Reflections about the functional potential of legume proteins：A Review

Reflections about the functional potential of legume proteins A ReviewK.D.Schwenke1IntroductionThe quality of a food protein is determined both by its nutri-tional and functional properties,the latter being defined as the “physical and chemical properties which affect the behaviour of proteins in food systems during processing,storage,pre-paration and consumption”[1].Protein functionality in food systems comprises accordingly those properties such as solubi-lity,water binding,emulsification,foaming,gelling,cohesion-adhesion,fat and flavour binding etc.These properties may be more important than nutritional properties (biological value,digestibility etc.)in the case,when the protein is applied not as main component but as an ingredient in a food system.Taking into account the multicomponent character of food systems,functionality should finally be considered as the result of inter-action of proteins with the other food components,may be with other proteins [2–6],may be with polysaccharides [7–10]or lipids [11–14].In plant protein containing systems,the interaction with low molecular mass compounds such as phe-nolics [15–17],phytic acid [18–20]and others is additionally to be taken into account.The interaction capacity is thus an important part of protein functionality.It is essentially determined by the conformation of the proteins and the chemical and steric properties of the protein surface.The elucidation of the dependence of func-tional properties on the structure of proteins,became therefore an exciting task in recent food protein research (see e.g.[21–26]).Some functional properties can be understood to be related to the “interaction capacity”of proteins.To these belong the solubility,water,fat and flavour binding as well as the surface active or “interfacial”properties,i.e.the foaming and emulsi-fying behaviour.They can be therefore regarded as manifesta-tions of the “functional potential”of the protein surface.On the other hand,viscosity and gelling behaviour etc.are mani-festations of hydrodynamic properties of protein macromole-cules that are affected largely by the actual shape and size ofthe macromolecules [22].Speaking about the “functional potential”of food proteins means that the complex of physico-chemical properties characteristic of the protein structure,including the protein capacity to conformational transition (“conformational potential”),can be realized in the course of a food system processing.In contrast to that,the term “func-tional properties”[1]characterizes the behaviour of a food sys-tem under conditions,which are determined by the require-ments of the practice.The term “techno-functional properties”may be therefore appropriate in some cases.The discussion in the present paper will be focussed on the concepts of evaluation of protein surface properties related to functional properties,i.e.the theoretical and practical estima-tion of surface hydrophobicity,on structural peculiarities of legume 11S and 7S storage proteins and on methods of mod-ification of their surface structure and conformation for under-standing of structure-functionality relationships and a better exploitation of the functional potential of these proteins.2Protein hydrophobicity and its relation to functionalityAttempts to relate the hydrophobic and hydrophilic amino acid content of proteins to their physical properties have been made already about forty years ago [27–30].The average hydrophobicity (H u ave )and the charge frequency were pro-posed by Bigelow [30]to be the most important molecular fea-tures that have the greatest influences on the physical proper-ties,such as solubility,of proteins.Since the solubility of a protein under given solution conditions can be understood as the manifestation of the equilibrium between the protein-sol-vent (hydrophilic)and the protein-protein (hydrophobic)inter-action,proteins having lower average hydrophobicity and higher charge frequency would have –according to Bigelow [30]–a higher solubility.This empirical relationship has been confirmed for most proteins.However,it does not explain the solubility characteristics of a number of proteins.In fact,it is the hydrophobicity and the charge distribution of the protein surface and not the average hydrophobicity or charge fre-quency of the molecule as a whole,which are critically impor-tant for its functionality.The average hydrophobicity accord-Correspondence to:Prof.Dr.K.D.Schwenke ,Institut für Angewandte Proteinchemie,e.V.,Forschungsgruppe Pflanzenprotein-Chemie,Stahnsdorfer Damm 81,D-14532Kleinmachnow,Germany (e-mail:schwenke@prochem-potsdam.de).The term “functional potential”was introduced to better approach to the understanding of the relationships between the structure and the functional properties of food proteins.Although in practice the complex of structural features of a protein contributes to its functionality,it is very useful to regard the functional potential of the protein surface on the one side and to look on special effects of protein conformation (role of compact or unfolded state)on the functionality on the other side.This point of view may help to design the best strategy of modification of the protein structure and functionality.The special situation of the oligomeric legume storage protein,i.e.legumin-like 11S globulins and vicilin-like 7S globulin,and the structural and functional modification of 11S globulin by limited tryptic hydrolysis and by acylation are dis-cussed in thispaper.ing to Bigelow may,however,serve as on indicator of struc-tural stability of a globular protein.The average hydrophobicity of most globular proteins including11S seed storage proteins range between3768and 4187J/res.(900and1000cal/res.)[30,31].Since a consider-able high part of hydrophobic amino acid residues serve to sta-bilize the inner part of the proteins,large discrepancies were found between the average hydrophobicity according to Bige-low and the surface hydrophobicity(S0)determined by fluores-cence probe techniques as introduced by Nakai et al.[32–34]. The experimentally determined“apparent hydrophobicity”S0of number of plant and animal proteins was shown to be cor-related with the emulsifying activity index(EAI)and the emul-sifying stability index(ESI)[35],whereas the foaming capac-ity(FC)and the foam stability(FS)showed a better correlation with the“exposable hydrophobicity”S e,i.e.the S0-value of unfolded proteins(eqn.1and2)[33,35,36].EAI=constant+0.214S0+0.932s–0.007s2(1)(S0[%–1]hydrophobicity,s[%]solubility index)FC=constant+25.93ln S e+1493g(2)(S e=S0of unfolded protein,g[mPa s]viscosity) These results indicate a more extensive uncoiling of protein molecules at an air/water interface than at an oil/water inter-face as reported by Graham et al.[37].As could be expected, S e was more closely related to the thermal properties(thicken-ing,heat coagulation and gelation)than S0[34,36].In these cases,the average hydrophobicity according to Bigelow (H u ave)may give a physically defined quantity(J/res.)for the prediction of functionality.The limits of application of fluorescence probe techniques for the estimation of protein surface hydrophobicity became evident,when the protein surface was modified by an increas-ing number of charged hydrophilic residues.Thus,increasing extent of succinylation of the11S globulin from faba bean (“legumin”)resulted in a slight increase and not in a decrease of the S0-value using ANS(1-anilino-8-naphthalene sulfonic acid),a commonly applied fluorescence probe[38,39].S0 decreased only,when an extremely high charge density was attained by the exhaustive acylation of both amino and hy-droxyl groups.In this case,the fluorescence probe is able to response to newly exposed hydrophobic clusters and not to give a response to the overall hydrophobicity/charge density ratio of the surface.The decrease of S0at excessive modifica-tion may be due to the effect of electrostatic repulsion between the negatively charged ANS molecules and succinyl residues and due to steric effects.In contrast to that,two-phase partition in a polymer–poly-mer(Ficoll/Dextran)-water system resulted in an increased hydrophilicity of the protein with increasing degree of succinylation well in accordance with the computed hydro-pathicity profiles[38].Partition in a suitable two-phase system seems to be,therefore,a useful completion of fluorescence probe methods for studying the protein surface properties. The surface functionality of protein largely depends on the extent of interaction of the protein surface with the surround-ing solvent and finally with an interface.The accessible sur-face area concept,introduced to help understand the role of hydrophobic interactions in protein stability[40],may also be helpful for understanding the surface activity.Despite the dif-ferences in the amino acid sequence and the secondary struc-ture the accessible surface(A n)area of globular proteins of a molecular mass of6000to35000has been shown to be related to the molecular mass[41,42]as follows:A n=11.1M2/3(3) A n is nearly exactly twice that of a sphere of the same mass and density[42].The excess area of proteins is largely attribut-able to the roughness of their surface.The correlation between A n and M shows that“the actual shape,more often an ellipsoid than a sphere,and the presence of crevices such as found at active centre,have only minor effects on the value of A n”[42]. Since the accessible surface area(A u)and the molecular mass of an unfolded polypeptide chain was shown to follow the rela-tion A u=1.44M[42],the net hydrophobic surface,i.e.A u–A n,buried during folding or gained by unfolding of monomeric globular proteins can be calculated[41].For larger globular proteins that have mostly an oligomeric structure such as the 11S and7S plant storage proteins,a significant fraction of their surface area is buried in subunit–subunit interfaces[43]. Correspondingly,the accessible surface area of the hexameric legumin-like11S globulins is considerably reduced by the subunit contact area.Intersubunit volumes unaccessible to the solvent should also be taken into consideration.The hypotheti-cal accessible surface area of the60kDa subunit,which do not exist in native state in a free form,amounts to170nm2and increases to864nm2after dissociation and unfolding.This large gain in accessible surface area enables the denatured pro-tein to interact in a manifold way.The higher the percentage of hydrophobic amino acid residues of the protein the higher is the tendency to form coagulated structures after heating, whereas proteins with a low content in hydrophobic amino acids such as gelatin tend to form translucent gel networks [44].(For a detailed discussion of the theory of gelation and the various interaction during gelation see[45]).3Structure and physico-chemical properties of legume storage proteins with regard to their functionalityThe11S and7S storage proteins of legumes are built up of polymorphic subunits encoded by multigene families[46,47]. They form,however,regular quaternary structures,hexameric in the case of legumin-like proteins and trimeric for vicilin-like proteins[46,47],whose association–dissociation behav-iour[25,48–50],along with tridimensional and surface struc-ture as well as the conformational stability[46,51–55]are most important for understanding the functionality of these proteins.Thus,the different surface structure and hydrophobic-ity of pea legumin and vicilin,to which the glycosylated state of the latter[46]considerably contributes,is reflected by rather different interfacial properties of both proteins[56].Moreover, despite an analogous subunit arrangement and a rather high similarity in the secondary structure7S globulins from various grain legumes can exhibit considerable differences in the sur-face hydrophobicity as shown for phaseolin from dry bean, vicilin from field pea and b-conglycinin from soybean[57]. Structural peculiarities of globulins of one and the same struc-tural type but of various botanical origin should be,therefore, taken into account,when their physico-chemical and func-tional properties are compared.On the other hand,compara-tive studies with homologous seed proteins are very useful to recognize structure-dependent regularities in the properties of 11S or7S globulins.In the case of11S globulins,the arrangement of the six sub-units is best approximated by the model of a trigonal antriprism [53],though the exact spatial structure using X-ray structural analysis could not be determined as yet due to often varying sub-unit composition with the subunit arising from different genes and due to some hypervariable regions,which may not adapt a defined secondary structure[54].It is the non-specific hydro-phobic interaction of the50–60kDa subunits that allows them to assemble in a legumin-like quaternary structure,even subunits from various11S globulins[53,58].The two disulphide-bridged polypeptide chains of these subunits fullfill different functions in stabilizing the globular structure.While the N-terminal regions of the30–40kDa a-chains and the20kDa b-chains have a con-served sequence forming two structural domains,the hydropho-bic C-terminal of the b-chains,which shows a certain sequence variability,is assumed to cause the non-specific subunit interac-tion[53].On the other hand,the strong hydrophilic C-terminal region of the a-chain is located at the surface of the protein mole-cule and protect the domains from the solvent[53].It is therefore of high importance for the solubility and interfacial properties of 11S globulins.Besides the properties of the protein surface,the rather com-pact oligomeric structure[46,51,53]and the dominating b-sheet conformation in the secondary structure[59]are to be considered as characteristic molecular features markedly influ-encing the functional properties of11S globulins.4Modification of the11S globulins by limited proteolysisLimited tryptic hydrolysis of11S globulins results in the splitting of the surface–exposed regions of a-chains,while the b-chains located in the inner part of the protein molecules remains intact[60–62].The loss of hydrophilic peptides leads to the reduction of the molecular mass from350–360kDa to 230kDa in soybean glycinin[63],240–250kDa in faba bean legumin[64,65]and260–270kDa in pea legumin[66].The resulting core(“glycinin-T”or“legumin-T”)are characterized by an increased average hydrophobicity[67,68]and by the maintenance of the principal quaternary structure of the11S globulins[61–63].The domain stabilizing b-sheet conforma-tion in the secondary structure of both pea and faba bean legu-min-T remained unchanged,which is also reflected by a high and even increased conformational stability[66,67].The sur-face activity of faba bean legumin increased considerably dur-ing the process of limited or even super limited proteolysis, i.e.when only few peptide bonds were cleaved[68,69].This phenomenon was used already to increase the foaming and emulsifying properties in soybean protein isolates[70,71]. These functional changes are highest,when the structural state of globulin-T is approached.The enzymatic breakdown of the latter during proceeding proteolysis impaired the surface func-tional properties.The presence of a sufficiently high molecular mass product is therefore a precondition for good surface func-tional properties,especially for the stabilization of emulsions and foams.The results obtained with enzymatically nicked legumin point however to a co-operative effect of both the core-protein(globulin-T)and the tryptically split peptides loosely bound by non-covalent interaction to the latter[68,69].The proteolytic modification results in a loosening of the a-chains that favours the processes which take place during the emulsifying,i.e.the interface adsorption and stabilization of oil droplets.Both the enhanced flexibility of the tryptically nicked a-chains and the increased hydrophobicity of the core protein should facilitate the anchoring of the protein at the oil/ water interface.One might assume that small peptides,which are able to reach the o/w interface very quickly,could act as pre-stabilizers of the very small oil droplets similarly as small detergents.The legumin-T reaching the interface later provides the long-term stabilization of the emulsions.Without any legu-min-T,emulsions could not be formed[67,68].5Changing the protein surface and conformation by chemical modificationThe various methods of chemical modification[72]provide manifold possibilities of modification of the protein structure well applicable to11S and7S storage proteins.Although the group specificity of the different reagents may be limited,it can be increased by careful selection of the reaction conditions.Acy-lation is a common method that allows a successive substitution of the positively charged amino groups by neutral(acetylation, lipophilization)or negatively charged(succinylation,maleyla-tion,citraconylation)residues[72].Thus,increasing or decreas-ing charge and hydrophobicity results in modified legume pro-teins with a large spectrum of physico-chemical and functional properties(see e.g.[39,73–82]).A considerable part of the sur-face exposed lysine residues in pea and faba bean legumin can be succinylated or acetylated without marked changes in the com-pact oligomeric structure[39,79].The changes in the interfacial and emulsifying properties[82–84]can in these cases be attribu-ted to the modified surface properties of the proteins.Proceeding succinylation results in a stepwise dissociation into the subunits and finally into their unfolding[39,79].The latter takes place most efficiently,when the most part(85–95%)of NH2-groups were acylated and the following succinylation of OH-groups of hydroxyamino acids additionally contributes to the electrostatic repulsion effects of the polypeptide chains.In contrast to succi-nylation,acetylation causes mainly an aggregation(oligomeriza-tion)of legumin due to the markedly increased hydrophobicity [81].The conformation of acetylated legumin subunits can be described as a structural state between a compact globular and an unfolded conformation.It might be approached by a molten glo-bule-like state,though the latter is strongly described only for small monomeric globulins[85].Such a partially unfolded but still globular conformation is attained in succinylated legumin only at medium degrees of modification[39].Excessive acetyla-tion or medium succinylation of faba bean legumin resulted in products with highest surface activity and best emulsifying prop-erties,the parameters of which were manifold increased[82–84].Indeed,it has been shown,that the molten globule-like con-formation ensures highest surface functionality[86].The enhanced flexibility of the polypeptide chains in this structural state allows a greater mobility at the interface and bet-ter penetration into the non-aqueous phase than the compact native structure.Moreover,the packing density of the partially unfolded but still globular modified legumin at the interface, which is related to the interfacial film stability,is higher than that of the completely unfolded protein,which tend to form statisti-cally aggregated products less suited for interface adsorption.The highly hydrophilic and partially unfolded succinylated faba bean protein isolates show excellent foaming properties in an unheated state,whereas heat denaturation is necessary for maximum foaming of the unmodified isolates[87].The loos-ening of the compact conformation,i.e.the increase in confor-mational flexibility,that can be caused in unmodified proteins at pH’s far enough from the I.P.under acidic or alkaline condi-tions,is induced after succinylation or related chemical modi-fication at moderate pH-conditions.The changed conforma-tion and increased hydrophilic properties of the succinylated protein isolates also results in the decrease of gelling tempera-ture and an improving of the properties of the formed gels, which are translucent in contrast to those from unmodified proteins[88].This kind of hydrophilization prevent the protein during the process of unfolding from undesired aggregation.It is thus a method of choice for the modification of plant pro-teins,when their purpose of application demands a highly unfolded but non-aggregated and well soluble protein.The application of modified plant protein isolates incoatings and adhesives[89]is a good example.Since the chemical modifi-cation of plant proteins has found its domain of application in the field of non-food use[90,91],the knowledge on structure-functionality relationships gained by various chemical reac-tions on legume storage proteins will find here its best utiliza-tion.6Conclusions and outlookThe polymorphic nature of the subunits of11S and7S glo-bulins may be a drawback for the study of structure-functional-ity relationships.To overcome this obstacle,homo-oligomeric proteins were prepared by reconstitution of the hexameric or trimeric structure using isolated and purified homogeneous subunits[92–94].In this way the mixed patterns of breakdown products resulting from the action of trypsin on the surface-exposed a-chains of faba bean legumin[62]could be unequi-vocally assigned to the A-type and B-type subunits,when reconstituted“pseudolegumins”consisting only of one of these subunit types were studied[95].Another approach to obtain a homohexameric legumin consists in the application of gene technique methods.The attempts to produce a uniform60kDa pea legumin subunit assembled to a homogeneous360kDa globulin in a transgenic plant are discussed in these Proceed-ings by Casey et al.[96].On the other hand,parameters of the surface hydrophobicity as well as the charge density,evaluated by using appropriate methods can provide sufficiently useful information to assign functionality to protein surface properties.For this purpose, the commonly used fluorescence probe techniques providing a relative hydrophobicity index(S0)only should be completed by methods,which result in well-defined physical(thermody-namic)quantities.Both ligand binding and partition methods may be in principle suitable techniques.However,an optimal approach is still to be developed.More advanced data process-ing techniques,e.g.multivariate analysis,are required as pro-posed by Nakai[97].The functional potential of legume proteins can only be fully exploited,when protein denaturation and irreversible aggrega-tion during isolate production are avoided.Interactions with polyphenolic compounds(see Kroll et al.in these Proceedings [98])or oxidized lipids[13]can drastically reduce the func-tionality of protein bined interaction with various non-protein seed components,e.g.phytic acid,and aggrega-tion of the protein during isoelectric precipitation mostly results in a considerable decrease of functional properties of isolates.In this context,the isoelectric pre-extraction of seed meals for removing soluble low molecular weight can result in a large loss 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