陽離子雙子表面活性劑誘導的α-CT超活性和構(gòu)象變化

白光月1,*劉君玲1王九霞2王玉潔2,*李艷娜1趙揚1,*姚美煥1

(1河南師范大學化學化工學院，精細化學品綠色制造河南省協(xié)同創(chuàng)新中心，綠色化學介質(zhì)與反應教育部重點實驗室，河南新鄉(xiāng)453007；2河南科技學院化學化工學院，河南新鄉(xiāng)453003)

報道了α-糜蛋白酶(α-CT)催化活性及其與陽離子雙子表面活性劑(N,N′-雙(十二烷基二甲基)-1,2-二溴化癸二銨，簡寫為12-10-12)相互作用熱力學的關(guān)系。酶活性通過紫外-可見吸收光譜法測量底物醋酸2-萘酯(2-NA)分解速率進行評估。在較短的培育時間內(nèi)，12-10-12能夠激活α-CT，得到催化2-NA分解的超活性，同時也加速了酶變性的動力學過程。在低于α-CT/12-10-12體系的臨界聚集濃度(cac12-10-12,CT)時，顯示一個鐘形的大的超活性區(qū)。酶活性隨時間變化的結(jié)果表明，由12-10-12激活的α-CT有高的酶活性和低的變性穩(wěn)定性。進而采用等溫滴定量熱(ITC)、穩(wěn)態(tài)熒光光譜和差示掃描量熱(DSC)技術(shù)研究了12-10-12誘導α-CT超活性的機理。結(jié)果表明酶的超活性來源于正電荷的12-10-12與α-CT相互作用對α-CT內(nèi)部結(jié)構(gòu)的擾動，使得酶的構(gòu)象變得比處于弱相互作用平衡的天然酶的構(gòu)象更加松弛，這有利于2-NA水解酸性產(chǎn)物釋放的動力學，而同時也導致了α-CT結(jié)構(gòu)的不穩(wěn)定性。

表面活性劑；α-糜蛋白酶；超活性；等溫滴定量熱；穩(wěn)態(tài)熒光；差示掃描量熱

1 Introduction

α-chymotrypsin(α-CT),like most other protein molecules, maintains its conformation mainly owing to the presence of abundanthydrogen bonds and hydrophobic interactions among the amino acid residuals in the native structure.Therefore,the effect of surfactant on its activity is attributed to the perturbation in protein′s conformation due to the interaction betweenα-CT and polar or apolar moiety of surfactant,notonly atthe active residues butalso atotherfunction groups.In many studies,α-CT is selected as a model enzyme in order to understand the possible mechanism on the interaction of surfactant with globular proteins in the presence of anionic,cationic and neutralsurfactants1－11.Itis well known thatanionic surfactant,sodium dodecylsulfate(SDS)inhibits the activity ofα-CT and many other globular proteins12,13,but cationic surfactants can activate their activity,as it has been reviewed that their superactivity and stabilization have been obtained in cationic surfactant solutions or some cosolvents1,2.These documented that the charge of surfactants seems to be a main factor controlling enzyme activity.On the other hand,itwas found that cetyltrimethylammonium bromide(CTAB)inhibits the hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide(GPNA)4－6, butitcan activate the enzyme in 80%for catalyzing hydrolysis of the substrate p-nitrophenylacetate9,demonstrating the dependence of the enzyme activity on the substrate.Itwas considered thatthe difference stems from the distribution of differentsubstrates between bulk buffer and micelles9.Dodecyltrimethylammonium bromide(DTAB)affects catalytic activity ofα-CT quite mildly for hydrolysis of 2-naphthylacetate(2-NA)7,14,but very strongly for hydrolysis of GPNA with a superactivity up to a factor of 13 times10.In the presence of cetyltripropylammonium bromide (CTPAB)and cetyltributylammonium bromide(CTBAB),a large superactivity was found and the highest superactivity was observed in CTBAB solution ata molarratio of surfactantto enzyme molecules of 5:1,where the corrected catalytic efficiency reaches a value 35 times higher than one in pure buffer4－6.Therefore,the charge as well as its hydrophobicity and/or size of head group of surfactant,play a criticalrole in enzyme-surfactantinteractions4－6,8.

So far,the large enzymatic superactivity was found only for a few systems including cationic surfactantas mentioned above,but the mechanism on superactivity activated by the surfactantis far from clarifying.Considering the restricted spacer length between two headgroups and much lower critical micelle concentration (cmc)ofgeminisurfactants,in presentwork,we chose decanediylα,ω-bis(dodecyldimethylammonium bromide)(assigned as 12-10-12)as a representative gemini surfactant to study its effect on activity and conformation ofα-CT.Because of the long spacer (―(CH2)10―),the hydrophobicity and size of double headgroups of 12-10-12 are larger than those of its corresponding monomer surfactant DTAB,which may be favorable for inducing superactivity ofα-CT.Thus for the mixed system 12-10-12/α-CT,we presented the results obtained from UV-Vis absorption spectrum, calorimetry(ITC and DSC),and steady state fluorescence,and discussed in detailthe effect of 12-10-12 on the enzyme activity. Further the interaction mechanism between 12-10-12 andα-CT was proposed.

2 Materials and methods

2.1 Materials

α-chymotrypsin(α-CT)was purchased from Aladdin(1000 u· mg－1),Na2HPO4·12H2O and NaH2PO4·2H2O from Sinopharm Chemical Reagent Co.(AR grade),2-naphthylacetate(2-NA) from Aladdin(＞98%)and 2-naphthyl(2-N)from TCI(＞99%). Phosphate buffer solution(PBS)of 10 mmol·L－1with pH 7.3 was prepared by dissolving Na2HPO4·12H2O and NaH2PO4·2H2O in double-distilled water(conductivity 1.2×10－6S·cm－1)produced by an Automatic distiller(SZ-93,ShanghaiYarong Biochemistry Instrument Factory,China)and all the aqueous solutions were prepared with the PBS except specially emphasized ones.Decanediyl-α,ω-bis(dodecyldimethylammonium bromide)(12-10-12) was synthesized and purified as described in our previous work15. Allchemicals for 12-10-12 synthesis were analyticalgrade.Allthe chemicals employed in the work were used as received.

2.2 Assessment for enzymatic activity ofα-CT

The activity ofα-CT was assessed by the rate of 2-NA hydrolysis both in buffer(10 mmol·L－1PBS of pH=7.3)and in buffered 12-10-12 solution14.The reaction was followed spectrophotometrically by the increase of 2-N absorbance as a function of time ata wavelength of 328 nm at(298.2±0.1)K.The UV-Vis absorption spectrum was recorded with a TU-1900 UV-Vis spectrophotometer(Purkinje General Instrument Co.,Ltd.,Beijing, China)thatwas equipped with quartz cuvettes with a lightpath of 10 mm.The measured extinction coefficient of 2-N was(1.68± 0.08)×103mol·L－1·cm－1,thatis similar to the previous value14.

2.3 ITCmeasurements

An isothermal titration calorimeter(Part No 3410,1 mL reaction vessels)with a Thermostat TAM III(4 channels,TA Instruments,USA)was used to determine the enthalpy changes ofsurfactantdilution and evaluate the interaction betweenα-CT and 12-10-12.The details of experimental procedure have been described previously16,17.Briefly,a titrating solution was automatically added in aliquots of 3－6μL from a gas tight Hamilton syringe controlled by a precision syringe pump with a control module(P/N 3810-5)through a thin stainless steelcapillary until the desired range of concentration had been covered.During the whole titration process,the system was stirred at80 r·min－1with a gold propeller,and the intervalbetween two injections was 8 min for the signal to return to the baseline.All measurements were performed in triple titrations at(298.15±0.01)K.

2.4 Steady-state fluorescence spectra

Fluorescence measurements were performed on A CARY Eclipse fluorescence spectrophotometer(Varian,America).In the measurement of the intrinsic fluorescence ofα-CT solution,a quartz cuvette of 10 mm path length was used and the excitation and emission slits were both fixed at 5.0 nm.The excitation wavelength was set at 295 nm,and emission spectrum was recorded from 300 to 450 nm.The scan rate was setto be 500 nm· min－1and the wavelength pitch of acquired spectrum was 1 nm. The fluorescence spectra of the bufferedα-CT solution with 12-10-12 of differentconcentrations were measured at298.2 K.

2.5 DSC measurements

DSC experiments were performed in a microcalorimeter(GE MicroCal VP-DSC,USA).Two fixed steelcells with a volume of 0.529 mL were filled with sample solution and reference solution of buffer(10 mmol·L－1PBS),respectively.The solutions were degassed with MicroCal ThermoVac before injecting into the individual cell at room temperature.The samples were equilibrated at the starting temperature for 15 min,and then the scan was performed for each sample ata scanning rate of 1°C·min－1over a temperature range of 10－95°C.The effectof buffer was corrected by running with buffer solution in both cells.The curves of heat capacity versus temperature(Cpvs T)can be corrected by subtracting the blank curve(PBS in both sample and reference cells)and baseline,using the software especially designed for MicroCal VP-DSC.Thereafter,the corrected curves were fitted with Gaussian function to obtain multi-peak fitting lines.The temperature Tmatthe extreme Cpcan be identified and the enthalpy changeΔH corresponding to each peak can be obtained by integral area of individualpeak.

Fig.1 Variation of the relative activity(V12-10-12/Vb)ofα-CT with concentration of 12-10-12(C12-10-12)The symbols indicate thatα-CT was incubated 10 min(●),3 h(○)and 21 h(Δ)in 12-10-12 solution.The concentrations of the other components except 12-10-12 were constant,0.020 g·L－1α-CT,0.097 mmol·L－12-NA, 10 mmol·L－1PBS(pH=7.3),respectively.

3 Results and discussion

3.1 Enzymatic activity ofα-CT in 12-10-12 solution

The initial rate of 2-NA hydrolysis ofα-CT catalysis was measured atthe constantconcentrations of 0.097 mmol·L－12-NA, 0.02 g·L－1α-CT and 10 mmol·L－1PBS(pH=7.3)and various 12-10-12 concentrations,which was followed by the change in the absorbance at328 nm.The plotof the absorbance vs time exhibits a good linearity in initial5 min.The relative activity(V12-10-12/Vb) ofα-CT was represented as a ratio of the initial hydrolysis rate (V12-10-12)under the different conditions(12-10-12 concentration and incubation time)to the catalytic rate(Vb)in pure buffer solution.The variation of V12-10-12/Vbofα-CT with the concentration of 12-10-12,C12-10-12,was shown in Fig.1,where the protein was incubated 0.17 h(10 min),3 h and 21 h in 12-10-12 solution, respectively.In the absence of 12-10-12,the ratio V12-10-12/Vbofα-CT decreases as incubation time increases,which reflects the common characteristics of enzymes in buffer solution.For the cases thatthe enzyme is incubated 10 min and 3 h,the ratio V12-10-12/ Vbin the presence of 12-10-12 is larger than in its absence(V12-10-12/ Vb＞1)in the studied concentration range of 12-10-12,and the large superactivity appears in a bell-shape in a concentration region of12-10-12 below its cmc12-10-12(0.13 mmol·L－1,see following section).The concentration range of the bell-shaped superactivity region becomes narrow with the increase of incubation time.The superactivity decreases with incubation time,and the maximum superactivity shifts toward low 12-10-12 concentration from V12-10-12/ Vb=1.9 at C12-10-12=0.044 mmol·L－1for the case of incubation of 10 min to V12-10-12/Vb=1.7 at C12-10-12=0.020 mmol·L－1for incubation of 3 h.When the enzyme was incubated 21 h,the significantinactivation was found whatever 12-10-12 was presentor not and the rate of enzymatic inactivation was faster than one in pure buffer solution,indicating thatthe high activity and low stability ofα-CT are correlated with the 12-10-12 molecules binding toα-CT surface.

In blank experiment withoutα-CT no detectable product 2-naphthyl(2-N)was found in 10 min in the absence or presence of gemini12-10-12,so thatthe self-hydrolysis of2-NAorthe micellar catalysis can be safely ruled out.On the other hand,the substrate distribution in surfactantaggregates may give rise to the decrease in the free substrate concentration and then the decrease in the relative activity when 12-10-12 concentration is above its cmc12-10-12.However,we can′t identify the effect of substrate distribution from the difference between the extinction coefficients of2-NAin buffer and in 12-10-12/buffer solution(data notshown) in this work9.We might consider,therefore,that the 12-10-12 clusters binding on the enzyme surface affectthe reaction kinetics.

In previous studies7,18,19,the mechanism of 2-NA hydrolysiscatalyzed byα-CT in DTAB solution was presented by following steps:

where S stands for the substrate,E for the enzyme,SE for the enzyme-substrate complex,S′E for the acyl-enzyme intermediate and P1and P2for the basic and acidic products,respectively. Further these authors proposed thatthe effect of surfactant DTAB on the enzymatic activity occurs in the laststep owing to the increase in the rate constant k3.In our studied system with gemini/ α-CT the release rate of the acidic productshould be correlated to the conformationalmodification induced by 12-10-12.Accordingly we will focus on the interaction between 12-10-12 andα-CT and the effect of 12-10-12 on the conformation ofα-CT in the following sections.

3.2 A calorimetric study on interaction ofα-CT with 12-10-12

The enthalpy of interaction between 12-10-12 andα-CT can be characterized by ITC and thus the events of the intermolecular interaction can be understood in terms of thermodynamics.The cmc12-10-12of 12-10-12 and its enthalpy of micellization(ΔHmic)in pure water were determined by ITC according to the reported method17as shown in Fig.2.It was found that the values of cmc12-10-12andΔHmicare(0.42±0.01)mmol·L－1and(－6.7±0.1) kJ·mol－1,respectively,which are consistent with literature values17,20.The cmc12-10-12,PBSvalue of 12-10-12 in 10 mmol·L－1PBS is 0.13 mmol·L－1,and the exothermicΔHmic,PBSis(－4.7±0.1)kJ· mol－1,being much smaller than in pure water.These results might stem together from the effects of ionic strength and the partial charge neutralization of 12-10-12 with phosphate anions(electrostatic attraction).In the bufferedα-CT solution,itwas observed that 12-10-12 molecules can aggregate to form micelles free or binding onto the protein′s amino residuals at a critical concentration of 12-10-12 assigned as cac12-10-12,CT.The results show thatin the presence ofα-CT in PBS the cac12-10-12,CTand the corresponding enthalpy of aggregation,ΔHagg,CTdoesn′t deviate significantly from the cmc12-10-12,PBSandΔHmic,PBS,respectively,indicating thatonly a few sites on the protein surface can bind with 12-10-12 molecules or clusters.

Considering thatthe bell-shaped superactivity region appears in dilute 12-10-12 concentration range,itis necessary to explore the interaction of 12-10-12 withα-CT in the same concentration range.Therefore,the titration of2.0 mmol·L－112-10-12 intoα-CT solution of different concentrations was carried out and the observed enthalpy curves were shown in Fig.3(I),where the dilution enthalpy curve for titration of 2.0 mmol·L－112-10-12 into buffer solution was also included for comparison.When there is noα-CT in the system,the first four injections in PBS lead to negative observed enthalpies,whereas the similar injections in pure water always have positive values21.It reflects thatthe equilibrium for the interaction of 12-10-12 cation with phosphate anion should shiftin the direction of the charge neutralization of 12-10-12 in the process of 12-10-12 dilution.The observed enthalpy of 12-10-12 dilution into PBS consists of(i)the enthalpy of demicellization of 12-10-12 micelles into monomers;(ii)dispersion of concentrated monomers;(iii)hydration of monomers and(iv)specially for the buffered 12-10-12,the exothermic interaction of cationic 12-10-12 with negative phosphate ions.

Fig.2 Variation of observed enthalpy per mole of12-10-12 with the concentration of 12-10-12 at298.15 K(■)in water;(○)in 10 mmol·L–1PBS(pH 7.3);(Δ)in 10 mmol·L－1PBS with 0.02 g·L－1α-CT in the cell.The C12-10-12in the syringe is 6.2 mmol·L－1.

Fig.3 ITC results for titration of 12-10-12 into α-CT solution at 298.15 K(I)Variation of observed enthalpy with the concentration of 12-10-12, (II)Difference in the observed enthalpy values in the presence ofα-CT and in its absence.Theα-CT concentration(g·L－1)in 10 mmol·L－1PBS(pH 7.3):(■)0; (○)0.05;(▲)0.10;(Δ)0.20;and(■)0.30,respectively. The C12-10-12in the syringe is 2.0 mmol·L－1

It was seen that before cac12-10-12,CT,all the observed enthalpy curves in the presence ofα-CT with Cα-CT=(0.05 to 0.30)g·L－1or (2.0 to 12)μmol·L－1deviate positively to some extent from thedilution enthalpy curve in PBS(Fig.3(I)).Thus the difference (Δ(ΔHobs))between the observed enthalpies in the presence ofα-CT and in its absence at each 12-10-12 concentration should highlight the interaction of 12-10-12 withα-CT.The plots of (Δ(ΔHobs))as a function of 12-10-12 concentration were drawn in Fig.3(II).When Cα-CTis 0.05 g·L－1,the enthalpy curve has a similar profile to one in the absence ofα-CT,and the positiveΔ(ΔHobs)was obtained,indicating thatan endothermic effectoccurs during the titration.For the other curves where Cα-CTis larger than 0.05 g·L－1, the observed enthalpies steeply increase for the initial several injections and then start to decrease at an extreme value Cn,at which a charge neutralization of the negatively charged amino residuals on theα-CT surface is reached through the electrostatic interaction.The interaction is called specific binding ofa few 12-10-12 molecules to the native protein22－24.In the whole studied concentration range of 12-10-12 main interaction components could include:(i)the dissociations of phosphate ions from the geminiheadgroups and/or hydrogen ions from the amino residuals on protein surface against the electrostatically attractive interaction between 12-10-12 andα-CT;(ii)possible hydrophobic interaction between surfactant alkyl chains and hydrophobic microdomain formed by hydrophobic side groups of amino residuals; (iii)the conformational change of protein against the intramolecular weak interactions(such as hydrogen bond)and(iv)hydrophobic interaction among surfactant alkyl chains owing to cluster formation on the protein surface.The surface charge of protein depends on pH value of aqueous media,which affects strongly the enthalpy of electrostatic interaction25.The used PBS has a pH value(pH=7.3)below the isoelectric point(pI=8.8)of α-CT14,suggesting that the electrostatic interaction only brings about a small exothermic effect.The hydrophobic interaction between 12-10-12 andα-CT should be weak as a consequence of the shortside chains of negatively charged amino residuals(Asp and Glu)26.Therefore,we could ascribe the positiveΔ(ΔHobs) mainly to the conformationalchange ofα-CT and the surfactant/ α-CT cluster formation,which will be further confirmed by fluorescence and DSC results(see following sections).

The charge neutralization concentration,Cnof 12-10-12 for the specific interaction(Fig.3(I))indicates the highest critical concentration where the surfactantmolecules bind on protein′s surface in the form of surfactantmonomer.After Cn12-10-12 molecules form clusters on protein′s surface by cooperative interaction of 12-10-12 molecules.According to mass balance of surfactant/α-CT interaction27,we have

Fig.4 Fluorescence spectra ofα-CT atthe different incubation time in PBS at298 KThe incubation time increases along the arrow direction:0.33,3.0,24,48 and 72 h,respectively,and the Cα-CTwas keptat0.02 g·L－1.

where N12-10-12is the binding molecule number of 12-10-12 perα-CT and Cmonois the concentration of free monomer 12-10-12. Linear fitting gives N12-10-12=(1.5±0.2)and Cmono=(0.0093± 0.0013)mmol·L－1.The nativeα-CT in neutralsolution(pH≈7) exists in monomer-hexamerequilibrium28,so thatitis possible that N12-10-12has a non-integer value.Here,it is necessary to recall activity assessment as shown in Fig.1.In the experimentof activity assessment,theα-CT concentration was keptat 0.02 g·L－1(0.008 μmol·L－1),and thus the Cnvalue of 12-10-12 for specific interaction is(0.0094±0.0013)mmol·L－1.For the relative activity curve incubated 10 min in 12-10-12 solution in Fig.1,the Cnvalue corresponds to the startof the bell-shaped superactivity region. Therefore,it is reasonable to drive a conclusion that the superactivity occurs when the cooperative interaction appears.As the 12-10-12 concentration increases,after formation of 12-10-12 clusters,Δ(ΔHobs)values for all the curves(Fig.3(II))decrease gradually and arrive at zero at aboutthe cmc12-10-12,PBSvalue(0.13 mmol·L－1).

3.3 Effect of 12-10-12 concentration onα-CT conformation

The fluorescentspectra ofα-CT were registered as a function of surfactant concentration for characterizing the effect of surfactant on the protein′s Trp fluorescence spectra.This will give information notonly on the changes in the protein′s conformation, but also changes in polarity caused by the binding of surfactant molecules owing to the high sensitivity of Trp fluorescence to even smallchanges in its polar environment.α-CT has eight Try residues,in which six are located at the surface,and other two buried in the interior of the native protein29,30.However,only two Trp residues presentaround 46%(Trp207)and 49%(Trp237)of exposed area to the solvent,while the other residues presenta low accessibility to aqueous solventwith less explored area than 20% of total area9.The fluorescence intensity at 335 nm decreases strongly,butits maximum emission wavelength does notchange after incubating from 3 to 24 h in PBS,as shown in Fig.4,reflecting the increase in the polarity of Trp environment31,32.For the longer incubation time the spectra losttheir originalcharacteristics owing to unfolding of the protein structure.The obtained results are consistent with the decrease of enzymatic activity with the incubation time,as we have known from Fig.1.

In a mixture of enzyme and surfactant,especially ionic surfactant,protein′s conformation often changes with unfolding or folding caused by disturbing its weak hydrogen bond network.The changes in emission spectra from Trp residuals can reflectprotein conformationaltransition,subunit association,ligand binding,or denaturation,all of which can affect the localenvironment sur-rounding the indole ring.For the case of the mixed system 12-10-12/α-CT,when the C12-10-12is 0.025 mmol·L－1(Fig.5(I)),the spectrum afterreaction of10 min with 12-10-12 overlaps with one of the nativeα-CT,indicating an almostunchanged tertiary conformation related to the Trp environment.Itis consistentwith the relative activity close to unit value at the same 12-10-12 concentration in Fig.1(at the left side of the bell-shape).When the incubation time increases until 190 h,the emission intensity decreases and the maximum emission wavelength shifts progressively from 335 to 350 nm,which corresponds to the diminishing and even complete loss of enzymatic activity with time.When the 12-10-12 concentration closes to or excesses its cac12-10-12,CTvalue (Fig.5(II)and Fig.5(III)),in the initial 30 min the spectra have an increasing maximum emission intensity at335 nm,following a red shift of the emission wavelength until 356 nm and an accompanying decrease in the fluorescence intensity.The increase in intensity ofα-CT fluorescence suggests that in the original conformation at least some of the Trp residues are located close to other residues,such as Arg residual,which can lead to internal quenching9.The conformational change resulted from the interaction with 12-10-12 monomers or micelle restrains the internal quenching effect.Therefore,the increasing intensity could signify thatprotein structure becomes more flexible,butthe Trp residuals are not exposed in water solvent.In comparison of the fluorescence results with the kinetic assessment and ITC studies,the interaction of 12-10-12 withα-CT leads to the flexible conformation ofα-CT and the rapid decomposition dynamics of the transient state of enzyme-substrate(see Eq.(1))in a short incubation time.Itshould be stressed that after incubation of 30 min, the fluorescence enhancementand red shiftcoexistfor both cases in Fig.5(II)and Fig.5(III).This confirms the appearance of the structuralunfolding.

Fig.5 Fluorescence spectra ofα-CT at different incubation time in different 12-10-12 concentrations at 298 KThe concentration of 12-10-12:(I)0.025 mmol·L－1,(II)0.073 mmol·L－1, (III)0.16 mmol·L－1.The incubation times(unitin h)were denoted as a,0.33 in buffer;b,0.17;c,2.0;d,3.0;e,23;f,47;g,96;h,144;i,190.The arrows point to the corresponding curve or the direction ofincrease in incubation time. Notes:before addition of12-10-12 the bufferedα-CT solution was incubated 0.33 h in PBS and the Cα-CTis keptat0.02 g·L－1.

3.4 Thermalstability ofα-CT

When a protein solution is gradually heated,the curve of its heat capacity vs temperature(Cpvs T)undergoes commonly a sharp peak,which was caused by a transition from its native state to a denatured one33,34.The corresponding transition enthalpy and temperature did characterize the stability of a protein,defined as the tendency to maintain a native conformation.Upon heating the thermal unfolding of the protein′s native structure occurs.The thermalstability ofα-CT was determined by DSC.Fig.6(I)shows DSC heating scans obtained forα-CT in the presence of 12-10-12. For the studied systems,the addition of 12-10-12 induces peak splitting from the lowestconcentration:two peaks appear,which can be fitted into the componentcurves by Gaussian function.The Tm,1and Tm,2values and the relative heat contribution to total transition can be obtained by integration.In Fig.6(II),we showed two typicaloriginalcurves together with the componentcurves as obtained from Gaussian fitting.Allthe transition temperatures and corresponding enthalpy changes for the studied systems with the differentconcentrations of 12-10-12 were collected in Table 1.

For pureα-CT,the Cpvs T curve presents two transitions:Tm,1corresponding to the main peak and Tm,2corresponding to the right shoulder.As the 12-10-12 concentration increases from(0.005 to 0.08)mmol·L－1,the both transition temperatures Tm,1and Tm,2shift together to low temperature and the enthalpy change(ΔH1)corresponding to the first transition decreases.When the 12-10-12 concentration exceeds 0.12 mmol·L－1,both Tm,1and Tm,2arrive at their respective constants.The transition temperatures seem to be related to the cmc12-10-12,PBSvalue(cmc12-10-12,PBS=0.13 mmol·L－1).It was found thatvariation ofΔH1values for the firsttransition with C12-10-12exhibits a break atcmc12-10-12,PBS,butfor the second one,ΔH2has an approximate constant value in the whole studied concentration range of 12-10-12,about(1.6±0.3)J·g－1(Table 1).Thedouble transition temperatures suggest a thermal denaturation mechanism ofα-CT with multi-transitions.

The temperature Tm,1indicates thata half of the native enzyme is denatured atthe temperature.The decrease in both Tm,1andΔH1below cmc12-10-12,PBS,should reflectthe change in the protein conformation.The enzyme could be in a conformation thathas weaker internalinteraction and then less conformationalstability than the native one.After cmc12-10-12,PBSthe approximtely constant Tm,1and ΔH1suggestthatan interaction of 12-10-12 withα-CT arrives at saturation.As shown in Fig.2,the cmc12-10-12,PBSis equal to the cac12-10-12,CT,so that the free 12-10-12 micelles start to form at cac12-10-12,CT.The second transition temperature Tm,2follows the denatured conformation ofα-CT after Tm,1.Itwas worth noting that the second transition has a constanttransition enthalpy independent on 12-10-12 concentration.Therefore,the transition has a reaction mechanism correlated only with the conformation of the protein itself.It is well-known thatα-CT tends to self-aggregate in aqueous solution and the aggregation number depends on pH value.At pH=7.3 for our systems the protein is in monomerhexamer equilibrium28.The second transition reflects most possibly the dissociation of the aggregates,which occurs always by following the denatruration process.Nevertheless,the decrease in Tm,1andΔH1with incresing the concentration of 12-10-12 suggests that the enzyme has a weakened conformational stability,which is consistent with the activity change(Fig.1)and red shift of the fluorescence spectra(Fig.5).

Fig.6 DSC curves forα-CT solutions incubated 20 min in buffer and 12-10-12/buffer(I)The 12-10-12 concentrations(mmol·L－1)increase along the arrow direction to be:0,0.005,0.01,0.02,0.04,0.08,0.12,0.16,0.20,0.25,0.28,respectively. (II)Two representative originalcurves were presented in symbol(○)for C12-10-12=0 and(Δ)for C12-10-12=0.08 mmol·L－1.Multi-peak fitting lines of experimentalcurve Cpvs T forα-CT/12-10-12 were obtained through Gaussian function and the corresponding temperatures were marked with Tm,1and Tm,2.

4 Conclusions

The superactivity ofα-CT for catalysis of 2-NA hydrolysis is induced by cationic gemini surfactant 12-10-12 both below and above cmc12-10-12,PBSin 10 mmol·L－1PBS in shortincubation time (3 h in this work)and the large superactivity in a bell-shape occurs below cmc12-10-12,PBS.In the bell-shaped region the interaction enthalpy between the protein and 12-10-12 is endothermic (Δ(ΔHobs)＞0).While the fluorescence intensity decreases,the maximum emission shifts to long wavelength as the incubation time increases and the denaturation temperature and enthalpy change reduce.Further combining results from the various methodologies suggestthatthe interaction of 12-10-12 withα-CT weakens the internalinteraction ofα-CT,which relaxes the tertiary conformation and diminishes the protein′s conformational stability,leading to much higher activity and a little faster denaturation.After cac12-10-12,CT(outof the bell-shaped region),12-10-12 bonding onα-CT reached a saturation concentration,the Trp fluorescence intensity is enhanced withoutred shift in short interval,buta large red shiftappears athigh intensity levelas the time increases,which means a flexible and instable conformation. The further evidences for the conformational change are lower temperature and smaller enthalpy change for its denaturation. Therefore,after cac12-10-12,CTthe surfactantaffects the conformation ofα-CT and its conformationalstability more significantly.Allthe results in this work together highlight the high activity and low conformationalstability ofα-CT in the presence ofcationic gemini surfactant 12-10-12,which will provide fresh insight into our current understanding for interaction mechanism and potential applications of enzyme/surfactantsystems.

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Enzymatic Superactivity and ConformationalChange of α-CT Induced by Cationic GeminiSurfactant

BAIGuang-Yue1,*LIU Jun-Ling1WANGJiu-Xia2WANGYu-Jie2,*LIYan-Na1ZHAOYang1,*YAO Mei-Huan1
(1Collaborative Innovation Centre of Henan Province for Green Manufacturing ofFine Chemicals,Key Laboratory of Green Chemical Media and Reactions,Ministry of Education,Schoolof Chemistry and Chemical Engineering, Henan NormalUniversity,Xinxiang 453007,Henan Province,P.R.China;2School of Chemistry and Chemical Engineering, Henan Institute of Science and Technology,Xinxiang 453003,Henan Province,P.R.China)

This work presents the correlation of the enzymatic activity ofα-chymotrypsin(α-CT)with the thermodynamics of interaction betweenα-CT and the cationic gemini surfactant decanediyl-α,ω-bis (dodecyldimethylammonium bromide)(12-10-12).The enzymatic activity was assessed by the rate of2-naphthyl acetate(2-NA)hydrolysis obtained from UV-Vis absorption spectra.The superactivity ofα-CT in the catalytic hydrolysis of 2-NA was obtained by activation with 12-10-12 in a short incubation time;the activatedα-CT showed faster denaturation kinetics.The larger superactivities appeared in a bell shape below the critical aggregation concentration(cac12-10-12,CT)ofthe mixed gemini/α-CT systems in buffered aqueous solution.The results obtained from the variation ofthe activity with the incubation time highlightthatthe protein incubated in 12-10-12 has a high catalysis activity and a weakened conformational stability.The mechanism of the superactivity ofα-CT in the presence of12-10-12 has been proposed by combining the results from isothermaltitration calorimetry(ITC),steady state fluorescence,and differential scanning calorimetry(DSC).The superactivity arises from perturbation ofthe internalstructure ofα-CT by an interaction between the positively charged 12-10-12 andα-CT,which makes the conformation ofα-CT looser than the native one,in the balance ofa weak interaction.Such a conformation is favorable for release of the acidic product of2-NA hydrolysis, whereas itsimultaneously leads to instability oftheα-CT structure.

Surfactant;α-chymotrypsin;Superactivity;Isothermaltitration calorimetry;Steady state fluorescence;Differentialscanning calorimetry

O642；O648

Banerjee,D.;Pal,S.K.Langmuir 2008,24,8163.

10.1021/ la8010184

doi:10.3866/PKU.WHXB201702089

Received:January 13,2017;Revised:February 5,2017;Published online:February 8,2017.

*Corresponding authors.BAIGuang-Yue,Email:baiguangyue@htu.cn;Tel:+86-373-3328622.WANG Yu-Jie,Email:yujiewang2001@163.com. ZHAO Yang,Email:zhaoyang@htu.cn.

The projectwas supported by the NationalNatural Science Foundation of China(21273061,21573061)and Scientific Research Projectof Higher Education of Henan Province,China(17A150032).

國家自然科學基金(21273061,21573061)和河南省高等學校重點科研項目(17A150032)資助?Editorialoffice of Acta Physico-Chimica Sinica