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Artocarpus hirsutalectinDifferential modes of chemical and thermal denaturationSushama M. Gaikwad, Madhura M. Gurjar* and M. Islam KhanDivision of Biochemical Sciences, National Chemical Laboratory, Pune, IndiaUnfolding, inactivation and dissociation o f the lectin fromArtocarpus hirsuta seeds were studied by chemical (guanidinehydrochloride, GdnHCl) and thermal denaturation. Con-formational transitions were monitored by intrinsic ﬂuor-escence and circular dichroism. The gradual red shift in t heemission maxima of the native p rotein from 33 5 to 356 nm,change in the e llipticity at 218 nm and simultaneous decreasein the sugar binding activity were observed with increasingconcentration o f G dnHCl in the pH range between 4.0 and9.0. The unfolding and inactivation by GdnHCl were par-tially reversible. Gel ﬁltration of the lectin in presence o f1–6MGdnHCl showed that the protein dissociates rever-sibly into partially unfolded dimer and then irreversibly intounfolded inactive monomer. Thermal denaturation wasirreversible. The l ectin loses activity rapidly above 45 °C.The exposure of h ydrophobic patches, distorted secondarystructure and formation o f insoluble aggregates of thethermally inactivated protein probably leads to the irre-versible d enaturation.Keywords: Artocarpus lectin; denaturation; intrinsic ﬂuores-cence; unfolding; aggregation.Proteins that bind carbohydrates speciﬁcally and reversiblyare termed as lectins. They occur ubiquitously in nature andhave diverse role in plants, animals and microbes. T herecognition of c arbohydrate m oieties b y l ectins h as import-ant applications in a number of biological processes such a scell–cell i nteractions, signal transduction, and cell growthand differentiation . a-Galac toside speciﬁc lectin presentin the s eeds of Artocarpus hirsuta [2–4], is a homotetramericprotein with molecular mass of 60 000 Da and highspeciﬁcity f or methyl a-D-galactopyranoside (Me a-gal).The folding pathways of oligomeric proteins involve bothintramolecular and intermolecular interactions. The dena-turation of pea and peanut lectins, both oligomeric proteins,has b een studied in great detail [ 5–7]. In t his paper we showthe progressive unfolding and inactivation of the lectin inpresence of GdnHCl and heat, and refolding and reactiva-tion under renaturing conditions.MATERIALS AND METHODSMaterialsGdnHCl was a product o f Sigma Chemical Co. A ll otherchemicals were of t he highest purity available. The l ectinfrom A. hirsuta was puriﬁed as described previously .Stocks of 7MGdnHCl were freshly prepared in appropriatebuffers and ﬁltered through 0.45-lm ﬁlter. Buffers usedwere acetate f or pH 4.0, citrate/phosphate for pH 5.0 and6.0, phosphate for p H 7.0 and Tris/HCl for pH 8.0 and 9.0(all 100 mM).Fluorescence studiesProtein samples (1.5 lM) were equilibrated for 4 h at thedesired denaturant concentration at 30 °C in the pH rangeof 4.0–9.0. Unfolding as a function of GdnHCl concentr a-tion was monitored by intrinsic tryptophan ﬂuorescenceemission in a 1-cm quartz cell in the 300–400 nm region,when excited at 280 nm, in a Perkin-Elmer LS 50Bspectroﬂuorimeter with attached circulating water bath.Excitation and emission band passes of 5 n m were u sed.Activity of the sample was measured at the same time.Unfolding as a function of temperature w as carried outby incubating the protein samples (1.5 lM) in duplicates atthe temperature from 30–70 °C for 15 min. One of theduplicates was u sed to r ecord the spectra and activity a t therespective temperature. The other sample was brought to35 °C, centrifuged to remove any particulate matter, spectrawere recorded and activity was estimated.Circular dichroism studiesFar UV CD (210–250 nm) spectra of the protein samples(15 lM) t reated with different concentrations GdnHCl inrespective buffers of pH 4.0–9.0 and incubated for 14 h wererecorded in a 1-mm path length cell, on a Jasco J715spectropolarimeter connected to a circulating water bath.For thermal denaturation studies, the protein sample wasincubated at various temperatures for 10 min and thespectra were recorded. The spectra were collected withresponse time of 4 s, and scan speed of 100 n mÆs)1.Eachdata point was an average of three accumulations.Correspondence to S. M. Gaikwad, Division of Biochemical Sciences,National Chemical Laboratory, Pune, 411008, India.Fax: + 9 1 20 5884032, Tel.: + 91 20 5893034,E-mail: firstname.lastname@example.orgAbbreviations: GdnHCl, guanidine hydrochloride; Me a-gal, methyla-D-galactopyranoside; ANS, 1-anilino-8-naphthalene sulfonate.*Present address: University of Medic ine and D e ntistry New J ersey,Robert Wood J ohn son Medical School, D ivision of Biochemistry(Research t ower), Piscataway, Ne w Jersey 0 8854, USA.(Received 21 December 2 001, acc epted 14 Ja nuary 2002)Eur. J. Biochem. 269, 1413–1417 (2002) Ó FEBS 2002Hydrophobic dye binding studies8-Anilino-1-naphthalene sulfonate (ANS) emission spectrawere recorded in the range of 400–500 nm with excitation at375 n m using slit widths of 5 nm. The changes in the ANSﬂuorescence induced by the binding to the lectin werefollowed by r ecording the spectra at constant concentrationof protein (1 lM)andANS(50lM), in different concentra-tions of GdnHCl (1–5M).Determination of the lectin activitySugar binding activity of the samples was measured by theenhancement in the intrinsic ﬂu orescence of the prote in at335 n m after addition of the nonﬂuorescent ligand Me a-gal(4 mM) at saturating c oncentration. Increase in the ﬂ uores-cence of the native lectin after adding Me a-gal was taken as100% activity .Light scattering studiesRayleigh light scattering experiments w ere carried out withthe s pectroﬂuorimeter t o f ollow p rotein aggregation duringGdnHCl and thermal denaturation. Both excitation andemission wavelengths were set at 400 nm and the timedependent change in scattering intensity was followed.Renaturation studiesTwo-hundred microliters aliquot was removed from thesamples treated with different c oncentrations of GdnHCl(3.0–5.0M)for4hat30°C a t p H 7 .0 and d iluted 1 0 t imeswith 100 mMbuffer of pH 7.0. After 30 min, the ﬂuores-cence spectra and activity of the original (treated w ithGdnHCl) a s well as diluted samples were recorded. Proteinsample without GdnHCl tre ated under identical conditionswas taken as control.The renaturation of thermally denatured protein wasfollowed b y c ooling t he heate d samples to 3 5 °C, re movingany p articulate matter by centrifuging, and t hen recordingthe ﬂuorescence spectra and the activity.Gel ﬁltration studiesLectin samples (10 lMin 100 lL) were incubated for 14 hwith GdnHCl (1.0–6.0M) a nd injected onto Protein Pak300SW HPLC column (7.8 · 300 mm) connected to aWaters HPLC syste m preequilibrated a nd eluted withdifferent concentrations of GdnHCl (1.0–6.0M)in100 mMbuffer of pH 7 .0 at a ﬂow rate of 0.5 mLÆmin)1.Elution was monitored by absorbance at 280 nm. Thestandard molecular mass markers run in the presence ofbuffer were, BSA (66 kDa), o valbumin (45 kDa), carbonicanhydrase (29 kDa) and cytochrome c ( 14.5 k Da).RESULTS AND DISCUSSIONUnfolding studiesThe ﬂuorescence emission spectrum of the native lectinshowed a maximum a t 335 nm which characterizes no n-polar environment o f t he tryptoph an residues. On dena-turation of the lectin with increasing concentration ofGdnHC l (1–5M) a lthough ﬂuorescence intensity at 335 nmdoes not change much, the ﬂuorescence at 356 n mincreases signiﬁcantly thus changing the emission maxi-mum from 335 to 356 nm (Fig. 1A) indicating that due tounfolding, most of the tryptophans in the protein aregetting exposed to th e solvent. The ratio of ﬂuorescenceintensities at 3 35 and 356 F(335/356) (Fig. 1B) decreasesfrom 1.35 to 0.82. Similar trend of denaturation withGdnHCl is observed in the pH range of 5.0–8.0, while it ismore drastic at pH 4.0 and 9.0. The concentration ofGdnHCl required for 50% unfolding of the proteinbetween the pH range 6.0–8.0 is higher (3.2M)thanatpH 4.0 and pH 9.0 (2.2M).The l ectin showed a typical far UV CD spectrumobserved for proteins with high proportion of b sheetcontent, with minimum at 218 nm . The relativepercentage of structural elements calculated using CDProsoftware package for analyzing protein CD spectra wasa helix 2%, b sheet 44%, turns 2 3% and random coil 30%for native protein. The CD spectra of the G dnHCl treatedprotein when analysed using the above programs did notshow any signiﬁcant change in the different structuralelements compared to the native protein, while there wasvisible difference in the respective CD spectra. This wasprobably due to the incompatibility o f the data with theprogrammes used. Because GdnHCl was interferingthe CD s pectra below 2 10 nm, d ata in the ran ge of210–250 nm could be collected. The negative ellipticity ofthe protein at 218 nm increases in 1–2MGdnHCl andthen decreases at h igher c oncentration ( Fig. 1C). Thechange in the structure at 1–2MGdnHCl is concomitantwith loss of activity and therefore cannot be a stableFig. 1. GdnHCl-induced unfolding of A. hirsuta lectin at 30 °C. Protein(1.5 lM) at the required GdnHCl concentration was incubated for 4 hand the ﬂuorescence emission spectra were re co rded between 300 and400 nm with the excitation wavelength of 280 nm (A), shift in theemission max (B), ratio F(335/356) (C), mean residue ellipticity at218 nm in far UV region and (D) activity of the GdnHCl treatedprotein. The s ym bols used for all the ﬁgure s a re p H 4.0 (.), pH 5.0(d), pH 6.0 ( m), pH 7.0 (,), pH 8.0 (s)andpH9.0(n).1414 S. M. Gaikwad et al. (Eur. J. Biochem. 269) Ó FEBS 2002conformation. When Rayleigh light scattering studies ofthe s amples were carried out, the sample incubated at 1MGdnHCl at 30 °C showed lower light scattering intensitythan the native p rotein. At 2.0MGdnHCl, there waslower light scattering than that with 1.0Mand at s tillhigher concentrations of GdnHCl, there was n o lightscattering at all (data not shown). Thus, the increase inthe negative ellipticity at 218 n m at low concentrationsof GdnHCl could be due to the solubilization of theaggregates in the protein. There is substantial loss inthe secondary structure of t he lectin as indicated b y thedecrease in the n egative ellipticity at 218 nm withincreasing concentration of GdnHCl. Similar trend wasobserved for denaturation between p H 5.0–8.0, while therate of unfolding was faster at pH 4 .0 and 9.0.The inactivation of the lectin was proportional to theconcentration of GdnHCl (Fig. 1D). The maximumenhancement in the intrinsic ﬂuorescence of the lectin dueto the binding of sugar, Me a-gal, taken as measure of100% activity of the lectin  determined at different pHwas diffe rent. T he percentage decrease in the enhancement,i.e. activity w ith increasing concentration o f GdnHCl,however, was equivalent in the pH range of 4.0–9.0 a ndthe loss in the activity of the lectin is concomitant with theunfolding of the protein. A t 3MGdnHCl, more than 5 0%activity was lost with 60% decrease in the ratio (F335/356)and 25–35% shift in the emission maximum.Refolding of the proteinRenaturation or re folding of t he protein w as measured asthe extent of reappearance of the original spectra (F 335/356) and recovery of the sugar binding activity. Afterdilution of the r eaction mixture containing lectin andGdnHCl (10 t imes), partial reactivation o f the lectin wasobserved. The lectin treated with 3 , 4, and 5MGdnHClhad 45, 13 and 7% activity, which increased to 75, 37,and 23%, respectively ( Table 1 ) on renaturation. Re fold-ing of the protein w as indicated by s ubstantial increase i nthe F(335/356) ratio. GdnHCl probably unfolds theprotein in such a way that substantial interactions arereformed after removal of the denaturant, leading to thesigniﬁcant reformation of the structure and regaining ofactivity.Gel ﬁltration studiesThe native protein gets dissociated ﬁrst into dimer (Mr30 000) and then into monomer (Mr14 000) with increasingconcentration of G dnHCl (Fig. 2). At 3–4MGdnHC l, asingle peak at 10.4 min appears that seems arise f romthe totally denatured monomer. Complete dissociation ofthe tetramer does not take place even at 6MGdnHCl. Theprotein components corresponding to the peaks 1, 2, 3 and 0were analysed separately for sugar binding activity. Peak 1was found to be the f olded and active fraction of the totalpopulation o f the lectin molecules t reated with GdnHCl.Peak 2 is p artially unfolded form of t he lectin with traces ofactivity. Peak 3 is unfolded, inactive monomer. Peak 0 is thetotally denatu red m onomer similar t o t hat observed in c aseof peanut lectin . The dissociation of the native protein inpresence of GdnHCl into dimer is reversible, that intomonomer is irreversible as observed by rechromatographyof the individual peaks on gel ﬁltration column underrenaturing conditions (data not shown). Based on thedissociation pattern, the following scheme can be written:T () D () M () M*where T is tetramer, D is dimer, M is monomer and M* istotally denatured monomer.The monomer seems to be unstable and the conforma-tional stability of the oligomer seems to be contributedwholly by the q uaternary i nteractions. I n case of p eanutlectin, folded m onomer is obtained after dissociation of theprotein  and the molten globule-like state of the monomerwas d etected during its unfolding , both of which retainthe sugar binding activity.Thermal denaturationThe A. hirsuta lectin loses sugar binding activity and startsprecipitating above 45 °C. The ﬂuorescence emission spec-trum broadens, but the emission maxima does not shiftfrom 335 to 356 even at 70 °C where almost totalinactivation of the lectin takes place. T he decrease in theratio F(335/356) observed for thermally denatured protein,from 1.36 (native) to 1.03 (70 °C, 15 min) (Table 1)was less than that observed with GdnHCl denaturation(at pH 7.0), 1.35 (native) to 0.82 (5MGdnHCl) ( Fig. 1B).Table 1. Eﬀect of treatment GdnHCl and thermal denaturation and renaturation on A. hirsuta lectin. The samples treated w ith GdnHCl were diluted10 times with 100 mMphosphate buﬀer, pH 7.0, the spectra were recorded and activity was estimated as described in Materials and methods. Thelectin samples i ncubated at respective temperatures were cooled to 35 °C, spectra were recorded and activity was estimated.TreatmentActivity (%) F 335/356On denaturation On Renaturation On denaturation On RenaturationLectin + GdnHCl (0M) 100 100 1.35 1.35Lectin + GdnHCl (3M) 45 75 1.15 1.26Lectin + GdnHCl (4M) 13 37 0.86 1.2Lectin + GdnHCl (5M) 7 23 0.82 1.16Lectin Þ 35 °C,15 min 100 100 1.36 1.36Lectin Þ 45 °C,15 min 75 70 1.26 1.18Lectin Þ 50 °C,15 min 53 35 1.23 1.1Lectin Þ 60 °C,15 min 13 5 1.09 0.98Lectin Þ 70 °C,15 min 7 0 1.03 0.95Ó FEBS 2002 Denaturation studies of Artocarpus hirsuta lectin (Eur. J. Biochem. 269) 1415On thermal denaturation, the p rotein forms insolubleaggregates before total unfolding and loses its sugarbinding activity. When the temperature of the samplesincubatedfrom45to70°C was brought down slowly to35 °C, the activity was not restored and no refolding wasobserved a s there is decrease in the F(335/356) ratio(Table 1) indicating that the thermal denaturation isirreversible. Because the protein s tarts a ggregating o nthermal denaturation, Rayleigh light scattering studies werecarried out. The protein shows higher light scatteringintensity a t 45 °C (Fig. 3A) which goes on increasing withfurther i ncrease i n the temperature.ANS binding studiesBinding of ANS to the proteins occurs upon the exposure ofhydrophobic clusters during the unfolding process. ANSdoes not bind to the native or t he denatured states of t heA. hirsuta lectin but binds at the intermediate stage (at 2MGdnHCl), showing increase in t he ﬂuorescence intensity,indicating temporary exposure of the hydrophobic patchesof the protein during unfolding (Fig. 3B). The p ossibility ofthe occurrence of the molten globule during unfolding ofA. hirsuta lectin as observed in t he peanut lectin , wasruled out because a signiﬁcant amount of th e tertiary andsecondary structure was intact. T he ANS binding to theprotein samples exposed to 50, 60, and 70 °C was more thanthose incubated a t 30 a nd 40 °C (Fig. 3C) i ndicating theexposure of hydrophobic patches are due to therm aldenaturation. The tendency of the protein to aggregateincreases a s the hydrophobic patches get exposed due tothermal denaturation. The CD s pectra of the proteinexposed at 45–70 °C for 10 min s hows progressive loss inthe secondary structure (Fig. 3D).There s eem to be two different modes o f d enaturation o fthe A. hirsuta lectin with GdnHCl and heat. The formerunfolds and inactivates the protein, allowing it to fold backand reactivate to certain extent after removal of the r eagent.Thermal denaturation l eads to unfolding and simultaneousformation of insoluble aggregates and is therefore irrevers-ible. Different modes of folding and unfolding observedunder different conditions could be due to the unusualFig. 2. Gel ﬁltration of A. hirsuta lectin in presence of GdnHCl in100 mMpotassium phosphate buﬀer (pH 7.0). Molarity of GdnHCl isindicated o n t he ﬁgure. Mrvalues of the standards used were as f o l-lows, 1, BSA 6 6 kDa, 2 , ov albumin, 45 kDa, 3, carbonic anhydrase,29 kDa and 4, cytochrome c, 14. 5 k Da.Fig. 3. Rayleigh light scattering (A) and ANS ﬂuorescence (B,C) studiesof A. hirsuta lectin. (A) The l ectin (1.5 lM) was incubated at d iﬀ erenttemperatures for 10 min an d the light scattering was m onitored bysetting kex ¼ kem ¼ 400nm1,50mMbuﬀerofpH7.0,2,30°C, 3,35 °C, 4, 40 °C and 5, 45 °C. (B) C hange in A NS ﬂuorescence in thepresence of A. hirsuta lectin an d GdnHCl. T he ﬂuorescence emissionspectra of the lectin (2.0 lM) in the presence of ANS (50 lM).(kex, 375 nm). Numbers on the curves indicate the molarity ofGdnHCl. (C) Change in ANS ﬂ uorescence in the p resence of t heA. hirsuta lectin a t various temperatures. T he spectra were t aken asdescribed in (B) prote in sam ples treated a t, 1, 30 °C, 2, 40 °C, 3, 50 °C,4, 60 °C, and 5, 70 °C. (D) N ear UV CD s pectra of A. hirsuta lectin(15 lM), lectin exposed at 1, 35 °C , 2, 45 °C, 3, 50 °C, 4, 55 °C, 5,60 °C, 6, 65 °C and 7, 70 °C f or 15 min.1416 S. M. Gaikwad et al. (Eur. J. Biochem. 269) Ó FEBS 2002folding and association of subunits of the lectin as comparedto other plant lectins [5,6].REFERENCES1. Lis, H. & Sharon, N. (1991) lectin–carbohydrate interactions. Curr.Opin. Struct. Biol. 1, 741–749.2. Gurjar, M.M., Khan, M.I. & Gaikwad, S.M. (1998) a-Galactosidebinding lectin from Artocarpus hirsuta: c haracterization of thesugar speciﬁcity and binding site. Biochim. Biophys. Acta 1381,256–264.3. Gaikwad, S.M., Gurjar, M.M. & Khan, M.I. (1998) Fluorimetricstudies on saccharide binding to the b asic lectin fr om Artocarpushirsuta Biochem. Mol . Biol. I nt. 46,1–9.4. Rao, K.N., Gurjar, M.M., Gaikwad, S.M., Khan, M.I. &Suresh, C .G. (1999) Crystallization and preliminary X-ray studiesof the basic lectin from the seeds of Artocarpus hirsuta. ActaCrystallo. D 55, 120 4–1205.5. Ahmed, N ., Srinivas, V .R., Reddy, G.B. & Surolia, A. (1998)Thermodynamic characterization of the confor mational stabilityof th e homodimeric protein, Pea lectin. Bi oc hemi stry 37, 16765–16772.6. Reddy, G .B., Srinivas, V.R., Ahmed, N . & Surolia, A. (1999)Molten globule like state of peanut l ectin monomer retains itscarbohydrate speciﬁcity. J. Biol. Chem. 274, 4500–4503.7. Reddy, G.B., Bharadwaj, S. & Surolia, A. (1999) Thermal stabilityand m ode o f oligomerization of the tetrameric Peanut agglutinin:a d iﬀerent scanning calorimetric stud y. Bioc hemistry 38 , 4 464–4470.Ó FEBS 2002 Denaturation studies of Artocarpus hirsuta lectin (Eur. J. Biochem. 269) 1417 . Artocarpus hirsuta lectin Differential modes of chemical and thermal denaturation Sushama M. Gaikwad, Madhura M. Gurjar* and M. Islam KhanDivision of. bind carbohydrates speciﬁcally and reversiblyare termed as lectins. They occur ubiquitously in nature and have diverse role in plants, animals and microbes.
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