International Journal of Biological Macromolecules 38 (2006) 289–295

Effect of organic solvents on the conformation and interactionof 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 2006Available online 29 March 2006

Abstract

Effect of six organic solvents—methanol, ethanol, propanol, dimethyl sulphoxide (DMSO), N,N-dimethyl formamide (DMF), and glycerolon the conformation and interaction of catalase and anticatalase antibodies were studied with the aim of identifying the solvents in whichantigen–antibody interactions are strong. The antigen binding activity of the antibodies in the various organic solvents increased in the followingorder: ethanol < methanol < no organic solvent < propanol < DMSO < DMF < glycerol. The structure of both the antibody and the antigen moleculewas affected significantly in 40% concentration of the organic solvents used in this study. Catalase activity was inhibited in DMSO. However, thee©

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eywords: Organic solvents; Antigen–antibody interaction; Conformation; Catalase

. Introduction

The ability of various organic solvents to interfere with thehysico-chemical properties of proteins is well known [1–4].he effects of such solvents are generally attributed to alterationf various non-covalent interactions in the protein, solvation ofonic groups and dipoles, hydrogen bonding and hydrophobicnteractions. The conformation that a protein attains in a solventepends on the ratio of hydrophobic and hydrophilic regions onts surface. For the stabilization of the native protein structure,his ratio should have a certain value. Changes in this ratio cause aearrangement of hydrogen bonds that results in conformationalhanges of the whole molecule [5,6]. Some organic solventsave been used earlier to change the conformation of proteinolecules for studying protein folding/unfolding. For example,

rifluoroethanol was used to induce formation of �-helices inarious proteins such as �-lactoglobulin [7], porin [8] and tumorecrosis factor [9]. The alcohol-induced denaturation of some

Abbreviations: DMSO, dimethyl sulphoxide; DMF, N,N-dimethyl for-amide; HRP, horse rabbit peroxidase; TMB, tetramethyl benzidine; ELISA,

proteins e.g. cytochrome c results in the appearance of a partiallystructured state resembling the molten globule [10]. The additionof DMSO into the medium for the renaturation of lysozymeincreases the rate of production of native like structures andformation of the final native state [11].

It is widely accepted, however, that the catalytic efficiency ofenzymatic reactions in organic media is comparable to, and insome cases higher, than that displayed in aqueous media, as longas the enzymes are surrounded by a thin film of water, whichis necessary for the retention of their catalytic activity [3,4,12].Thus organic phase enzyme electrodes have been developed foruse in organic phases [13]. The operation of such devices canoffer several advantages, such as extended concentration range,prevention of undesirable side reactions, improved operationalstability and simplified immobilization techniques.

In contrast to the above mentioned studies, there are onlya few investigations on the behaviour of antibodies in organicsolvents [14–17]. It was demonstrated that the binding of a hap-ten, 4-aminobiphenyl, to its monoclonal antibody was strong andspecific in such solvents [14]. The inhibition of binding betweentestosterone and its antibody in a variable range of water misci-

nzyme-linked immunosorbent assay; PBS, phosphate buffer saline; CD, circu-ar dichroism∗ Corresponding author. Tel.: +91 571 2720 388; fax: +91 571 272 1776.

E-mail address: hinayounus@rediffmail.com (H. Younus).

ble solvents was found not to be correlated with some solventproperties, such as dielectric constant, polarity index and dipolemoment, but was found to be inversely correlated to the molecu-lar mass of the solvent [15]. A study on the effect of nine organic

141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2006.03.023

290 Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295

solvents on lysozyme-antilysozyme precipitin reaction showedthat these solvents destabilize the antigen–antibody complex[18]. Data on functional activation of protein molecules aftertheir structural perturbation are very scarce [17,19].

Several proteins are extracted and utilized in organic solvents[20–23]. However, studies on the conformation of proteins inorganic solvents are rare. In the present study, the effect of sixorganic solvents (methanol, ethanol, propanol, DMSO, DMFand glycerol) on the conformation and interaction of antigen andthe antibody molecule were studied. The aim was to identify thesolvents in which antigen–antibody interactions are strong. Suchsolvents may be utilized in immunoassays for the identificationof trace amounts of antigen/antibody. Catalase was chosen asthe test antigen because its activity may be easily monitoredand its activity is reasonably stable in both aqueous and organicsolvents [4,24]. The concentration of the organic solvents usedwas that which is generally employed for isolation and duringchemical modification of proteins [25,26].

2. Materials and methods

2.1. Materials

Bovine liver catalase [9001-05-2] (EC 1.11.1.6) was pur-chased from Sigma (St. Louis, MO). It was homogenous onthe basis of size and was used in the experiments withoutfwaMrtli

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sisted of H2O2, the enzyme, and the appropriate concentrationof DMSO or DMF in a total volume of 1 ml of sodium phos-phate buffer, pH 7.0. The degradation of H2O2 was measured asdescribed above.

2.2.3. Immunization of rabbitsHealthy rabbits were injected subcutaneously with 500 �g of

catalase using freund’s adjuvant. The animals were boosted onday 21 and subsequently bled after a week for monitoring theproduction of catalase specific antibodies.

2.2.4. Enzyme-linked immunosorbent assay (ELISA)The generation of catalase specific antibodies was measured

in the sera of catalase immunized rabbits by the ELISA. Ninety-six well microtitre plates were coated overnight with 100 �l ofcatalase (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 inPBS-Tween 20) was applied to the wells and the plates incubatedat 37 ◦C for 2 h. After removal of the blocking buffer, seriallydiluted test and control sera were added and binding allowed toproceed at 37 ◦C for 2 h. The microtitre plates were washed andincubated with 100 �l of HRP conjugated goat anti-rabbit IgG at37 ◦C for 1 h. After the usual washing steps, the peroxidase reac-tion was initiated by the addition of the substrate TMB/H2O2,arrested by the addition of 4.0 N H SO and absorbance mea-sa

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urther purification. H2O2, methanol, ethanol, DMSO, DMFere obtained from Qualigens fine chemicals, India. Glycerol

nd propanol were supplied by Sisco Research Lab., India.icrotitre plates were purchased from Granier, USA. Horse

abbit peroxidase (HRP) conjugated goat anti-rabbit IgG andetramethyl benzidine (TMB)/H2O2 were the product of Geneiaboratories, India. All the other chemicals used were of analyt-cal grade.

.2. Methods

.2.1. Assay for catalase activityA standard curve for determining the concentration of H2O2

as constructed by taking increasing concentration of H2O2 inseries of tubes in 1 ml of 20 mM sodium phosphate buffer,

H 7.0. Then 2 ml of dichromate/acetic acid reagent (5% potas-ium dichromate and acetic acid in the ratio of 1:3) was addedo each tube and they were incubated at 100 ◦C for 10 min. Areen solution of chromic acetate appeared, whose absorbanceas measured at 570 nm. A standard curve of the absorbance

t 570 nm versus concentration of H2O2 was plotted. The activ-ty of catalase was measured using the above reaction mixture.oth H2O2 and the enzyme were taken in 1 ml volume of the

odium phosphate buffer, pH 7.0. The reaction was started byhe addition of the enzyme, followed by incubation at 30 ◦C formin. The degradation of H2O2 was measured using the above

tandard curve.

.2.2. Catalase activity measurement in organic solventsCatalase activity was determined in increasing concentration

f DMSO or DMF (0–60%). The standard reaction mixture con-

2 4ured at 492 nm in an ELISA reader. Catalase was immunogenicnd readily elicited the formation of antibodies in rabbits.

.2.5. Purification of antibodiesThe rabbit serum samples that exhibited a good titre of anti-

atalase antibodies were saturated with ammonium sulphate to0% concentration. The precipitate thus obtained was separatedut by centrifugation at 5000 rpm for 15 min and dissolved ininimal volume of sodium phosphate buffer (20 mM, pH 7.0)

nd then was dialyzed against the same buffer. The antibod-es were further purified by ion exchange chromatography onEAE-cellulose matrix equilibrated with the same buffer. The

mpurities bound to the matrix while pure IgG was left in theupernatant. The purity of the IgG was ascertained by SDS-AGE. Two bands were visible in the SDS-PAGE of IgG andheir molecular mass corresponded to those of heavy (50 kda)nd light chain (25 kda) of IgG. The cross-reactivity of the puri-ed IgG with catalase was also proved by ELISA.

.2.6. Measurement of antigen–antibody interactions inrganic solvents by ELISA

Ninety-six well microtitre plates were coated overnight with00 �l of catalase (10 �g/ml) in 0.05 M bicarbonate buffer, pH.6 at 4 ◦C. After extensive washing with PBS-Tween 20, 100 �lf blocking buffer (5% skimmed milk in PBS-Tween 20) waspplied to the wells and the plates incubated at 37 ◦C for 2 h.eanwhile, anticatalase IgG was incubated in 40% organic sol-

ents (methanol, ethanol, propanol, DMSO, DMF, and glycerol)or 1 h at room temperature. After removal of blocking bufferrom the wells of the plate, increasing amount of the IgG (incu-ated in the different organic solvents) in the range of 10–100 ng

Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295 291

was added to the microtitre plate and incubated at 37 ◦C for2 h. Then, the microtitre plates were washed and incubated with100 �l of HRP conjugated goat anti-rabbit IgG at 37 ◦C for 1 h.After the usual washing steps, the peroxidase reaction was initi-ated by the addition of the substrate TMB/H2O2, arrested by theaddition of 4.0 N H2SO4 and absorbance measured at 492 nm.

2.2.7. Fluorescence measurementsFluorescence measurements were carried out on a Shimadzu

spectrofluorimeter, model RF-540 equipped with a data recorderDR-3 at 25 ± 0.1 ◦C. The fluorescence was recorded in the wave-length range 300–400 nm, after exciting the protein solution at280 nm for total protein fluorescence. The slits were set at 10 nm,for both excitation and emission. The path length of the samplecuvette was 1 cm.

2.2.8. Circular dichroism (CD) measurementsCD measurements were made on a Jasco spectropolarimeter,

model J-720 equipped with a microcomputer. The instrumentwas calibrated with d-10-camphorsulphonic acid. All the mea-surements were carried out at 25 ◦C. Far UV (200–250 nm) andnear UV (250–300 nm) CD spectra were taken at 0.5 mg/ml pro-tein concentration with a 1 cm path length cell. The results areexpressed as CD (mdeg), which is defined as:

CD(mdeg) = MRE(10 × n × 1 × Cp)

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Fig. 1. Antigen–antibody interaction in organic solvents. The binding of catalaseto 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 duplicatesand averages from three independent experiments are shown.

with antitestosterone antibodies in various organic solvents [15],and the binding of ferritin with three monoclonal antibodies inorganic solvents [17].

The ELISA results suggested the possibility of conforma-tional changes in the antibody molecule after incubation in theorganic solvents, resulting in an increase or decrease in its affin-ity for the antigen. However, the observed effects could alsobe due to the conformational changes in the antigen moleculein these solvents. Hence, the conformational changes inducedin the anticatalase antibodies and the catalase molecules afterincubation in 40% organic solvent were studied by fluorescenceand CD spectroscopy. The solutions of the proteins in 40% con-centration of the organic solvent used in these studies were clearand no aggregate formation was observed.

The fluorescence spectra of anticatalase antibodies incu-bated in 40% organic solvents i.e. methanol, ethanol, propanol,DMSO, DMF, and glycerol was compared with the spectrumof the antibodies in buffer without any added organic solvent(Fig. 2A, Table 1). The spectrum of the antibody in buffer (withno organic solvent) shows emission maximum at 338 nm. Thespectrum of the antibodies in methanol, DMSO and DMF showsfluorescence quenching and a red shift in the emission maxi-mum. This suggests an increase in the polar microenvironment ofthe aromatic fluorophores due to unfolding of the protein whichresults in the exposure of the aromatic fluorophores to the surfaceof the protein. The spectrum of the antibodies in glycerol showsflftdoac

here MRE is the mean residual ellipticity in deg/cm2/dmol, ns the number of amino acid residues, 1 is the path length of theell and Cp is the mole fraction.

. Results and discussion

The interaction of anticatalase antibodies with catalase in therganic solvents as determined by ELISA showed that incuba-ion of the antibodies with 40% methanol or ethanol resulted indecrease in their antigen-binding activity (Fig. 1). However,

ncubation of the antibodies with 40% propanol, DMSO, DMF,r glycerol resulted in a substantial increase in their antigen-inding activity. The order of the antigen binding activity ofhe antibodies in buffer with different organic solvents and inhat without any added organic solvent was observed to be asollows:

glycerol > DMF > DMSO > propanolno > organic solvent

> methanol > ethanol.

Similar results were also obtained when the concentration ofrganic solvents taken was 10%. However, the magnitude of thehange in antigen binding activity was very less.

The dielectric constant and the dipole moment of the organicolvents used decreases as follows;

MSO > glycerol > DMF > methanol > ethanol > propanol

The interaction of anticatalase antibodies with catalase in therganic solvents used in this study is uncorrelated to the solventroperties (i.e. dielectric constant and dipole moment). Similarbservations were noted in the case of interaction of testosterone

uorescence quenching and a blue shift. This suggests that a con-ormational change has occurred in the molecule that increaseshe non-polar microenvironment of the aromatic fluorophoresue to increased compactness of the protein and the embeddingf the fluorophores into the hydrophobic core of the molecule,nd perhaps this change in conformation brings the fluorophoresloser to the intermolecular quenchers (presumably disulphide

292 Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295

Fig. 2. Fluorescence spectra of anticatalase antibodies (panel A) and catalase (panel B) in organic solvents. The emission spectra were recorded after exciting theproteins 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. Thespectra 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 fluorescence of nativeimmunoglobulins [27–29]. The spectrum of the antibodies inethanol shows fluorescence enhancement and a blue shift imply-ing that conformational change has occurred in the moleculeresulting in increased compactness of the protein. The spectrumof the antibodies in propanol shows fluorescence enhancementbut no shift in the emission maximum, therefore, perhaps theconformational change is such that it results in a spatial sep-aration of the aromatic fluorophores from the intramolecularquenchers. These fluorescence spectral studies suggest that theconformation of the antibodies was partially distorted in 40%concentration of all the six organic solvents used in this study.

Similarly, the fluorescence spectra of catalase in the variousorganic solvents were studied (Fig. 2B, Table 1). The spectrum ofcatalase in buffer (with no organic solvent) shows emission max-imum at 330 nm. The spectra of catalase in methanol and DMFshowed a red shift but no significant change in fluorescence,implying that some unfolding has taken place. The spectrumof catalase in ethanol shows fluorescence enhancement and no

shift in emission maximum, implying that the conformationalchange in the molecule is such that it results in a spatial separa-tion of the fluorophores from the intramolecular quenchers. Thespectra of catalase in propanol, DMSO, and glycerol shows flu-orescence enhancement and a red shift, implying that unfoldingof the molecule has taken place, which results in the spatial sep-aration of the fluorophores from the intramolecular quenchers.The results suggest that the conformation of the antigen was alsopartially 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 thespectrum of the antibodies in buffer without any added organicsolvent (Fig. 3A). The spectrum of antibodies in buffer withoutany organic solvent shows a maximum around 288 nm corre-sponding to the aromatic residues in the protein i.e. the tryp-tophans and tyrosines. The spectrum also shows a minimumaround 260 nm which is due to the disulphide linkages in theprotein. It was observed that the spectra of antibodies in all thesix organic solvents were different from that in buffer (with no

Table 1The effect on the relative fluorescence and the emission maximum of the anticatalase antibodies and catalase separately incubated in the various organic solvents ascompared to those in buffer (with no organic solvent)

Organic solvent Anticatalase antibodies Catalase

Relative fluorescence Shift in emission maximum Relative fluorescence Shift in emission maximum

MEPDDG )

ethanol Quenching Red shift (1 nm)thanol Enhancement Blue shift (9 nm)ropanol Enhancement No shiftMSO Slight Quenching Red shift (2 nm)MF Quenching Red shift (2 nm)lycerol Quenching Blue shift (18 nm

Insignificant change Red shift (10 nm)Enhancement No shiftEnhancement Red shift (10 nm)Enhancement Red shift (8 nm)Insignificant change Red shift (1 nm)Enhancement Red shift (9 nm)

Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295 293

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 mMsodium 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 haveundergone a change in their tertiary structure in 40% concen-tration of the organic solvent taken in these studies. The farUV-CD spectrum of antibodies in buffer (with no organic sol-vent) shows a minimum at 214 nm (Fig. 3B). Analysis of the CDspectra reveals that the �-sheet is the most dominant secondarystructure element present in native IgG. These results are similarto those reported in literature [30,31]. The far UV-CD spectra ofthe antibodies in all the organic solvents were different from thatin buffer (with no organic solvent), implying that the secondarystructural elements have also undergone a change. Therefore,both the tertiary and the secondary structure of the antibodies arepartially disordered in all the organic solvents taken in this study.

The near UV-CD spectrum of catalase in buffer (with noorganic solvent) shows a maximum around 288 nm correspond-ing to the aromatic residues in the protein (Fig. 4A). The farUV-CD spectrum of catalase in buffer (with no organic solvent)shows a large sharp minimum at 213 nm and two small minimaat 205 and 221 nm. The far UV-CD spectrum of catalase seemsto 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 catalasein all the organic solvents were different from that in buffer(with no organic solvent), implying that both the tertiary andthe secondary structure of catalase is partially disordered in allthe organic solvents used in this study.

F rganicb of m

ig. 4. Near UV-CD (panel A) and far-UV-CD (panel B) spectra of catalase in ouffer, pH 7.0 were recorded in buffer without any organic solvent (1), with 40%

solvents. The CD spectra of catalase (0.5 mg/ml) in 10 mM sodium phosphateethanol (2), ethanol (3), propanol (4), DMSO (5), DMF (6), and glycerol (7).

294 Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295

Fig. 5. Catalase activity in organic solvents. The activity of catalase was deter-mined in varying concentration of DMSO (1) and DMF (2) as described inmethods. The experiment was done in duplicates and averages from two inde-pendent experiments are shown.

Since the beginning of 1980s, it has been clearly shown thatenzymes can be used in organic solvents with great efficiency[32]. The use of organic solvents as reaction media for biocat-alytic reactions has proven to be an extremely useful approachto expanding the range and efficiency of practical applicationsof biocatalysis [3]. The advantages of using organic solventsinclude, for example, increased solubility of hydrophobic sub-strates and favourable shifts of reaction equilibrium. The activityof catalase was measured in the various organic solvents used inthis study. However, methanol, ethanol, propanol and glycerolinterfered in the assay, therefore, the activity was determined inDMSO and DMF only (Fig. 5). The enzyme activity decreasedslowly with increasing DMSO concentration till 40% of theorganic solvent in the reaction mixture. After this the decreasein 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 onantigen–antibody interactions and their conformation. It wasremarkable to observe that the enzyme was activated in DMFupto 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 recentstudy that, in tetrahydrofuran, dioxane, and acetone, catalasebreaks down tert-butyl hydroperoxide several fold faster thanin pure water [4]. The rate of catalase-catalyzed production oftert-butyl from tert-butyl hydroperoxide increased more than4uraaev

Changes in protein structure induced by chemical or physicalagents are usually associated with a complete or partial loss ofthe biological activity. Data on functional activation are veryscarce [17,19,26]. Many methods for chemical modification ofproteins require organic solvents to be added because of thehydrophobicity of many chemical modifiers. The addition ofsmall quantities of organic solvents is usually presumed to haveno effect on both the conformation and function of a protein.However, our findings suggest that this presumption cannot begeneralized.

The findings of this study show that the antigen–antibodyinteractions were much stronger in 40% glycerol, DMF, DMSO,and propanol than in buffer without any added organic sol-vent. However, the interactions were weaker in 40% methanoland in ethanol. Similar results on antigen–antibody interactionswere also obtained at 10% concentration of the organic sol-vents, although, the magnitude of the change was comparativelyvery less. Our studies on the conformation of anticatalase anti-bodies and catalase in these organic solvents suggest that theabove mentioned differences in the antigen–antibody interac-tions arise due to the conformational changes that are inducedboth in the antibody and the antigen molecule in organic sol-vents. Changes in properties of proteins in water-organic mediaare induced by changes in the hydrogen bonding property ofwater molecules at high and low concentrations of organic sol-vents due to alterations in the system of hydrogen bonds andhcioa4fdammbFdntgiteiffolavncim

00-fold in ethanol. These findings suggest that water is not anique medium in its ability to support molecular interactionsequired for folding of the polypeptide chain into a catalyticctive conformation. Organic solvents seriously affect catalasectivity. Therefore, the results of any kinetic study using thisnzyme will depend very much on the content of organic sol-ents used in the assay.

ydrophobic interactions in protein molecules [33–35]. Thesehanges cause a rearrangement of hydrogen bonds that resultsn conformational changes of the whole molecule [5]. It wasbserved that both the secondary and tertiary structure of thentibody and the antigen molecule undergoes a change when0% concentration of the organic solvent is used. These con-ormational changes presumably involve small and relativelyisordered polypeptide segments of the molecule, that are rel-tively autonomous because of their weak interaction with theicroenvironment. In immunoglobulins, two types of structureeet the mentioned requirements; CDR loops of the antigen-

inding regions and the “hinge” region connecting the Fab andc fragments of antibodies [36]. The antibody conformers pro-uced in glycerol, DMF, DMSO, and propanol differ from theative molecules by the higher affinity for the antigen. Whilehese in methanol and ethanol have lower affinity for the anti-en. The solvent dependence of antibody/antigen activation andnhibition was an unexpected finding. Not only the concentra-ion but also chemical features of the organic solvents weressential for conformational changes in the structure involvedn the production of the activated or partially deactivated con-ormers of antibodies. In this study we have also shown thatunctional effects induced by the organic solvents also dependn the antigen as the conformation and the activity of cata-ase in organic solvents was significantly affected. The studylso supports the fact that enzymes can be used in organic sol-ents and in some cases with good efficiency. The knowledge inon-aqueous enzymology is however still largely empirical andonsiderable research is required to explain the enzyme behav-or in non-aqueous environment for improvement and intelligent

anipulation of catalyst properties. At present, limited knowl-

Mohd. Rehan, H. Younus / International Journal of Biological Macromolecules 38 (2006) 289–295 295

edge on mechanism of conformational changes in proteins inorganic solvents prevents to unequivocally explain some find-ings of our study i.e. the reason for solvent dependence forthe activation or inhibition of antigen–antibody interaction andenzyme activity is unclear.

Acknowledgments

Facilities provided by Aligarh Muslim University are grate-fully acknowledged. The work was also supported by the depart-ment of Science and Technology, Government of India, under itsFIST programme, and the University Grants Commission, Indiaunder its special assistance programme.

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