M bicarbonate buffer, pH 9.6 at 4 C. After extensive washing with PBS-Tween 20, 100 l of blocking buffer (5% skimmed milk in PBS-Tween 20) was applied to the wells and the plates incubated at 37 C for 2 h. Meanwhile, anticatalase IgG was incubated in 40% organic solvents (methanol, ethanol, propanol, DMSO, DMF, and glycerol) for 1 h at room temperature. After removal of blocking buffer from the wells of the plate, increasing amount of the IgG (incubated in the different organic solvents) in the range of 10100 ng

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was added to the microtitre plate and incubated at 37 C for 2 h. Then, the microtitre plates were washed and incubated with 100 l of HRP conjugated goat anti-rabbit IgG at 37 C for 1 h. After the usual washing steps, the peroxidase reaction was initiated by the addition of the substrate TMB/H2 O2 , arrested by the addition of 4.0 N H2 SO4 and absorbance measured at 492 nm. 2.2.7. Fluorescence measurements Fluorescence measurements were carried out on a Shimadzu spectrouorimeter, model RF-540 equipped with a data recorder DR-3 at 25 0.1 C. The uorescence was recorded in the wavelength range 300400 nm, after exciting the protein solution at 280 nm for total protein uorescence. The slits were set at 10 nm, for both excitation and emission. The path length of the sample cuvette was 1 cm. 2.2.8. Circular dichroism (CD) measurements CD measurements were made on a Jasco spectropolarimeter, model J-720 equipped with a microcomputer. The instrument was calibrated with d-10-camphorsulphonic acid. All the measurements were carried out at 25 C. Far UV (200250 nm) and near UV (250300 nm) CD spectra were taken at 0.5 mg/ml protein concentration with a 1 cm path length cell. The results are expressed as CD (mdeg), which is dened as: CD(mdeg) = MRE(10 n 1 Cp ) Where MRE is the mean residual ellipticity in deg/cm2 /dmol, n is the number of amino acid residues, 1 is the path length of the cell and Cp is the mole fraction. 3. Results and discussion The interaction of anticatalase antibodies with catalase in the organic solvents as determined by ELISA showed that incubation of the antibodies with 40% methanol or ethanol resulted in a decrease in their antigen-binding activity (Fig. 1). However, incubation of the antibodies with 40% propanol, DMSO, DMF, or glycerol resulted in a substantial increase in their antigenbinding activity. The order of the antigen binding activity of the antibodies in buffer with different organic solvents and in that without any added organic solvent was observed to be as follows: glycerol > DMF > DMSO > propanolno > organic solvent > methanol > ethanol. Similar results were also obtained when the concentration of organic solvents taken was 10%. However, the magnitude of the change in antigen binding activity was very less. The dielectric constant and the dipole moment of the organic solvents used decreases as follows; DMSO > glycerol > DMF > methanol > ethanol > propanol. The interaction of anticatalase antibodies with catalase in the organic solvents used in this study is uncorrelated to the solvent properties (i.e. dielectric constant and dipole moment). Similar observations were noted in the case of interaction of testosterone

Fig. 1. Antigenantibody interaction in organic solvents. The binding of catalase to anticatalase IgG was measured in buffer without any organic solvent (1), 40% of methanol (2), ethanol (3), propanol (4), DMSO (5), DMF (6), and glycerol (7) by ELISA as described in methods. The experiment was done in duplicates and averages from three independent experiments are shown.

with antitestosterone antibodies in various organic solvents [15], and the binding of ferritin with three monoclonal antibodies in organic solvents [17]. The ELISA results suggested the possibility of conformational changes in the antibody molecule after incubation in the organic solvents, resulting in an increase or decrease in its afnity for the antigen. However, the observed effects could also be due to the conformational changes in the antigen molecule in these solvents. Hence, the conformational changes induced in the anticatalase antibodies and the catalase molecules after incubation in 40% organic solvent were studied by uorescence and CD spectroscopy. The solutions of the proteins in 40% concentration of the organic solvent used in these studies were clear and no aggregate formation was observed. The uorescence spectra of anticatalase antibodies incubated in 40% organic solvents i.e. methanol, ethanol, propanol, DMSO, DMF, and glycerol was compared with the spectrum of the antibodies in buffer without any added organic solvent (Fig. 2A, Table 1). The spectrum of the antibody in buffer (with no organic solvent) shows emission maximum at 338 nm. The spectrum of the antibodies in methanol, DMSO and DMF shows uorescence quenching and a red shift in the emission maximum. This suggests an increase in the polar microenvironment of the aromatic uorophores due to unfolding of the protein which results in the exposure of the aromatic uorophores to the surface of the protein. The spectrum of the antibodies in glycerol shows uorescence quenching and a blue shift. This suggests that a conformational change has occurred in the molecule that increases the non-polar microenvironment of the aromatic uorophores due to increased compactness of the protein and the embedding of the uorophores into the hydrophobic core of the molecule, and perhaps this change in conformation brings the uorophores closer to the intermolecular quenchers (presumably disulphide

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Fig. 2. Fluorescence spectra of anticatalase antibodies (panel A) and catalase (panel B) in organic solvents. The emission spectra were recorded after exciting the proteins at 280 nm. Slits were 10 nm for both excitation and emission. The concentration of protein taken was 0.5 mg/ml in sodium phosphate buffer, pH 7.0. The spectra were recorded for each of the protein in buffer without any organic solvent (1), with 40% of methanol (2), ethanol (3), propanol (4), DMSO (5), DMF (6), and glycerol (7).

groups) that are quenchers of the intrinsic uorescence of native immunoglobulins [2729]. The spectrum of the antibodies in ethanol shows uorescence enhancement and a blue shift implying that conformational change has occurred in the molecule resulting in increased compactness of the protein. The spectrum of the antibodies in propanol shows uorescence enhancement but no shift in the emission maximum, therefore, perhaps the conformational change is such that it results in a spatial separation of the aromatic uorophores from the intramolecular quenchers. These uorescence spectral studies suggest that the conformation of the antibodies was partially distorted in 40% concentration of all the six organic solvents used in this study. Similarly, the uorescence spectra of catalase in the various organic solvents were studied (Fig. 2B, Table 1). The spectrum of catalase in buffer (with no organic solvent) shows emission maximum at 330 nm. The spectra of catalase in methanol and DMF showed a red shift but no signicant change in uorescence, implying that some unfolding has taken place. The spectrum of catalase in ethanol shows uorescence enhancement and no

shift in emission maximum, implying that the conformational change in the molecule is such that it results in a spatial separation of the uorophores from the intramolecular quenchers. The spectra of catalase in propanol, DMSO, and glycerol shows uorescence enhancement and a red shift, implying that unfolding of the molecule has taken place, which results in the spatial separation of the uorophores from the intramolecular quenchers. The results suggest that the conformation of the antigen was also partially disordered in all the organic solvents taken in this study. The near UV-CD spectra of antibodies in methanol, ethanol, propanol, DMSO, DMF, and glycerol was compared with the spectrum of the antibodies in buffer without any added organic solvent (Fig. 3A). The spectrum of antibodies in buffer without any organic solvent shows a maximum around 288 nm corresponding to the aromatic residues in the protein i.e. the tryptophans and tyrosines. The spectrum also shows a minimum around 260 nm which is due to the disulphide linkages in the protein. It was observed that the spectra of antibodies in all the six organic solvents were different from that in buffer (with no

Table 1 The effect on the relative uorescence and the emission maximum of the anticatalase antibodies and catalase separately incubated in the various organic solvents as compared to those in buffer (with no organic solvent) Organic solvent Anticatalase antibodies Relative uorescence Methanol Ethanol Propanol DMSO DMF Glycerol Quenching Enhancement Enhancement Slight Quenching Quenching Quenching Shift in emission maximum Red shift (1 nm) Blue shift (9 nm) No shift Red shift (2 nm) Red shift (2 nm) Blue shift (18 nm) Catalase Relative uorescence Insignicant change Enhancement Enhancement Enhancement Insignicant change Enhancement Shift in emission maximum Red shift (10 nm) No shift Red shift (10 nm) Red shift (8 nm) Red shift (1 nm) Red shift (9 nm)

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Fig. 3. Near UV-CD (Panel A) and far-UV-CD (Panel B) spectra of anticatalase antibodies in organic solvents. The CD spectra of antibodies (0.5 mg/ml) in 10 mM sodium phosphate buffer, pH 7.0 were recorded in buffer without any organic solvent (1), with 40% of methanol (2), ethanol (3), propanol (4), DMSO (5), DMF (6), and glycerol (7).

organic solvent). Therefore, it implies that the antibodies have undergone a change in their tertiary structure in 40% concentration of the organic solvent taken in these studies. The far UV-CD spectrum of antibodies in buffer (with no organic solvent) shows a minimum at 214 nm (Fig. 3B). Analysis of the CD spectra reveals that the -sheet is the most dominant secondary structure element present in native IgG. These results are similar to those reported in literature [30,31]. The far UV-CD spectra of the antibodies in all the organic solvents were different from that in buffer (with no organic solvent), implying that the secondary structural elements have also undergone a change. Therefore, both the tertiary and the secondary structure of the antibodies are partially disordered in all the organic solvents taken in this study.

The near UV-CD spectrum of catalase in buffer (with no organic solvent) shows a maximum around 288 nm corresponding to the aromatic residues in the protein (Fig. 4A). The far UV-CD spectrum of catalase in buffer (with no organic solvent) shows a large sharp minimum at 213 nm and two small minima at 205 and 221 nm. The far UV-CD spectrum of catalase seems to be contributed by all the secondary structural elements i.e. -helices, -sheets, -turn and randomly coiled conformations (Fig. 4B). Both the near and the far UV-CD spectra of catalase in all the organic solvents were different from that in buffer (with no organic solvent), implying that both the tertiary and the secondary structure of catalase is partially disordered in all the organic solvents used in this study.

Fig. 4. Near UV-CD (panel A) and far-UV-CD (panel B) spectra of catalase in organic solvents. The CD spectra of catalase (0.5 mg/ml) in 10 mM sodium phosphate buffer, pH 7.0 were recorded in buffer without any organic solvent (1), with 40% of methanol (2), ethanol (3), propanol (4), DMSO (5), DMF (6), and glycerol (7).

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Fig. 5. Catalase activity in organic solvents. The activity of catalase was determined in varying concentration of DMSO (1) and DMF (2) as described in methods. The experiment was done in duplicates and averages from two independent experiments are shown.

Since the beginning of 1980s, it has been clearly shown that enzymes can be used in organic solvents with great efciency [32]. The use of organic solvents as reaction media for biocatalytic reactions has proven to be an extremely useful approach to expanding the range and efciency of practical applications of biocatalysis [3]. The advantages of using organic solvents include, for example, increased solubility of hydrophobic substrates and favourable shifts of reaction equilibrium. The activity of catalase was measured in the various organic solvents used in this study. However, methanol, ethanol, propanol and glycerol interfered in the assay, therefore, the activity was determined in DMSO and DMF only (Fig. 5). The enzyme activity decreased slowly with increasing DMSO concentration till 40% of the organic solvent in the reaction mixture. After this the decrease in the activity was rapid and the enzyme retained only 8% activity in 60% DMSO. The activity was about 79% in 40% DMSO which was the concentration taken for our studies on antigenantibody interactions and their conformation. It was remarkable to observe that the enzyme was activated in DMF upto 50% of its concentration, after which the activity decreased. The activity in 5% DMF was 38% more than that in buffer (with no organic solvent). It has also been shown in a recent study that, in tetrahydrofuran, dioxane, and acetone, catalase breaks down tert-butyl hydroperoxide several fold faster than in pure water [4]. The rate of catalase-catalyzed production of tert-butyl from tert-butyl hydroperoxide increased more than 400-fold in ethanol. These ndings suggest that water is not a unique medium in its ability to support molecular interactions required for folding of the polypeptide chain into a catalytic active conformation. Organic solvents seriously affect catalase activity. Therefore, the results of any kinetic study using this enzyme will depend very much on the content of organic solvents used in the assay.

Changes in protein structure induced by chemical or physical agents are usually associated with a complete or partial loss of the biological activity. Data on functional activation are very scarce [17,19,26]. Many methods for chemical modication of proteins require organic solvents to be added because of the hydrophobicity of many chemical modiers. The addition of small quantities of organic solvents is usually presumed to have no effect on both the conformation and function of a protein. However, our ndings suggest that this presumption cannot be generalized. The ndings of this study show that the antigenantibody interactions were much stronger in 40% glycerol, DMF, DMSO, and propanol than in buffer without any added organic solvent. However, the interactions were weaker in 40% methanol and in ethanol. Similar results on antigenantibody interactions were also obtained at 10% concentration of the organic solvents, although, the magnitude of the change was comparatively very less. Our studies on the conformation of anticatalase antibodies and catalase in these organic solvents suggest that the above mentioned differences in the antigenantibody interactions arise due to the conformational changes that are induced both in the antibody and the antigen molecule in organic solvents. Changes in properties of proteins in water-organic media are induced by changes in the hydrogen bonding property of water molecules at high and low concentrations of organic solvents due to alterations in the system of hydrogen bonds and hydrophobic interactions in protein molecules [3335]. These changes cause a rearrangement of hydrogen bonds that results in conformational changes of the whole molecule [5]. It was observed that both the secondary and tertiary structure of the antibody and the antigen molecule undergoes a change when 40% concentration of the organic solvent is used. These conformational changes presumably involve small and relatively disordered polypeptide segments of the molecule, that are relatively autonomous because of their weak interaction with the microenvironment. In immunoglobulins, two types of structure meet the mentioned requirements; CDR loops of the antigenbinding regions and the hinge region connecting the Fab and Fc fragments of antibodies [36]. The antibody conformers produced in glycerol, DMF, DMSO, and propanol differ from the native molecules by the higher afnity for the antigen. While these in methanol and ethanol have lower afnity for the antigen. The solvent dependence of antibody/antigen activation and inhibition was an unexpected nding. Not only the concentration but also chemical features of the organic solvents were essential for conformational changes in the structure involved in the production of the activated or partially deactivated conformers of antibodies. In this study we have also shown that functional effects induced by the organic solvents also depend on the antigen as the conformation and the activity of catalase in organic solvents was signicantly affected. The study also supports the fact that enzymes can be used in organic solvents and in some cases with good efciency. The knowledge in non-aqueous enzymology is however still largely empirical and considerable research is required to explain the enzyme behavior in non-aqueous environment for improvement and intelligent manipulation of catalyst properties. At present, limited knowl-

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edge on mechanism of conformational changes in proteins in organic solvents prevents to unequivocally explain some ndings of our study i.e. the reason for solvent dependence for the activation or inhibition of antigenantibody interaction and enzyme activity is unclear. Acknowledgments Facilities provided by Aligarh Muslim University are gratefully acknowledged. The work was also supported by the department of Science and Technology, Government of India, under its FIST programme, and the University Grants Commission, India under its special assistance programme. References
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