Enzymes in the Environment: Activity, Ecology and Applications - Chapter 11 ppsx

11 Enzyme Adsorption on Soil Mineral Surfaces and Consequences for the Catalytic Activity Herve ´ Quiquampoix Institut National de la Recherche Agronomique, Montpellier, France Sylvie Servagent-Noinville and Marie-He ´ le ` ne Baron Centre National de la Recherche Scientifique, Universite ´ Paris VI, Thiais, France I. INTRODUCTION Soil enzymes are either actively secreted by living microorganisms and plant roots or released after the death of soil biota by cell lysis. One class of enzymes, the hydrolases, have a very important role in the biogeochemical cycles of major elements (C, N, P, and S) since their substrates in soil are mainly in a polymerized form. Usually microorganisms (and plant roots) cannot take up macromolecules directly from the external medium. There are few exceptions to this, such as the uptake of fragments of deoxyribonucleic acid (DNA) or plasmids, that lead to transformation in bacteria. In general, the membrane transport systems are specific and recognize the universal biological monomers, such as amino acids, sugars, and nucleotides; low number oligomers, such as cellobiose or maltose; or mineral ionic groups that can be released by enzymatic hydrolysis of organic molecules, such as orthophosphate and sulfate. Extracellular enzymes perform three main functions in soil: (1) they reach substrates in pores with dimensions roughly 100 times smaller than those of bacteria; (2) they hydrolyze these substrates and make them soluble and consequently able to diffuse back to the microorganisms or plant roots; and at the same time (3) they transform polymers into monomers or oligomers that can be recognized and taken up by membrane transport systems to undergo intracellular metabolism. As enzymes are proteins, they share with this class of macromolecules their strong affinity with interfaces. Proteins are potentially flexible polypeptide chains, even if they have a stable folded configuration in solution, with individual amino acids whose lateral chains have various physicochemical properties: hydrophilic or hydrophobic; negatively, neutrally, or positively charged. From a thermodynamic point of view, these properties give rise to both enthalpic (related to intermolecular forces) and entropic (related to the spatial arrangement of the molecules) contributions to the interactions with surfaces. The strong and often largely irreversible adsorption of enzymes on the mineral phase of the soil Copyright © 2002 Marcel Dekker, Inc. has important consequences, not only for their mobility, but also for their survival and catalytic activity. The most well-known effect of the adsorption of enzymes on negatively charged surfaces, such as clay minerals, is a shift of their optimal catalytic activity towards a higher pH range (1–4). A second general effect, which can be extended to all proteins, is that the maximal adsorption is observed near the isoelectric point (i.e.p.) of the enzyme. McLaren et al. described these two properties in the 1950s (5–7). However, until the beginning of the 1990s, no effort, supported by independent structural study of the ad- sorbed enzymes or proteins by modern physical methods, was made to propose a mecha- nism explaining both observations. II. NATURE OF THE DRIVING FORCES LEADING TO ADSORPTION OF ENZYMES ON SURFACES Studies on the quantity of protein adsorbed on surfaces cannot be separated from the study of their conformation on these surfaces. The reason is that a modification toward a more disordered structure contributes to the driving forces of adsorption, since it increases the entropy of the system and thus decreases the Gibbs energy. The modification of conforma- tion also can have an effect on the maximal quantity of protein adsorbed, since conforma- tional changes may affect the area occupied by each single protein on the surface. The spontaneous adsorption of proteins at constant temperature and pressure leads to a decrease of the Gibbs energy of the system, according to the second law of thermody- namics (8–13). The Gibbs energy, G, depends on enthalpy, H, which is a measure of the potential energy (energy that has to be supplied to separate the molecular constituents from one another), and entropy, S, which is related to the disorder of the system: ∆ ads G ϭ ∆ ads H Ϫ T∆ ads S Ͻ 0 where T is the absolute temperature and ∆ ads is the change in the thermodynamic functions resulting from adsorption. Some difficulties arise in the analysis of these processes because enthalpic effects, related to intermolecular forces, and entropic effects, related to the spatial arrangements of molecules, are not totally independent. Intermolecular forces influence the distribution of molecules, and the potential energy also is also dependent on the molecular structure of the system. A. Enthalpic Effects 1. Coulombic Interactions The electrical charge of proteins results from the ionization of the carboxylic, tyrosyl, amine, imine, and imidazole groups of the side chains of some amino acids. The electrical charge of mineral surfaces can result from pH-independent isomorphic substitutions in the crystal lattice, as in some clays (basal surfaces of illite or montmorillonite), or from pH-dependent ionization of hydroxyls (edge sites of clays, oxyhydroxides). Coulombic forces are very strong and long-range intermolecular forces. They can be screened by the ions in the solution. As all electrical charges have to be compensated by an equal number of electrical charges of the opposite sign, a diffuse double layer is established around the Copyright © 2002 Marcel Dekker, Inc. macromolecules and mineral surfaces. The electrostatic interactions between proteins and surfaces thus can be analyzed as an overlap of their electrical double layers (8,10). The electrostatic part of the Gibbs energy is given by the isothermal and isobaric work of charging the electrical double layer: G el ϭ Ύ σ 0 φ(σ)dσ where φ is the variable electrostatic potential and σ is the variable surface density during the charging process. 2. Lifshitz–van der Waals Interactions Contrary to the Coulombic interactions, van der Waals forces act on all molecules, even if they are electrically neutral. They are short-range forces and are composed of three different components. The main component are the dispersion (or London) forces, which originate from the instantaneous dipolar moment resulting from the fluctuation of the electrons around the nuclear protons. The electric field created induces, by polarization, a dipole moment in nearby molecules, which, in turn, creates an instantaneous attractive interaction. The two other components are the induction (or Debye) forces, related to the interaction between a polar molecule and a nonpolar molecule, and the orientation (or Keesom) forces, related to the interaction between two polar molecules. B. Entropic Effects 1. Hydrophobic Interactions The stability of proteins in solution results mainly from the shielding of amino acids with a hydrophobic side chain in the core of the protein from contact with water. It is due to the hydrophobic effect that causes water molecules around a nonpolar group to establish more hydrogen bonds among themselves than around a polar group. This process maxi- mizes the mutual association of water molecules by hydrogen bonds and results in an increased order of the surrounding water, and thus a favorable decrease in entropy of the system. Sometimes hydrophobic interactions are involved in interactions of proteins with hydrophilic mineral surfaces. An example is given by the higher affinity of a hydrophobic methylated derivative of bovine serum albumin (BSA) for the hydrophilic montmorillonite surface than the native, less hydrophobic BSA (14). The explanation is that the adsorption of proteins is accompanied by the exchange of charge-compensating cations on the clay, which are the true hydrophilic centers, leaving a hydrophobic siloxane layer (15). Thus, the montmorillonite surface presents hydrophilic properties for molecules whose adsorp- tion does not result in the removal of charge-compensating cations, and hydrophobic prop- erties for molecules that replace the charge-compensating cations. 2. Modifications in Protein Molecular Structure The entropic contribution to adsorption also can result from a modification of the confor- mation of the protein. This phenomenon is related to an increase of the rotational freedom of the peptide bonds engaged in secondary structures, such α helices and β sheets. The ordered secondary structures are an important part of the densely packed hydrophobic core of proteins. After adsorption, internal hydrophobic amino acids can reach more exter- nal positions in contact with the surface, since the amino acids remain shielded from Copyright © 2002 Marcel Dekker, Inc. contact with the water molecules of the surrounding solvent phase. If a decrease of internal ordered secondary structures accompanies this process, it results in an increase of confor- mational entropy. The gain of conformational entropy, S conf , can be calculated from the assumption that four different conformations are possible for peptide units in random structures as compared with only one in α helices and β sheets: ∆ ads S conf ϭ R ln 4 n where R is the molar gas constant and n is the number of peptide units involved in the transfer from an ordered secondary structure to a random secondary structure (9,10). III. EXPERIMENTAL EVIDENCE FOR pH-DEPENDENT CHANGES IN THE STRUCTURE OR ORIENTATION OF ADSORBED PROTEINS The previous thermodynamical considerations indicate that modifications in conformation are an important parameter to consider in the adsorption of proteins, since the entropic effect related to these structural changes is itself a factor of adsorption. These three-dimen- sional changes affect the catalytic activity of the adsorbed enzymes. A major difficulty in the evaluation of the extent of such events is that no experimen- tal method allows the direct measurement of the conformation of proteins in an adsorbed state. Only two methods are suitable for the determination of the tertiary structure of the proteins, and neither can be employed when proteins are adsorbed. One method, X-ray diffraction, necessitates the preparation of protein crystals, which is impossible for ad- sorbed proteins. The other, nuclear magnetic resonance (NMR) spectroscopy, is confined to molecules with a sufficiently high tumbling rate to obtain spectra with narrow linewidth peaks, a condition not compatible with the adsorption on a surface of larger dimension than the protein itself since even a small adsorbed molecule experiences the slower rotational movement of the mineral colloid. Without dramatic advances in solid-state NMR spectroscopy, information on the conformation can be deduced only from lower levels of structural information than the tertiary structure, such as the secondary structure and the specific interfacial area occupied by adsorbed proteins. Two spectroscopical approaches that permit such investigations are now discussed. A. Study of the Interfacial Area of Protein-Surface Contact by Nuclear Magnetic Resonance Spectroscopy The study of adsorption isotherms of proteins on clay mineral surfaces has been disap- pointing with regard to the interpretation of the adsorbed enzyme activity. The idea that can be advanced is that the quantity of protein adsorbed is by itself insufficient to describe a complex phenomenon that can involve at least four other parameters: (1) the orientation of the protein on the surface (this is important as proteins are rarely perfect spheres and are more often described as ellipsoids with a long and a large axis; thus an end-on adsorp- tion involves a higher quantity of protein adsorbed than a side-on adsorption); (2) a possi- ble unfolding of the protein on the surface changing the interfacial area between individual protein and surface and the quantity of protein adsorbed at saturation; (3) the surface coverage at saturation, which could be less than 100% for packing reasons, if the adsorp- tion is irreversible; and (4) the possibility of a multilayer adsorption. Thus, it is always Copyright © 2002 Marcel Dekker, Inc. possibletofindseveralexplanationstointerpretaproteinadsorptionisotherm,withno experimentalevidenceavailabletochooseamongthem.TheadvantageoftheNMR methodisthatitsimultaneouslygivesthequantityofadsorbedprotein,thesurfacecover- ageofthesolidbytheprotein,andthemonolayerormultilayermodeofadsorption(16). Onlyknowledgeofthesethreefactorsallowsapossibleunfoldingoftheproteinsonthe claysurfacestobedetectedandquantified. 1.NuclearMagneticResonanceDetectionoftheExchangeofa ParamagneticCationonProteinAdsorptiononClays Theprincipleofthemethod(16)isbasedonthefactthattheadsorptionofproteinson clayscausesthereleaseofcharge-compensatingcations(7,17).Italsousesthesensitivity oftherelaxationtimesT 1 andT 2 ofnuclearspinstoparamagneticcationsinNMRspectros- copy(18,19). Asmallquantity(between3and20µMdependingonthepH)ofaparamagnetic cation,Mn 2ϩ ,isaddedtoasodium-saturatedmontmorillonitesuspension(1gL Ϫ1 )with a10-mMconcentrationoforthophosphate.Thesuspensionisstudiedby 31 PNMRspec- troscopy.Aninterestingphenomenonisobserved:(1)theMn 2ϩ cationsthatareadsorbed ontheclaysurfacedonotinteractatallwiththeorthophosphate,asshownbythecompari- sonbetweentheclaysuspensionandsupernatantafterremovaloftheclaybycentrifuga- tion;and(2)theMn 2ϩ cationsinsolutioninteractwiththeorthophosphate,leadingtoa linearincreaseofthelinewidthathalfheight,∆ν 1/2 ,oftheorthophosphatepeakonthe NMRspectrum.Thislasteffectistheresultoftheparamagneticcontributiontothede- creaseofthespin–spinrelaxationtime,T 2 ,oftheorthophosphatesignal.Whenagiven quantityofproteinisintroducedintothissuspension,itdisturbstheequilibriumbetween theparamagneticMn 2ϩ adsorbedontheclaysurfaceandthatinsolution.Theanalysisof theresultinglinewidthoftheorthophosphosphatesignalgivesthequantityofcations exchangedonadsorption. Witha300-MHzNMRspectrometer,themeasurementtakesafewminutes;with a500-MHzspectrometer,1minissufficient(evenlessifhigherconcentrationsofortho- phosphateareused).Asnocentrifugationisrequiredwiththismethod,thisshorttimeof signalacquisitioniscompatiblewithkineticstudies.Theresultsareexpressedas∆ν P , whichisthedifferencebetween∆ν 1/2 inthesystemwithparamagneticcationsand∆ν 1/2 inacontrolofthesamecomposition,(butwithoutparamagneticcations)dividedbythe concentrationofparamagneticcations.Thesurfacecoverageoftheclaybytheprotein canbededucedfromthefractionofMn 2ϩ released.Theknowledgeofboththequantity ofproteinadsorbedandthesurfacecoverageofthesolidallowsthecalculationofthe interfacialareaofcontactbetweenasingleproteinmoleculeandtheclaysurfaceatdiffer- entpHandionicstrengths. 2.ConformationalChangesonAdsorptionofaSoftProtein,Bovine SerumAlbumin a.DescriptionoftheProgressiveSurfaceCoverageoftheClayFigure1shows the evolution of ∆ν p , i.e., the release of the paramagnetic cation Mn 2ϩ , when the total quantity of bovine serum albumin (BSA) introduced in the clay suspension increases, and at a pH corresponding to the i.e.p. of the BSA (pH 4.7). The increase is linear, followed by a plateau. The plateau corresponds to the saturation of the montmorillonite surface, as shown by the comparison with the measurement, by UV adsorption (A 279 nm), of the BSA in the supernatant solution after centrifugation. The linear increase of ∆ν p before the pla- Copyright © 2002 Marcel Dekker, Inc. Figure1EffectoftheadditionofbovineserumalbuminonthereleaseofMn 2ϩ ,asdetectedby itsline-broadeningeffect∆νponorthophosphatebyNMR,andontheUVabsorptionA 279 nmof theprotein.Whenpresent,themontmorillonitesuspensionisat1gdm Ϫ3 ,pH4.65.(Adaptedfrom Ref.16.) teauindicatesthattheadsorbedproteinsalwayshavethesameinterfacialareaofcontact withtheclaysurface,whateverthesurfacecoverage.Nochange,fromaside-ontoan end-onstate,orfromanunfoldedtoamorenativestate,resultingfromanincreaseof lateralrepulsionswithpackingcanbeinvoked.Ifthereweresuchachangeinthemode ofadsorption,theamountofparamagneticcationreleasedperunitmassofproteinwould begreateratlowsurfacecoveragethanathighsurfacecoverage,andthiswouldbeseen asaconvexityratherthanalinearityofthecurve. b.MonolayerModeofAdsorptionThecomparisonbetweentheMn 2ϩ exchange dataandthedepletiondatainFigure1showsalsothatthemaximaladsorptionofBSA correspondstoamonolayer.Indeed,onlythecontactoftheproteinwiththeclaysurface canleadtotheexchangeofthecharge-compensatingcations.Asecondlayerwouldin- volveaprotein–proteincontact,withnoreleaseofMn 2ϩ .Thus,theoccurrenceofthe breaksinbothcationexchangeandproteindepletioncurvesatthesameproteinconcentra- tioniscompatibleonlywithamonolayerofproteinontheclaysurface. c.MaximumofAdsorptionatthei.e.p.Thedata,suchasthosepresentedin Fig.1,havebeencollectedoveralargepHrange.Theyallhavethesamegeneralaspect; onlytwoparametersvary.Fig.2showstheevolutionwithpHofthesetwoparameters: the plateau amount of BSA adsorbed on montmorillonite, measured by either NMR or the depletion method, and the maximal fraction of Mn 2ϩ that is displaced. As often is observed, the maximal amount of protein adsorbed occurs near the i.e.p. of the protein, which is 4.7 for BSA. Several hypotheses have been advanced to explain this phenomenon. One class of hypotheses is based on the same (symmetrical) mechanisms above and below the i.e.p. to explain the decrease of adsorption. They can be based on the effect of lateral electrostatic repulsions between the adsorbed proteins, which increase as the pH is more Copyright © 2002 Marcel Dekker, Inc. Figure 2 Effect of pH on the maximal amount of bovine serum albumin adsorbed on montmoril- lonite and on the clay surface coverage followed by the release of Mn 2ϩ on protein adsorption. (Adapted from Ref. 16.) distant from the i.e.p. (20–22). Alternatively, they can be based on a decrease in the structural stability of the protein when the net electric charge increases, leading to an unfolding of the protein (7,8,10). In addition, different (asymmetrical) mechanisms can be invoked above and below the i.e.p., and the comparison of the NMR exchange data and the protein adsorption data shows that such an asymmetrical mechanism is involved in the adsorption of BSA on montmorillonite, as explained later. d. Repulsive Electrostatic Interactions and Protein Unfolding Figure 2 shows that, above the i.e.p., the maximal amount of BSA adsorbed and the fraction of Mn 2ϩ exchanged decrease in exactly the same proportion. This good correlation between the quantity of protein adsorbed and the surface coverage of the clay surface supports a mecha- nism based on an increase of the electrostatic repulsion between the protein, whose net negative charge increases with pH above the i.e.p., and the montmorillonite, which carries a permanent negative charge. Figure 2 also shows that, below the i.e.p., the maximal amount of BSA adsorbed decreases, but the fraction of Mn 2ϩ exchanged remains nearly constant. Below the i.e.p. of the BSA (pH 4.7) there is no important variation in the number of positively charged side chains of the protein because the pKa of histidine (His), lysine (Lys), and arginine (Arg) is approximately pH 7, 10, and 12, respectively. The constant proportion of the cation exchanged by the BSA, despite a decreasing quantity adsorbed, can be explained only by an unfolding of the protein on the clay surface, moving more positively charged side chains of His, Lys, and Arg to near the surface. The increase of the specific interfacial area of the protein resulting from this unfolding is compatible with a smaller quantity adsorbed at constant surface coverage. It can be calculated from the data reported in Fig. 2 that the interfacial surface area occupied by a molecule of BSA on montmorillonite is 60 nm 2 at pH 4.5 and increases to 120 nm 2 at pH 3.0. Again, electro- static interactions appear to be the main driving force, but here they are attractive since the protein becomes more positively charged as the pH decreases below the i.e.p. and the montmorillonite remains negatively charged. BSA is a soft protein since, even at the i.e.p., the interfacial area of 60 nm 2 is higher than would be expected from the X-ray structure Copyright © 2002 Marcel Dekker, Inc. oftheanalogoushumanserumalbumin,whichhastheshapeofanequilateraltriangle withsidesof8nmandadepthof3nm(23,24).Ifnomodificationofconformationhad occuredonadsorptionatthei.e.p.,theinterfacialareaofcontactshouldhavebeen28 nm 2 foraside-onadsorption.ThisishalfofthevalueobtainedbyNMRatpH4.5. B.StudyoftheModificationinSecondaryStructuresbyFourier TransformInfraredSpectroscopy Althoughthedeterminationofthetertiarystructureofadsorbedproteinsisimpossible withthepresentstateofscientificknowledge,thestudyofchangesintherepartitionof thedifferentsecondarystructuresonadsorptionispossible.Thisdeterminationallowsthe deductionoftheoccurrenceofamodifiedconformation,anditalsoallowsdirectcalcula- tionofthecomponentoftheGibbsenergyofadsorptionthatisrelatedtothesestructural changes.Circulardichroismandinfraredspectroscopycanbeusedtoinvestigatethesec- ondarystructureofproteins.Circulardichroismhas,nevertheless,limitationsinturbid suspensionsbecauseoflight-scatteringeffectsandcanbeappliedonlytoparticleswith asizebelow30nm(25–27).Fouriertransforminfraredspectroscopy(FTIR)doesnot havethisdisadvantageandcanbeappliedtomoreturbidsuspensionsofclaysofagreater size. 1.FourierTransformInfraredSpectralAnalysis TransmissionFTIRspectrainthe1800-to1500-cm Ϫ1 regiongiveinformationonthe protonationstate,thesecondarystructure,andthesolvationoftheprotein.Allsamples werepreparedin 2 H 2 Omediuminordertoshiftthespectralabsorptiondomainofwater molecules,boundtothepolypeptidebackboneofthestudiedprotein,outoftheAmideI andIIspectralrange.Aphosphatebuffer(Na 2 H 2 PO 4 )wasusedatafinalconcentration of0.055molL Ϫ1 in 2 H 2 O.Severalp 2 Hvaluesinthe4–12rangewereobtainedbyadding 2 HClorNaO 2 H(11).Thestateoftheproteininsolutionisobtainedfromthespectral differencebetweentheproteininsolutionandthecorrespondingbuffer;thespectraldiffer- encebetweenthesolidprotein–claymixtureandthecorrespondingclaysuspensionreveals thestateoftheadsorbedprotein.Spectraldecompositioncouldbeachievedbysecond- derivative,curvatureanalysis,orself-deconvolutionprocedures.Thesamenumberofprin- cipalcomponentsoftheoverallspectrumrange(1500–1800cm Ϫ1 )wasobtainedatsimilar wavenumbers(Ϯ1cm Ϫ1 orless).Aleast-squareiterativecurve-fittingprogram(Leven- berg–Marquardt)wasappliedtofittheoverallspectrumwiththefoundnumberofprinci- palcomponents.Thefixedparameters(frequency,IRbandprofile)foranyspectraldecom- positionallowsacomparativequantitativeanalysisofintensitychangesforeach componentfromonespectrumtoanother.Examplesofinitialdifferencespectraandspec- traldecompositionofBSAinsolutionandadsorbedonmontmorillonitearereproduced inFig.3. The assignments specific for the Amide I′/I region are deduced from the literature and our own experiments on model amides, polypeptides, and proteins (28–40). The area of each Amide I component is expressed as a percentage of the sum of the areas of all Amide I components. Intensities (percentage peptide CO) are used to deduce the propor- tion of peptide units involved in the various solvated structural domains of the polypeptide backbone. The solvation parameter is given by the percentage of N 2 H. The level of exchange at a given time depends on the rate at which water molecules gain access to internal Copyright © 2002 Marcel Dekker, Inc. Figure3FTIR-vibrationalabsorptionspectra(1750–1500cm Ϫ1 )andcomputeddecomposition ofspectralprofilesforBSAinsolution(left)oradsorbedonmontmorillonite(right)atp 2 Hϭ5.6. (AdaptedfromRef.43.) peptidegroupsintheproteincore(28–32,41).Theprotonationparameterisgivenby COO Ϫ fractions(percentage)deducedfrommeasurementsoftheareaoftheν(CO) COOH absorptionfortheremainingCOOHspecies(withrespecttotheoverallAmideIintensity atagivenp 2 H).Theserelativeareasareexpressedwithrespecttothecorresponding relativeareas(percentage)obtainedatlowp 2 HwhenAspandGlusidechainsareall fullyprotonated(100%COOH). 2.ConformationalChangesonAdsorptionofaSoftProtein,Bovine SerumAlbumin AdsorptiononmontmorillonitesurfacesimpliespH-dependentchangesinBSAsolvation, unfoldingofhelicaldomains,aswellaschangesinhydrationandself-associateddomains (42,43). a.BSASolvationFigure4showstheadsorptioneffectsinducedbythenega- tivelychargedmontmorillonitesurfaceontheBSAsolvationwithp 2 H,proteinconcentra- tion,andtime.Theadsorptioneffectsalreadyareestablishedafter10min;therelative intensityissimplymorepronouncedat2hours.Foracidicp 2 H,theweakerexchange afteradsorptionsuggeststhattheelectronegativesurfaceprotectssomedomainofthe protein.Incontrast,inthei.e.p.range,adsorptionincreasestheNH/N 2 Hexchange,and athigherp 2 H,therateofwaterdiffusionisnolongerinfluencedbyadsorption. b.BSAProtonationBSAadsorptiononmontmorilloniteleadstotheproton- ationoftheionizablecarboxylicgroupsoftheprotein,asparticacid(Asp),andglutamic acid(Glu),atleasttop 2 H6.5(Fig.5).Variousreasonsmayexplainsuchashiftofthe apparent pK a of Asp and Glu. In the primary structure of BSA, some Asp and Glu side chains are adjacent to R ϩ functions. Embedded among positively charged side chains inter- acting with the electronegative clay or embedded in self-associated domains, external Asp and Glu side chains are assumed to become indifferent to buffer. Moreover, the electroneg- ative charge of the clay surface could favor a protonation of the Asp and Glu carboxylates to decrease the coulombic repulsion between the protein and the surface, as observed by titration on other systems (8,10). Copyright © 2002 Marcel Dekker, Inc. Figure4p 2 H-DependentNH/N 2 Hexchange(expressedasN 2 H%)ofBSAin 2 H 2 Oat10min and2h.∆N 2 H%representsthechangeinproteinsolvationforBSAadsorbedonmontmorillonite withrespecttothesolution.(AdaptedfromRef.43.) c.HelixUnfoldingMontmorilloniteinducesimportantunfoldingofhelicaldo- mainsofBSA(Fig.6).Afteradsorptiononmontmorillonite,thelargelyp 2 H-independent external helix unfolding is related to new orientations for the Lys ϩ and Arg ϩ side chains forced close to the negative clay surface. In contrast, unfolding in internal and packed helices is largely p 2 H-dependent. This change could be related to the disruption of some Figure 5 Effect of BSA adsorption on montmorillonite on the p 2 H-dependent Asp and Glu depro- tonation. (Adapted from Ref. 43.) Copyright © 2002 Marcel Dekker, Inc. [...]... parameters: (1) the deprotonation of the carboxylic side chains; (2) the deprotonation of two His side chains, increasing both protein flexibility and hydration; and (3) a local β sheet folding that results from the formation of a salt bridge between the Ile -1 6 (isoleucine) end chain aminium group and the Asp-194 side chain carboxylate For pH Ͼ 10, the Figure 7 Catalytic activity of α-chymotrypsin in the presence... cannot explain the inactivation of the enzyme in the 5–9 pH range by these weak structural changes alone If the tertiary structure of α-chymotrypsin, as determined by X-ray diffraction studies, is taken as relatively invariant on adsorption and if the time dependence of the Amide II intensity is analyzed for varying pH, information on the pH dependence of the α-chymotrypsin orientation on the montmorillonite... positively charged His -4 0 and His -5 7 imidazole and Ala -1 49 (alanine) end chain aminium that control the initial specific recognition of the substrate by the enzyme At pH higher than 8.5, when His-40, His-57, and Ala-149 are deprotonated, the enzyme is adsorbed with a different orientation, which allows a recovery of activity, similar to that measured in solution in the same pH range, since the catalytic site... limits the access of the substrate to this site Such a case has been described for the interaction of α-chymotrypsin with montmorillonite (32) 4 pH-Dependent Modifications of Conformation In contrast to the three preceding models, which assume that the enzymes retain the same conformation in the adsorbed state and in solution, another model is based on a pH-dependent unfolding of the enzyme on the surface... observed for the decreases of the Amide II bands for both adsorbed and solution states Unfolding of bundled/internal helical domains increases water diffusion inside the core of the protein (Fig 6) d Hydrated and Self-Associated Domains Helix unfolding increases the amount of self-association, free polar CO, and hydrated peptide CO Among the peptide units that Copyright © 2002 Marcel Dekker, Inc are unfolded,... obtained (32) The kinetics of the NH/N 2 H exchange measured by the Amide II intensity indicates which class of amino Copyright © 2002 Marcel Dekker, Inc acids are protected from water contact The analysis of the results shows that most of the inhibition would aries from a steric hindrance by the clay of the substrate access to the α-chymotrypsin catalytic site This is due to an interaction involving... give the pH of the catalytic reaction on the x axis (Adapted from Ref 52.) the reversal of the unfolding of the adsorbed enzyme therefore would be greater than the thermal energy, kT, available to the system 4 Inadequacy of the Interfacial pH Hypothesis The irreversible effect of pH changes on adsorbed enzyme activity is not the only fact that mitigates against the interfacial pH interpretation of the. .. this question since they, respectively, give information on the interfacial area of the surface in contact with the protein and on the secondary structure of adsorbed proteins It has been shown that both pH-dependent modification of conformation and pH-dependent orientation of the catalytic site of the enzyme can explain the alkaline pH shift of the enzyme activity on electronegative soil mineral surfaces... between the positively charged enzyme at this pH and the neutral polymer This mechanism is supported by the absence of inhibition of the β-d-glucosidase at pH 4 by a lysozyme–montmorillonite complex In this case, it is the strong interaction of the lysozyme with the clay surface, the high i.e.p of the protein, and its positive charge over the entire pH range studied that prevents exchange with the β-d-glucosidase... Marcel Dekker, Inc Figure 11 Effect of pH on the relative catalytic activity R in the adsorbed state and on the relative quantity F in the nonadsorbed state of two β-d-glucosidases from Aspergillus niger and sweet almond (Adapted from Ref 51.) negatively charged enzyme and the electronegative clay surface In contrast, when the pH decreases below the i.e.p., the relative activity, R, of the enzyme decreases . Inc. oftheanalogoushumanserumalbumin,whichhastheshapeofanequilateraltriangle withsidesof8nmandadepthof3nm(23,24).Ifnomodificationofconformationhad occuredonadsorptionatthei.e.p.,theinterfacialareaofcontactshouldhavebeen28 nm 2 foraside-onadsorption.ThisishalfofthevalueobtainedbyNMRatpH4.5. B.StudyoftheModificationinSecondaryStructuresbyFourier TransformInfraredSpectroscopy Althoughthedeterminationofthetertiarystructureofadsorbedproteinsisimpossible withthepresentstateofscientificknowledge,thestudyofchangesintherepartitionof thedifferentsecondarystructuresonadsorptionispossible.Thisdeterminationallowsthe deductionoftheoccurrenceofamodifiedconformation,anditalsoallowsdirectcalcula- tionofthecomponentoftheGibbsenergyofadsorptionthatisrelatedtothesestructural changes.Circulardichroismandinfraredspectroscopycanbeusedtoinvestigatethesec- ondarystructureofproteins.Circulardichroismhas,nevertheless,limitationsinturbid suspensionsbecauseoflight-scatteringeffectsandcanbeappliedonlytoparticleswith asizebelow30nm(25–27).Fouriertransforminfraredspectroscopy(FTIR)doesnot havethisdisadvantageandcanbeappliedtomoreturbidsuspensionsofclaysofagreater size. 1.FourierTransformInfraredSpectralAnalysis TransmissionFTIRspectrainthe1800-to1500-cm Ϫ1 regiongiveinformationonthe protonationstate,thesecondarystructure,andthesolvationoftheprotein.Allsamples werepreparedin 2 H 2 Omediuminordertoshiftthespectralabsorptiondomainofwater molecules,boundtothepolypeptidebackboneofthestudiedprotein,outoftheAmideI andIIspectralrange.Aphosphatebuffer(Na 2 H 2 PO 4 )wasusedatafinalconcentration of0.055molL Ϫ1 in 2 H 2 O.Severalp 2 Hvaluesinthe4–12rangewereobtainedbyadding 2 HClorNaO 2 H (11) .Thestateoftheproteininsolutionisobtainedfromthespectral differencebetweentheproteininsolutionandthecorrespondingbuffer;thespectraldiffer- encebetweenthesolidprotein–claymixtureandthecorrespondingclaysuspensionreveals thestateoftheadsorbedprotein.Spectraldecompositioncouldbeachievedbysecond- derivative,curvatureanalysis,orself-deconvolutionprocedures.Thesamenumberofprin- cipalcomponentsoftheoverallspectrumrange(1500–1800cm Ϫ1 )wasobtainedatsimilar wavenumbers(Ϯ1cm Ϫ1 orless).Aleast-squareiterativecurve-fittingprogram(Leven- berg–Marquardt)wasappliedtofittheoverallspectrumwiththefoundnumberofprinci- palcomponents .The xedparameters(frequency,IRbandprofile)foranyspectraldecom- positionallowsacomparativequantitativeanalysisofintensitychangesforeach componentfromonespectrumtoanother.Examplesofinitialdifferencespectraandspec- traldecompositionofBSAinsolutionandadsorbedonmontmorillonitearereproduced inFig.3. The. (2) a possi- ble unfolding of the protein on the surface changing the interfacial area between individual protein and surface and the quantity of protein adsorbed at saturation; (3) the surface coverage. contrast to the three preceding models, which assume that the enzymes retain the same conformation in the adsorbed state and in solution, another model is based on a pH-depen- dent unfolding of the enzyme